chapter
N
ucleotides have a variety of roles in cellular metab-
olism. They are the energy currency in metabolic
transactions, the essential chemical links in the re-
sponse of cells to hormones and other extracellular stim-
uli, and the structural components of an array of en-
zyme cofactors and metabolic intermediates. And, last
but certainly not least, they are the constituents of nu-
cleic acids: deoxyribonucleic acid (DNA) and ribonu-
cleic acid (RNA), the molecular repositories of genetic
information. The structure of every protein, and ulti-
mately of every biomolecule and cellular component, is
a product of information programmed into the nu-
cleotide sequence of a cell’s nucleic acids. The ability to
store and transmit genetic information from one gener-
ation to the next is a fundamental condition for life.
This chapter provides an overview of the chemical
nature of the nucleotides and nucleic acids found in
most cells; a more detailed examination of the function
of nucleic acids is the focus of Part III of this text.
8.1 Some Basics
Nucleotides, Building Blocks of Nucleic Acids The amino acid
sequence of every protein in a cell, and the nucleotide
sequence of every RNA, is specified by a nucleotide se-
quence in the cell’s DNA. A segment of a DNA molecule
that contains the information required for the synthesis
of a functional biological product, whether protein or
RNA, is referred to as a gene. A cell typically has many
thousands of genes, and DNA molecules, not surpris-
ingly, tend to be very large. The storage and transmis-
sion of biological information are the only known func-
tions of DNA.
RNAs have a broader range of functions, and sev-
eral classes are found in cells. Ribosomal RNAs
(rRNAs) are components of ribosomes, the complexes
that carry out the synthesis of proteins. Messenger
RNAs (mRNAs) are intermediaries, carrying genetic
information from one or a few genes to a ribosome,
where the corresponding proteins can be synthesized.
Transfer RNAs (tRNAs) are adapter molecules that
faithfully translate the information in mRNA into a
specific sequence of amino acids. In addition to these
major classes there is a wide variety of RNAs with spe-
cial functions, described in depth in Part III.
Nucleotides and Nucleic Acids Have Characteristic
Bases and Pentoses
Nucleotides have three characteristic components:
(1) a nitrogenous (nitrogen-containing) base, (2) a pen-
tose, and (3) a phosphate (Fig. 8–1). The molecule with-
out the phosphate group is called a nucleoside. The
nitrogenous bases are derivatives of two parent com-
pounds, pyrimidine and purine. The bases and pentoses
of the common nucleotides are heterocyclic compounds.
The carbon and nitrogen atoms in the parent structures
are conventionally numbered to facilitate the naming
and identification of the many derivative compounds.
The convention for the pentose ring follows rules out-
lined in Chapter 7, but in the pentoses of nucleotides
NUCLEOTIDES AND
NUCLEIC ACIDS
8.1 Some Basics 273
8.2 Nucleic Acid Structure 279
8.3 Nucleic Acid Chemistry 291
8.4 Other Functions of Nucleotides 300
A structure this pretty just had to exist.
—James Watson, The Double Helix, 1968
8
273
and nucleosides the carbon numbers are given a prime
(H11032) designation to distinguish them from the numbered
atoms of the nitrogenous bases.
The base of a nucleotide is joined covalently (at N-1
of pyrimidines and N-9 of purines) in an N-H9252-glycosyl
bond to the 1H11032 carbon of the pentose, and the phosphate
is esterified to the 5H11032 carbon. The N-H9252-glycosyl bond is
formed by removal of the elements of water (a hydroxyl
group from the pentose and hydrogen from the base),
as in O-glycosidic bond formation (see Fig. 7–31).
Both DNA and RNA contain two major purine bases,
adenine (A) and guanine (G), and two major pyrim-
idines. In both DNA and RNA one of the pyrimidines is
cytosine (C), but the second major pyrimidine is not
the same in both: it is thymine (T) in DNA and uracil
(U) in RNA. Only rarely does thymine occur in RNA or
uracil in DNA. The structures of the five major bases
are shown in Figure 8–2, and the nomenclature of their
corresponding nucleotides and nucleosides is summa-
rized in Table 8–1.
Nucleic acids have two kinds of pentoses. The re-
curring deoxyribonucleotide units of DNA contain 2H11032-
deoxy-D-ribose, and the ribonucleotide units of RNA
contain D-ribose. In nucleotides, both types of pentoses
are in their H9252-furanose (closed five-membered ring)
form. As Figure 8–3 shows, the pentose ring is not pla-
nar but occurs in one of a variety of conformations gen-
erally described as “puckered.”
Figure 8–4 gives the structures and names of the
four major deoxyribonucleotides (deoxyribonucleo-
side 5H11032-monophosphates), the structural units of DNAs,
and the four major ribonucleotides (ribonucleoside 5H11032-
monophosphates), the structural units of RNAs. Specific
long sequences of A, T, G, and C nucleotides in DNA are
the repository of genetic information.
Although nucleotides bearing the major purines and
pyrimidines are most common, both DNA and RNA also
Chapter 8 Nucleotides and Nucleic Acids274
FIGURE 8–1 Structure of nucleotides. (a) General structure showing
the numbering convention for the pentose ring. This is a ribonu-
cleotide. In deoxyribonucleotides the OOH group on the 2H11032 carbon
(in red) is replaced with OH. (b) The parent compounds of the pyrim-
idine and purine bases of nucleotides and nucleic acids, showing the
numbering conventions.
(b)
(a)
FIGURE 8–2 Major purine and pyrimidine bases of nucleic acids.
Some of the common names of these bases reflect the circumstances
of their discovery. Guanine, for example, was first isolated from guano
(bird manure), and thymine was first isolated from thymus tissue.
FIGURE 8–3 Conformations of ribose. (a) In solution, the straight-
chain (aldehyde) and ring (H9252-furanose) forms of free ribose are in equi-
librium. RNA contains only the ring form, H9252-D-ribofuranose. Deoxy-
ribose undergoes a similar interconversion in solution, but in DNA
exists solely as H9252-2H11032-deoxy-D-ribofuranose. (b) Ribofuranose rings in
nucleotides can exist in four different puckered conformations. In all
cases, four of the five atoms are in a single plane. The fifth atom
(C-2H11032 or C-3H11032) is on either the same (endo) or the opposite (exo) side
of the plane relative to the C-5H11032 atom.
8.1 Some Basics 275
CH
2
O
H11002
O
OH
H
P
CH
3
O
H11002
HN
N
H
H
H
H
O
T, dT, dTMP
Deoxythymidine
Nucleotide: Deoxyadenylate
(deoxyadenosine
5H11032-monophosphate)
Deoxyguanylate
(deoxyguanosine
5H11032-monophosphate)
Deoxythymidylate
(deoxythymidine
5H11032-monophosphate)
Deoxycytidylate
(deoxycytidine
5H11032-monophosphate)
Symbols: A, dA, dAMP
Nucleoside: Deoxyadenosine
O
G, dG, dGMP
Deoxyguanosine
O
C, dC, dCMP
Deoxycytidine
(a) Deoxyribonucleotides
O
O
CH
2
N
O
H11002
O
OH
H
P
NH
2
O
H11002
N
N
N
H
H
H
H
O
O
CH
2
O
H11002
O
OH
H
P
HN
H
2
N
O
H11002
N
N
N
H
H
H
H
O
O
CH
2
O
H11002
O
OH
H
P
NH
2
O
H11002
N
N
H
H
H
H
O
O
O
O
CH
2
N
O
H11002
O
OH
H
P
NH
2
O
H11002
N
N
N
H
H
H
O
O
CH
2
O
H11002
O
OH
H
P
HN
H
2
N
O
H11002
N
N
N
H
H
H
O
O
CH
2
O
H11002
O
OH
H
P
O
H11002
N
N
H
H
H
O
O
(b) Ribonucleotides
U, UMP C, CMP
Uridine
Nucleotide: Adenylate (adenosine
5H11032-monophosphate)
Guanylate (guanosine
5H11032-monophosphate)
Uridylate (uridine
5H11032-monophosphate)
Cytidylate (cytidine
5H11032-monophosphate)
Symbols: A, AMP
Nucleoside: Adenosine
G, GMP
Guanosine Cytidine
CH
2
O
H11002
O
OH
H
P
NH
2
O
H11002
N
N
H
H
H
O
O
O
OH OH OH OH
H
O
O
FIGURE 8–4 Deoxyribonucleotides and ribonucleotides of nucleic
acids. All nucleotides are shown in their free form at pH 7.0. The nu-
cleotide units of DNA (a) are usually symbolized as A, G, T, and C,
sometimes as dA, dG, dT, and dC; those of RNA (b) as A, G, U, and
C. In their free form the deoxyribonucleotides are commonly abbre-
viated dAMP, dGMP, dTMP, and dCMP; the ribonucleotides, AMP,
GMP, UMP, and CMP. For each nucleotide, the more common name
is followed by the complete name in parentheses. All abbreviations
assume that the phosphate group is at the 5H11032 position. The nucleoside
portion of each molecule is shaded in light red. In this and the fol-
lowing illustrations, the ring carbons are not shown.
TABLE 8–1 Nucleotide and Nucleic Acid Nomenclature
Base Nucleoside Nucleotide Nucleic acid
Purines
Adenine Adenosine Adenylate RNA
Deoxyadenosine Deoxyadenylate DNA
Guanine Guanosine Guanylate RNA
Deoxyguanosine Deoxyguanylate DNA
Pyrimidines
Cytosine Cytidine Cytidylate RNA
Deoxycytidine Deoxycytidylate DNA
Thymine Thymidine or deoxythymidine Thymidylate or deoxythymidylate DNA
Uracil Uridine Uridylate RNA
Note: “Nucleoside” and “nucleotide” are
generic terms that include both ribo- and
deoxyribo- forms. Also, ribonucleosides and
ribonucleotides are here designated simply
as nucleosides and nucleotides (e.g., ribo-
adenosine as adenosine), and deoxyribo-
nucleosides and deoxyribonucleotides as
deoxynucleosides and deoxynucleotides
(e.g., deoxyriboadenosine as deoxyadeno-
sine). Both forms of naming are accept-
able, but the shortened names are more
commonly used. Thymine is an exception;
“ribothymidine” is used to describe its
unusual occurrence in RNA.
contain some minor bases (Fig. 8–5). In DNA the most
common of these are methylated forms of the major
bases; in some viral DNAs, certain bases may be hy-
droxymethylated or glucosylated. Altered or unusual
bases in DNA molecules often have roles in regulating
or protecting the genetic information. Minor bases of
many types are also found in RNAs, especially in tRNAs
(see Fig. 26–24).
The nomenclature for the minor bases can be con-
fusing. Like the major bases, many have common names—
hypoxanthine, for example, shown as its nucleoside ino-
sine in Figure 8–5. When an atom in the purine or
pyrimidine ring is substituted, the usual convention (used
here) is simply to indicate the ring position of the sub-
stituent by its number—for example, 5-methylcytosine,
7-methylguanine, and 5-hydroxymethylcytosine (shown
as the nucleosides in Fig. 8–5). The element to which
the substituent is attached (N, C, O) is not identified.
The convention changes when the substituted atom is
exocyclic (not within the ring structure), in which case
the type of atom is identified and the ring position to
which it is attached is denoted with a superscript. The
amino nitrogen attached to C-6 of adenine is N
6
; simi-
larly, the carbonyl oxygen and amino nitrogen at C-6
and C-2 of guanine are O
6
and N
2
, respectively. Examples
of this nomenclature are N
6
-methyladenosine and N
2
-
methylguanosine (Fig. 8–5).
Cells also contain nucleotides with phosphate
groups in positions other than on the 5H11032 carbon (Fig.
8–6). Ribonucleoside 2H11541,3H11541-cyclic monophosphates
are isolatable intermediates, and ribonucleoside 3H11541-
monophosphates are end products of the hydrolysis
of RNA by certain ribonucleases. Other variations are
adenosine 3H11032,5H11032-cyclic monophosphate (cAMP) and
guanosine 3H11032,5H11032-cyclic monophosphate (cGMP), consid-
ered at the end of this chapter.
Phosphodiester Bonds Link Successive Nucleotides
in Nucleic Acids
The successive nucleotides of both DNA and RNA are
covalently linked through phosphate-group “bridges,” in
which the 5H11032-phosphate group of one nucleotide unit is
Chapter 8 Nucleotides and Nucleic Acids276
(a)
(b)
FIGURE 8–5 Some minor purine and pyrimidine bases, shown as the
nucleosides. (a) Minor bases of DNA. 5-Methylcytidine occurs in the
DNA of animals and higher plants, N
6
-methyladenosine in bacterial
DNA, and 5-hydroxymethylcytidine in the DNA of bacteria infected
with certain bacteriophages. (b) Some minor bases of tRNAs. Inosine
contains the base hypoxanthine. Note that pseudouridine, like uridine,
contains uracil; they are distinct in the point of attachment to the
ribose—in uridine, uracil is attached through N-1, the usual attach-
ment point for pyrimidines; in pseudouridine, through C-5.
FIGURE 8–6 Some adenosine monophosphates. Adenosine 2H11032-
monophosphate, 3H11032-monophosphate, and 2H11032,3H11032-cyclic monophosphate
are formed by enzymatic and alkaline hydrolysis of RNA.
joined to the 3H11032-hydroxyl group of the next nucleotide,
creating a phosphodiester linkage (Fig. 8–7). Thus
the covalent backbones of nucleic acids consist of al-
ternating phosphate and pentose residues, and the ni-
trogenous bases may be regarded as side groups joined
to the backbone at regular intervals. The backbones of
both DNA and RNA are hydrophilic. The hydroxyl
groups of the sugar residues form hydrogen bonds with
water. The phosphate groups, with a pK
a
near 0, are
completely ionized and negatively charged at pH 7, and
the negative charges are generally neutralized by ionic
interactions with positive charges on proteins, metal
ions, and polyamines.
All the phosphodiester linkages have the same ori-
entation along the chain (Fig. 8–7), giving each linear
nucleic acid strand a specific polarity and distinct 5H11032 and
3H11032 ends. By definition, the 5H11541 end lacks a nucleotide at
the 5H11032 position and the 3H11541 end lacks a nucleotide at the
3H11032 position. Other groups (most often one or more phos-
phates) may be present on one or both ends.
The covalent backbone of DNA and RNA is subject
to slow, nonenzymatic hydrolysis of the phosphodiester
bonds. In the test tube, RNA is hydrolyzed rapidly un-
der alkaline conditions, but DNA is not; the 2H11032-hydroxyl
groups in RNA (absent in DNA) are directly involved in
the process. Cyclic 2H11032,3H11032-monophosphate nucleotides
are the first products of the action of alkali on RNA and
are rapidly hydrolyzed further to yield a mixture of 2H11032-
and 3H11032-nucleoside monophosphates (Fig. 8–8).
The nucleotide sequences of nucleic acids can be
represented schematically, as illustrated on the follow-
ing page by a segment of DNA with five nucleotide units.
The phosphate groups are symbolized by PH22071, and each
deoxyribose is symbolized by a vertical line, from C-1H11032
at the top to C-5H11032 at the bottom (but keep in mind that
8.1 Some Basics 277
O
H11002
RNA
CH
2
O
H11002
O
H
P
H
OH
H
O
3H11032
5H11032
U
H
O
CH
2
O
H11002
O
H
P
HH
O
O
3H11032
5H11032
G
H
O
CH
2
O
H11002
O
H
P
HH
O
O
3H11032
5H11032
H
O
H
O
5H11032 End
O
H11002
CH
2
O
H11002
O
H
P
H
H
H
O
3H11032
5H11032
A
H
O
CH
2
O
H11002
O
H
P
H
H
H
O
O
3H11032
5H11032
T
H
O
CH
2
O
H11002
O
H
P
H
H
H
O
O
3H11032
5H11032
G
H
O
H
O
5H11032 End
3H11032 End3H11032 End
C
5H11541
3H11541
DNA
Phospho-
diester
linkage
OH
OH
FIGURE 8–7 Phosphodiester linkages in the covalent backbone of
DNA and RNA. The phosphodiester bonds (one of which is shaded in
the DNA) link successive nucleotide units. The backbone of alternat-
ing pentose and phosphate groups in both types of nucleic acid is
highly polar. The 5H11032 end of the macromolecule lacks a nucleotide at
the 5H11032 position, and the 3H11032 end lacks a nucleotide at the 3H11032 position.
H
P
H
H
H
O
H11002
OH
2H11032,3H11032-Cyclic
monophosphate
derivative
O
O
CH
2
O
H
P
H
H
H
O
O
Base
1
O
O
H11002
O
H
CH
2
O
H
P
H
H
H
O
O
Base
2
O
H11002
O
H
OP
H11002
O
CH
2
H
H
H
H
O
O
Base
2
O
H
OP
H11002
O
OH
H11001
Base
1
OP
O
H11002
O
Mixture of 2H11032- and
3H11032-monophosphate
derivatives
CH
2
H11002
O
O
RNA Shortened
RNA
H
2
O
O
RNA
Shortened
RNA
FIGURE 8–8 Hydrolysis of RNA under alkaline
conditions. The 2H11032 hydroxyl acts as a nucleophile
in an intramolecular displacement. The 2H11032,3H11032-cyclic
monophosphate derivative is further hydrolyzed to
a mixture of 2H11032- and 3H11032-monophosphates. DNA,
which lacks 2H11032 hydroxyls, is stable under similar
conditions.
the sugar is always in its closed-ring H9252-furanose form in
nucleic acids). The connecting lines between nucleotides
(which pass through PH22071) are drawn diagonally from the
middle (C-3H11032) of the deoxyribose of one nucleotide to
the bottom (C-5H11032) of the next.
By convention, the structure of a single strand of nu-
cleic acid is always written with the 5H11032 end at the left
and the 3H11032 end at the right—that is, in the 5H11032 n 3H11032 di-
rection. Some simpler representations of this pentade-
oxyribonucleotide are pA-C-G-T-A
OH
, pApCpGpTpA,
and pACGTA.
A short nucleic acid is referred to as an oligonu-
cleotide. The definition of “short” is somewhat arbi-
trary, but polymers containing 50 or fewer nucleotides
are generally called oligonucleotides. A longer nucleic
acid is called a polynucleotide.
The Properties of Nucleotide Bases Affect
the Three-Dimensional Structure of Nucleic Acids
Free pyrimidines and purines are weakly basic com-
pounds and are thus called bases. They have a variety
of chemical properties that affect the structure, and
ultimately the function, of nucleic acids. The purines
and pyrimidines common in DNA and RNA are highly
conjugated molecules (Fig. 8–2), a property with im-
portant consequences for the structure, electron distri-
bution, and light absorption of nucleic acids. Resonance
among atoms in the ring gives most of the bonds par-
tial double-bond character. One result is that pyrim-
idines are planar molecules; purines are very nearly
planar, with a slight pucker. Free pyrimidine and purine
bases may exist in two or more tautomeric forms de-
pending on the pH. Uracil, for example, occurs in lac-
tam, lactim, and double lactim forms (Fig. 8–9). The
structures shown in Figure 8–2 are the tautomers that
predominate at pH 7.0. As a result of resonance, all nu-
cleotide bases absorb UV light, and nucleic acids are
characterized by a strong absorption at wavelengths
near 260 nm (Fig. 8–10).
The purine and pyrimidine bases are hydrophobic
and relatively insoluble in water at the near-neutral pH
of the cell. At acidic or alkaline pH the bases become
charged and their solubility in water increases. Hy-
drophobic stacking interactions in which two or more
bases are positioned with the planes of their rings par-
allel (like a stack of coins) are one of two important
modes of interaction between bases in nucleic acids. The
stacking also involves a combination of van der Waals
and dipole-dipole interactions between the bases. Base
stacking helps to minimize contact of the bases with wa-
ter, and base-stacking interactions are very important in
stabilizing the three-dimensional structure of nucleic
acids, as described later.
Chapter 8 Nucleotides and Nucleic Acids278
Uracil
FIGURE 8–9 Tautomeric forms of uracil. The lactam form predomi-
nates at pH 7.0; the other forms become more prominent as pH de-
creases. The other free pyrimidines and the free purines also have tau-
tomeric forms, but they are more rarely encountered.
14,000
12,000
10,000
8,000
6,000
4,000
2,000
280
Molar extinction
coefficient,
H9280
Wavelength (nm)
230 240 250 260 270
Molar extinction
coefficient at 260 nm,
H9280
260
(M
H110021
cm
H110021
)
AMP
GMP
UMP
dTMP
CMP
15,400
11,700
9,900
9,200
7,500
FIGURE 8–10 Absorption spectra of the
common nucleotides. The spectra are
shown as the variation in molar extinction
coefficient with wavelength. The molar
extinction coefficients at 260 nm and
pH 7.0 (H9255
260
) are listed in the table. The
spectra of corresponding ribonucleotides
and deoxyribonucleotides, as well as the
nucleosides, are essentially identical. For
mixtures of nucleotides, a wavelength of
260 nm (dashed vertical line) is used for
absorption measurements.
The most important functional groups of pyrim-
idines and purines are ring nitrogens, carbonyl groups,
and exocyclic amino groups. Hydrogen bonds involving
the amino and carbonyl groups are the second impor-
tant mode of interaction between bases in nucleic acid
molecules. Hydrogen bonds between bases permit a
complementary association of two (and occasionally
three or four) strands of nucleic acid. The most impor-
tant hydrogen-bonding patterns are those defined by
James D. Watson and Francis Crick in 1953, in which A
bonds specifically to T (or U) and G bonds to C (Fig.
8–11). These two types of base pairs predominate in
double-stranded DNA and RNA, and the tautomers
shown in Figure 8–2 are responsible for these patterns.
It is this specific pairing of bases that permits the du-
plication of genetic information, as we shall discuss later
in this chapter.
SUMMARY 8.1 Some Basics
■ A nucleotide consists of a nitrogenous base
(purine or pyrimidine), a pentose sugar, and
one or more phosphate groups. Nucleic acids
are polymers of nucleotides, joined together by
phosphodiester linkages between the 5H11032-
hydroxyl group of one pentose and the 3H11032-
hydroxyl group of the next.
■ There are two types of nucleic acid: RNA and
DNA. The nucleotides in RNA contain ribose,
and the common pyrimidine bases are uracil
and cytosine. In DNA, the nucleotides contain
2H11032-deoxyribose, and the common pyrimidine
bases are thymine and cytosine. The primary
purines are adenine and guanine in both RNA
and DNA.
8.2 Nucleic Acid Structure
The discovery of the structure of DNA by Watson and
Crick in 1953 was a momentous event in science, an
event that gave rise to entirely new disciplines and in-
fluenced the course of many established ones. Our pres-
ent understanding of the storage and utilization of a
cell’s genetic information is based on work made possi-
ble by this discovery, and an outline of how genetic in-
formation is processed by the cell is now a prerequisite
for the discussion of any area of biochemistry. Here, we
concern ourselves with DNA structure itself, the events
8.2 Nucleic Acid Structure 279
3H11032
C
C
C
CG
G
G
G
A
A
A
A
A
T
T
T
T
T
5H11032
5H11032 3H11032
10.8 ?
N
C
O
C
N
C
H
C
C
H
C
N
C
N
C
11.1
?
2.8
?
3.0 ?
H
N
C
O
CH
3
C
O
N
H
N
C
H
C
CH
N
C
C
N
C
H
N
C
N
O
N
H
H
H
H
2.9 ?
3.0 ?
2.9 ?
Adenine
Thymine
Guanine
Cytosine
N
H
C-1H11032
C-1H11032
C-1H11032
H
H
N
C
N
C-1H11032
FIGURE 8–11 Hydrogen-bonding patterns in the base pairs defined
by Watson and Crick. Here as elsewhere, hydrogen bonds are
represented by three blue lines.
James Watson Francis Crick
that led to its discovery, and more recent refinements
in our understanding. RNA structure is also introduced.
As in the case of protein structure (Chapter 4), it
is sometimes useful to describe nucleic acid structure
in terms of hierarchical levels of complexity (primary,
secondary, tertiary). The primary structure of a nucleic
acid is its covalent structure and nucleotide sequence.
Any regular, stable structure taken up by some or all of
the nucleotides in a nucleic acid can be referred to as
secondary structure. All structures considered in the re-
mainder of this chapter fall under the heading of sec-
ondary structure. The complex folding of large chro-
mosomes within eukaryotic chromatin and bacterial
nucleoids is generally considered tertiary structure; this
is discussed in Chapter 24.
DNA Stores Genetic Information
The biochemical investigation of DNA began with
Friedrich Miescher, who carried out the first systematic
chemical studies of cell nuclei. In 1868 Miescher isolated
a phosphorus-containing substance, which he called
“nuclein,” from the nuclei of pus cells (leukocytes) ob-
tained from discarded surgical bandages. He found
nuclein to consist of an acidic portion, which we know
today as DNA, and a basic portion, protein. Miescher
later found a similar acidic substance in the heads of
sperm cells from salmon. Although he partially purified
nuclein and studied its properties, the covalent (pri-
mary) structure of DNA (as shown in Fig. 8–7) was not
known with certainty until the late 1940s.
Miescher and many others suspected that nuclein
(nucleic acid) was associated in some way with cell in-
heritance, but the first direct evidence that DNA is the
bearer of genetic information came in 1944 through a
discovery made by Oswald T. Avery, Colin MacLeod, and
Maclyn McCarty. These investigators found that DNA
extracted from a virulent (disease-causing) strain of the
bacterium Streptococcus pneumoniae, also known as
pneumococcus, genetically transformed a nonvirulent
strain of this organism into a virulent form (Fig. 8–12).
Chapter 8 Nucleotides and Nucleic Acids280
(a)
(b)
(c)
(d)
(e)
FIGURE 8–12 The Avery-MacLeod-McCarty experiment. (a) When
injected into mice, the encapsulated strain of pneumococcus is lethal,
(b) whereas the nonencapsulated strain, (c) like the heat-killed en-
capsulated strain, is harmless. (d) Earlier research by the bacteriolo-
gist Frederick Griffith had shown that adding heat-killed virulent bac-
teria (harmless to mice) to a live nonvirulent strain permanently
transformed the latter into lethal, virulent, encapsulated bacteria.
(e) Avery and his colleagues extracted the DNA from heat-killed vir-
ulent pneumococci, removing the protein as completely as possible,
and added this DNA to nonvirulent bacteria. The DNA gained en-
trance into the nonvirulent bacteria, which were permanently trans-
formed into a virulent strain.
Avery and his colleagues concluded that the DNA ex-
tracted from the virulent strain carried the inheritable ge-
netic message for virulence. Not everyone accepted these
conclusions, because protein impurities present in the
DNA could have been the carrier of the genetic informa-
tion. This possibility was soon eliminated by the finding
that treatment of the DNA with proteolytic enzymes did
not destroy the transforming activity, but treatment with
deoxyribonucleases (DNA-hydrolyzing enzymes) did.
A second important experiment provided inde-
pendent evidence that DNA carries genetic information.
In 1952 Alfred D. Hershey and Martha Chase used ra-
dioactive phosphorus (
32
P) and radioactive sulfur (
35
S)
tracers to show that when the bacterial virus (bacterio-
phage) T2 infects its host cell, Escherichia coli, it is
the phosphorus-containing DNA of the viral particle, not
the sulfur-containing protein of the viral coat, that en-
ters the host cell and furnishes the genetic information
for viral replication (Fig. 8–13). These important early
experiments and many other lines of evidence have
shown that DNA is the exclusive chromosomal compo-
nent bearing the genetic information of living cells.
DNA Molecules Have Distinctive Base Compositions
A most important clue to the structure of DNA came
from the work of Erwin Chargaff and his colleagues in
the late 1940s. They found that the four nucleotide
bases of DNA occur in different ratios in the DNAs of
different organisms and that the amounts of certain
bases are closely related. These data, collected from
DNAs of a great many different species, led Chargaff to
the following conclusions:
1. The base composition of DNA generally varies
from one species to another.
2. DNA specimens isolated from different tissues of
the same species have the same base composition.
3. The base composition of DNA in a given species
does not change with an organism’s age,
nutritional state, or changing environment.
4. In all cellular DNAs, regardless of the species, the
number of adenosine residues is equal to the
number of thymidine residues (that is, A H11005 T),
and the number of guanosine residues is equal to
the number of cytidine residues (G H11005 C). From
these relationships it follows that the sum of the
purine residues equals the sum of the pyrimidine
residues; that is, A H11001 G H11005 T H11001 C.
These quantitative relationships, sometimes called
“Chargaff’s rules,” were confirmed by many subsequent
researchers. They were a key to establishing the three-
dimensional structure of DNA and yielded clues to how
genetic information is encoded in DNA and passed from
one generation to the next.
32
P experiment
35
S experiment
Radioactive
DNA
Nonradioactive
coat
Nonradioactive
DNA
Radioactive
coat
Injection
Blender
treatment
shears off
viral heads
Separation
by
centrifugation
Radioactive Not radioactive
Phage
Radioactive
Not
radioactive
Bacterial
cell
FIGURE 8–13 The Hershey-Chase experiment. Two batches of iso-
topically labeled bacteriophage T2 particles were prepared. One was
labeled with
32
P in the phosphate groups of the DNA, the other with
35
S in the sulfur-containing amino acids of the protein coats (capsids).
(Note that DNA contains no sulfur and viral protein contains no phos-
phorus.) The two batches of labeled phage were then allowed to in-
fect separate suspensions of unlabeled bacteria. Each suspension of
phage-infected cells was agitated in a blender to shear the viral cap-
sids from the bacteria. The bacteria and empty viral coats (called
“ghosts”) were then separated by centrifugation. The cells infected with
the
32
P-labeled phage were found to contain
32
P, indicating that the
labeled viral DNA had entered the cells; the viral ghosts contained no
radioactivity. The cells infected with
35
S-labeled phage were found to
have no radioactivity after blender treatment, but the viral ghosts con-
tained
35
S. Progeny virus particles (not shown) were produced in both
batches of bacteria some time after the viral coats were removed, in-
dicating that the genetic message for their replication had been in-
troduced by viral DNA, not by viral protein.
DNA Is a Double Helix
To shed more light on the structure of DNA, Rosalind
Franklin and Maurice Wilkins used the powerful method
of x-ray diffraction (see Box 4–4) to analyze DNA fibers.
They showed in the early 1950s that DNA produces a
characteristic x-ray diffraction pattern (Fig. 8–14).
From this pattern it was deduced that DNA molecules
are helical with two periodicities along their long axis,
a primary one of 3.4 ? and a secondary one of 34 ?. The
problem then was to formulate a three-dimensional
model of the DNA molecule that could account not only
for the x-ray diffraction data but also for the spe-
cific A H11005 T and G H11005 C base equivalences discovered by
Chargaff and for the other chemical properties of DNA.
In 1953 Watson and Crick postulated a three-
dimensional model of DNA structure that accounted for
all the available data. It consists of two helical DNA
chains wound around the same axis to form a right-
handed double helix (see Box 4–1 for an explanation of
the right- or left-handed sense of a helical structure).
The hydrophilic backbones of alternating deoxyribose
and phosphate groups are on the outside of the double
helix, facing the surrounding water. The furanose ring
of each deoxyribose is in the C-2H11032 endo conformation.
The purine and pyrimidine bases of both strands are
stacked inside the double helix, with their hydrophobic
and nearly planar ring structures very close together
and perpendicular to the long axis. The offset pairing of
the two strands creates a major groove and minor
groove on the surface of the duplex (Fig. 8–15). Each
nucleotide base of one strand is paired in the same plane
with a base of the other strand. Watson and Crick found
that the hydrogen-bonded base pairs illustrated in Fig-
ure 8–11, G with C and A with T, are those that fit best
within the structure, providing a rationale for Chargaff’s
rule that in any DNA, G H11005 C and A H11005 T. It is important
to note that three hydrogen bonds can form between G
and C, symbolized GqC, but only two can form between
A and T, symbolized AUT. This is one reason for the
finding that separation of paired DNA strands is more
difficult the higher the ratio of GqC to AUT base pairs.
Other pairings of bases tend (to varying degrees) to
destabilize the double-helical structure.
When Watson and Crick constructed their model,
they had to decide at the outset whether the strands
of DNA should be parallel or antiparallel—whether
their 5H11032,3H11032-phosphodiester bonds should run in the same
or opposite directions. An antiparallel orientation pro-
duced the most convincing model, and later work with
DNA polymerases (Chapter 25) provided experimental
evidence that the strands are indeed antiparallel, a find-
ing ultimately confirmed by x-ray analysis.
To account for the periodicities observed in the x-
ray diffraction patterns of DNA fibers, Watson and Crick
manipulated molecular models to arrive at a structure
Chapter 8 Nucleotides and Nucleic Acids282
FIGURE 8–14 X-ray diffraction pattern of DNA. The spots forming a
cross in the center denote a helical structure. The heavy bands at the
left and right arise from the recurring bases.
FIGURE 8–15 Watson-Crick model for the structure of DNA. The
original model proposed by Watson and Crick had 10 base pairs, or
34 ? (3.4 nm), per turn of the helix; subsequent measurements revealed
10.5 base pairs, or 36 ? (3.6 nm), per turn. (a) Schematic represen-
tation, showing dimensions of the helix. (b) Stick representation show-
ing the backbone and stacking of the bases. (c) Space-filling model.
Rosalind Franklin,
1920–1958
Maurice Wilkins
in which the vertically stacked bases inside the double
helix would be 3.4 ? apart; the secondary repeat dis-
tance of about 34 ? was accounted for by the presence
of 10 base pairs in each complete turn of the double
helix. In aqueous solution the structure differs slightly
from that in fibers, having 10.5 base pairs per helical
turn (Fig. 8–15).
As Figure 8–16 shows, the two antiparallel polynu-
cleotide chains of double-helical DNA are not identical
in either base sequence or composition. Instead they are
complementary to each other. Wherever adenine oc-
curs in one chain, thymine is found in the other; simi-
larly, wherever guanine occurs in one chain, cytosine is
found in the other.
The DNA double helix, or duplex, is held together
by two forces, as described earlier: hydrogen bonding
between complementary base pairs (Fig. 8–11) and
base-stacking interactions. The complementarity be-
tween the DNA strands is attributable to the hydrogen
bonding between base pairs. The base-stacking interac-
tions, which are largely nonspecific with respect to the
identity of the stacked bases, make the major contribu-
tion to the stability of the double helix.
The important features of the double-helical model
of DNA structure are supported by much chemical and
biological evidence. Moreover, the model immediately
suggested a mechanism for the transmission of genetic
information. The essential feature of the model is the
complementarity of the two DNA strands. As Watson and
Crick were able to see, well before confirmatory data be-
came available, this structure could logically be replicated
by (1) separating the two strands and (2) synthesizing
a complementary strand for each. Because nucleotides
in each new strand are joined in a sequence specified by
the base-pairing rules stated above, each preexisting
strand functions as a template to guide the synthesis of
one complementary strand (Fig. 8–17). These expecta-
tions were experimentally confirmed, inaugurating a rev-
olution in our understanding of biological inheritance.
DNA Can Occur in Different Three-Dimensional Forms
DNA is a remarkably flexible molecule. Considerable ro-
tation is possible around a number of bonds in the
sugar–phosphate (phosphodeoxyribose) backbone, and
thermal fluctuation can produce bending, stretching, and
unpairing (melting) of the strands. Many significant de-
viations from the Watson-Crick DNA structure are found
in cellular DNA, some or all of which may play impor-
tant roles in DNA metabolism. These structural varia-
tions generally do not affect the key properties of DNA
defined by Watson and Crick: strand complementarity,
8.2 Nucleic Acid Structure 283
FIGURE 8–16 Complementarity of strands in the DNA double helix.
The complementary antiparallel strands of DNA follow the pairing
rules proposed by Watson and Crick. The base-paired antiparallel
strands differ in base composition: the left strand has the composition
A
3
T
2
G
1
C
3
; the right, A
2
T
3
G
3
C
1
. They also differ in sequence when
each chain is read in the 5H11032 n 3H11032 direction. Note the base equiva-
lences: A H11005 T and G H11005 C in the duplex.
FIGURE 8–17 Replication of DNA as suggested by Watson and Crick.
The preexisting or “parent” strands become separated, and each is the
template for biosynthesis of a complementary “daughter” strand (in red).
antiparallel strands, and the requirement for APT and
GqC base pairs.
Structural variation in DNA reflects three things:
the different possible conformations of the deoxyribose,
rotation about the contiguous bonds that make up the
phosphodeoxyribose backbone (Fig. 8–18a), and free
rotation about the C-1H11032–N-glycosyl bond (Fig. 8–18b).
Because of steric constraints, purines in purine nu-
cleotides are restricted to two stable conformations with
respect to deoxyribose, called syn and anti (Fig. 8–18b).
Pyrimidines are generally restricted to the anti confor-
mation because of steric interference between the sugar
and the carbonyl oxygen at C-2 of the pyrimidine.
The Watson-Crick structure is also referred to as B-
form DNA, or B-DNA. The B form is the most stable
structure for a random-sequence DNA molecule under
physiological conditions and is therefore the standard
point of reference in any study of the properties of DNA.
Two structural variants that have been well character-
ized in crystal structures are the A and Z forms. These
three DNA conformations are shown in Figure 8–19,
with a summary of their properties. The A form is fa-
vored in many solutions that are relatively devoid of wa-
ter. The DNA is still arranged in a right-handed double
helix, but the helix is wider and the number of base pairs
per helical turn is 11, rather than 10.5 as in B-DNA. The
Chapter 8 Nucleotides and Nucleic Acids284
FIGURE 8–18 Structural variation in DNA. (a) The conformation of a nucleotide in
DNA is affected by rotation about seven different bonds. Six of the bonds rotate freely.
The limited rotation about bond 4 gives rise to ring pucker, in which one of the atoms in
the five-membered furanose ring is out of the plane described by the other four. This
conformation is endo or exo, depending on whether the atom is displaced to the same
side of the plane as C-5H11032 or to the opposite side (see Fig. 8–3b). (b) For purine bases in
nucleotides, only two conformations with respect to the attached ribose units are
sterically permitted, anti or syn. Pyrimidines generally occur in the anti conformation.
FIGURE 8–19 Comparison of A, B, and Z forms of DNA. Each struc-
ture shown here has 36 base pairs. The bases are shown in gray, the
phosphate atoms in yellow, and the riboses and phosphate oxygens
in blue. Blue is the color used to represent DNA strands in later chap-
ters. The table summarizes some properties of the three forms of DNA.
A form B form Z form
Helical sense Right handed Right handed Left handed
Diameter H1101126 ? H1101120 ? H1101118 ?
Base pairs per helical
turn 11 10.5 12
Helix rise per base pair 2.6 ? 3.4 ? 3.7 ?
Base tilt normal to the
helix axis 20° 6° 7°
Sugar pucker conformation C-3H11032 endo C-2H11032 endo C-2H11032 endo for
pyrimidines;
C-3H11032 endo for
purines
Glycosyl bond conformation Anti Anti Anti for pyrimidines;
syn for purines
plane of the base pairs in A-DNA is tilted about 20H11034 with
respect to the helix axis. These structural changes
deepen the major groove while making the minor groove
shallower. The reagents used to promote crystallization
of DNA tend to dehydrate it, and thus most short DNA
molecules tend to crystallize in the A form.
Z-form DNA is a more radical departure from the B
structure; the most obvious distinction is the left-
handed helical rotation. There are 12 base pairs per hel-
ical turn, and the structure appears more slender and
elongated. The DNA backbone takes on a zigzag ap-
pearance. Certain nucleotide sequences fold into left-
handed Z helices much more readily than others. Promi-
nent examples are sequences in which pyrimidines
alternate with purines, especially alternating C and G or
5-methyl-C and G residues. To form the left-handed
helix in Z-DNA, the purine residues flip to the syn
conformation, alternating with pyrimidines in the anti
conformation. The major groove is barely apparent in
Z-DNA, and the minor groove is narrow and deep.
Whether A-DNA occurs in cells is uncertain, but there
is evidence for some short stretches (tracts) of Z-DNA
in both prokaryotes and eukaryotes. These Z-DNA tracts
may play a role (as yet undefined) in regulating the ex-
pression of some genes or in genetic recombination.
Certain DNA Sequences Adopt Unusual Structures
A number of other sequence-dependent structural vari-
ations have been detected within larger chromosomes
that may affect the function and metabolism of the DNA
segments in their immediate vicinity. For example,
bends occur in the DNA helix wherever four or more
adenosine residues appear sequentially in one strand.
Six adenosines in a row produce a bend of about 18H11034.
The bending observed with this and other sequences may
be important in the binding of some proteins to DNA.
A rather common type of DNA sequence is a palin-
drome. A palindrome is a word, phrase, or sentence
that is spelled identically read either forward or back-
ward; two examples are ROTATOR and NURSES RUN.
The term is applied to regions of DNA with inverted
repeats of base sequence having twofold symmetry
over two strands of DNA (Fig. 8–20). Such sequences
are self-complementary within each strand and there-
fore have the potential to form hairpin or cruciform
(cross-shaped) structures (Fig. 8–21). When the in-
verted repeat occurs within each individual strand of
the DNA, the sequence is called a mirror repeat.
Mirror repeats do not have complementary sequences
within the same strand and cannot form hairpin or cru-
ciform structures. Sequences of these types are found
8.2 Nucleic Acid Structure 285
FIGURE 8–20 Palindromes and mirror repeats. Palindromes are se-
quences of double-stranded nucleic acids with twofold symmetry. In
order to superimpose one repeat (shaded sequence) on the other, it
must be rotated 180H11034 about the horizontal axis then 180H11034 about the
vertical axis, as shown by the colored arrows. A mirror repeat, on the
other hand, has a symmetric sequence within each strand. Superim-
posing one repeat on the other requires only a single 180H11034 rotation
about the vertical axis.
FIGURE 8–21 Hairpins and cruciforms. Palindromic DNA (or RNA)
sequences can form alternative structures with intrastrand base pair-
ing. (a) When only a single DNA (or RNA) strand is involved, the
structure is called a hairpin. (b) When both strands of a duplex DNA
are involved, it is called a cruciform. Blue shading highlights asym-
metric sequences that can pair with the complementary sequence ei-
ther in the same strand or in the complementary strand.
in virtually every large DNA molecule and can encom-
pass a few base pairs or thousands. The extent to which
palindromes occur as cruciforms in cells is not known,
although some cruciform structures have been demon-
strated in vivo in E.coli. Self-complementary sequences
cause isolated single strands of DNA (or RNA) in solu-
tion to fold into complex structures containing multiple
hairpins.
Several unusual DNA structures involve three or even
four DNA strands. These structural variations merit
investigation because there is a tendency for many of
them to appear at sites where important events in DNA
metabolism (replication, recombination, transcription)
are initiated or regulated. Nucleotides participating in a
Watson-Crick base pair (Fig. 8–11) can form a number
of additional hydrogen bonds, particularly with func-
tional groups arrayed in the major groove. For example,
a cytidine residue (if protonated) can pair with the
guanosine residue of a GqC nucleotide pair, and a
thymidine can pair with the adenosine of an AUT pair
(Fig. 8–22). The N-7, O
6
, and N
6
of purines, the atoms
that participate in the hydrogen bonding of triplex DNA,
are often referred to as Hoogsteen positions, and the
non-Watson-Crick pairing is called Hoogsteen pairing,
after Karst Hoogsteen, who in 1963 first recognized the
potential for these unusual pairings. Hoogsteen pairing
allows the formation of triplex DNAs. The triplexes
shown in Figure 8–22 (a, b) are most stable at low pH
Chapter 8 Nucleotides and Nucleic Acids286
CH
3
CH
3
O
N
O
H
N
N
H H
N
H
N
N
C-1H11032
1H11032-C
C-1H11032
N
NN
O
O
TAT
(a)
N
N
H11001
O
H
N
O
H
H
H
N
H
N
N
C-1H11032
1H11032-C
C-1H11032
N
H
H
N
NN
O
H
N
CGC
H11001
H
Guanosine tetraplex
(c)
H
H
N
N
O
C-1H11032
N
N
N
H
H
H
N
N
N N
1H11032-C
N O
C-1H11032
N
N
N
N
O
NH
H
H
O
C-1H11032
N
H
H
N
NN
N
H
Parallel Antiparallel
(e)
FIGURE 8–22 DNA structures containing three or four DNA strands.
(a) Base-pairing patterns in one well-characterized form of triplex
DNA. The Hoogsteen pair in each case is shown in red. (b) Triple-
helical DNA containing two pyrimidine strands (poly(T)) and one
purine strand (poly(A)) (derived from PDB ID 1BCE). The dark blue
and light blue strands are antiparallel and paired by normal Watson-
Crick base-pairing patterns. The third (all-pyrimidine) strand (purple)
is parallel to the purine strand and paired through non-Watson-Crick
hydrogen bonds. The triplex is viewed end-on, with five triplets shown.
Only the triplet closest to the viewer is colored. (c) Base-pairing pat-
tern in the guanosine tetraplex structure. (d) Two successive tetraplets
from a G tetraplex structure (derived from PDB ID 1QDG), viewed
end-on with the one closest to the viewer in color. (e) Possible vari-
ants in the orientation of strands in a G tetraplex.
because the CqG
H11554
C
H11001
triplet requires a protonated cy-
tosine. In the triplex, the pK
a
of this cytosine is H110227.5,
altered from its normal value of 4.2. The triplexes also
form most readily within long sequences containing only
pyrimidines or only purines in a given strand. Some
triplex DNAs contain two pyrimidine strands and one
purine strand; others contain two purine strands and
one pyrimidine strand.
Four DNA strands can also pair to form a tetraplex
(quadruplex), but this occurs readily only for DNA se-
quences with a very high proportion of guanosine
residues (Fig. 8–22c, d). The guanosine tetraplex, or G
tetraplex, is quite stable over a wide range of condi-
tions. The orientation of strands in the tetraplex can
vary as shown in Figure 8–22e.
A particularly exotic DNA structure, known as
H-DNA, is found in polypyrimidine or polypurine tracts
that also incorporate a mirror repeat. A simple example
is a long stretch of alternating T and C residues (Fig.
8–23). The H-DNA structure features the triple-stranded
form illustrated in Figure 8–22 (a, b). Two of the three
strands in the H-DNA triple helix contain pyrimidines
and the third contains purines.
In the DNA of living cells, sites recognized by many
sequence-specific DNA-binding proteins (Chapter 28)
are arranged as palindromes, and polypyrimidine or
polypurine sequences that can form triple helices or
even H-DNA are found within regions involved in the
regulation of expression of some eukaryotic genes. In
principle, synthetic DNA strands designed to pair with
these sequences to form triplex DNA could disrupt gene
expression. This approach to controlling cellular me-
tabolism is of growing commercial interest for its po-
tential application in medicine and agriculture.
Messenger RNAs Code for Polypeptide Chains
We now turn our attention briefly from DNA structure
to the expression of the genetic information that it con-
tains. RNA, the second major form of nucleic acid in
cells, has many functions. In gene expression, RNA acts
as an intermediary by using the information encoded in
DNA to specify the amino acid sequence of a functional
protein.
Given that the DNA of eukaryotes is largely con-
fined to the nucleus whereas protein synthesis occurs
on ribosomes in the cytoplasm, some molecule other
than DNA must carry the genetic message from the nu-
cleus to the cytoplasm. As early as the 1950s, RNA was
considered the logical candidate: RNA is found in both
the nucleus and the cytoplasm, and an increase in pro-
tein synthesis is accompanied by an increase in the
amount of cytoplasmic RNA and an increase in its rate
of turnover. These and other observations led several
researchers to suggest that RNA carries genetic infor-
mation from DNA to the protein biosynthetic machin-
ery of the ribosome. In 1961 Fran?ois Jacob and Jacques
Monod presented a unified (and essentially correct) pic-
ture of many aspects of this process. They proposed the
name “messenger RNA” (mRNA) for that portion of the
total cellular RNA carrying the genetic information from
DNA to the ribosomes, where the messengers provide
the templates that specify amino acid sequences in
polypeptide chains. Although mRNAs from different
genes can vary greatly in length, the mRNAs from a par-
ticular gene generally have a defined size. The process
of forming mRNA on a DNA template is known as
transcription.
In prokaryotes, a single mRNA molecule may code for
one or several polypeptide chains. If it carries the code
for only one polypeptide, the mRNA is monocistronic;
8.2 Nucleic Acid Structure 287
FIGURE 8–23 H-DNA. (a) A sequence of alternating T and C residues
can be considered a mirror repeat centered about a central T or C.
(b) These sequences form an unusual structure in which the strands
in one half of the mirror repeat are separated and the pyrimidine-
containing strand (alternating T and C residues) folds back on the other
half of the repeat to form a triple helix. The purine strand (alternating
A and G residues) is left unpaired. This structure produces a sharp
bend in the DNA.
if it codes for two or more different polypeptides, the
mRNA is polycistronic. In eukaryotes, most mRNAs
are monocistronic. (For the purposes of this discussion,
“cistron” refers to a gene. The term itself has historical
roots in the science of genetics, and its formal genetic
definition is beyond the scope of this text.) The mini-
mum length of an mRNA is set by the length of the
polypeptide chain for which it codes. For example, a
polypeptide chain of 100 amino acid residues requires
an RNA coding sequence of at least 300 nucleotides, be-
cause each amino acid is coded by a nucleotide triplet
(this and other details of protein synthesis are discussed
in Chapter 27). However, mRNAs transcribed from DNA
are always somewhat longer than the length needed sim-
ply to code for a polypeptide sequence (or sequences).
The additional, noncoding RNA includes sequences that
regulate protein synthesis. Figure 8–24 summarizes the
general structure of prokaryotic mRNAs.
Many RNAs Have More Complex
Three-Dimensional Structures
Messenger RNA is only one of several classes of cellu-
lar RNA. Transfer RNAs serve as adapter molecules in
protein synthesis; covalently linked to an amino acid at
one end, they pair with the mRNA in such a way that
amino acids are joined to a growing polypeptide in the
correct sequence. Ribosomal RNAs are components of
ribosomes. There is also a wide variety of special-func-
tion RNAs, including some (called ribozymes) that have
enzymatic activity. All the RNAs are considered in de-
tail in Chapter 26. The diverse and often complex func-
tions of these RNAs reflect a diversity of structure much
richer than that observed in DNA molecules.
The product of transcription of DNA is always
single-stranded RNA. The single strand tends to assume
a right-handed helical conformation dominated by base-
stacking interactions (Fig. 8–25), which are stronger be-
tween two purines than between a purine and pyrimi-
dine or between two pyrimidines. The purine-purine
interaction is so strong that a pyrimidine separating two
purines is often displaced from the stacking pattern so
that the purines can interact. Any self-complementary
sequences in the molecule produce more complex struc-
tures. RNA can base-pair with complementary regions
of either RNA or DNA. Base pairing matches the pat-
tern for DNA: G pairs with C and A pairs with U (or with
the occasional T residue in some RNAs). One difference
is that base pairing between G and U residues—unusual
in DNA—is fairly common in RNA (see Fig. 8–27). The
paired strands in RNA or RNA-DNA duplexes are an-
tiparallel, as in DNA.
RNA has no simple, regular secondary structure
that serves as a reference point, as does the double he-
lix for DNA. The three-dimensional structures of many
RNAs, like those of proteins, are complex and unique.
Weak interactions, especially base-stacking interactions,
play a major role in stabilizing RNA structures, just as
they do in DNA. Where complementary sequences are
present, the predominant double-stranded structure is
an A-form right-handed double helix. Z-form helices
have been made in the laboratory (under very high-salt
or high-temperature conditions). The B form of RNA
has not been observed. Breaks in the regular A-form he-
lix caused by mismatched or unmatched bases in one
or both strands are common and result in bulges or in-
ternal loops (Fig. 8–26). Hairpin loops form between
nearby self-complementary sequences. The potential for
base-paired helical structures in many RNAs is exten-
sive (Fig. 8–27), and the resulting hairpins are the most
common type of secondary structure in RNA. Specific
Chapter 8 Nucleotides and Nucleic Acids288
FIGURE 8–24 Prokaryotic mRNA. Schematic diagrams show (a)
monocistronic and (b) polycistronic mRNAs of prokaryotes. Red seg-
ments represent RNA coding for a gene product; gray segments rep-
resent noncoding RNA. In the polycistronic transcript, noncoding RNA
separates the three genes.
FIGURE 8–25 Typical right-handed stacking pattern of single-
stranded RNA. The bases are shown in gray, the phosphate atoms in
yellow, and the riboses and phosphate oxygens in green. Green is used
to represent RNA strands in succeeding chapters, just as blue is used
for DNA.
short base sequences (such as UUCG) are often found
at the ends of RNA hairpins and are known to form par-
ticularly tight and stable loops. Such sequences may act
as starting points for the folding of an RNA molecule
into its precise three-dimensional structure. Important
additional structural contributions are made by hydro-
gen bonds that are not part of standard Watson-Crick
base pairs. For example, the 2H11032-hydroxyl group of ribose
can hydrogen-bond with other groups. Some of these
properties are evident in the structure of the phenyl-
alanine transfer RNA of yeast—the tRNA responsible for
inserting Phe residues into polypeptides—and in two
RNA enzymes, or ribozymes, whose functions, like those
of protein enzymes, depend on their three-dimensional
structures (Fig. 8–28).
The analysis of RNA structure and the relationship
between structure and function is an emerging field of
inquiry that has many of the same complexities as the
analysis of protein structure. The importance of under-
standing RNA structure grows as we become increas-
ingly aware of the large number of functional roles for
RNA molecules.
8.2 Nucleic Acid Structure 289
G
U
C
A
C
C
A
G
U
G
C
A
A
C
A
G
A
G
A
G
C
A
A
C
A
G
U
G
A
180
A
G
G
C
G
C
120
A
C
G
G
G
C
G
C
C
C
A
240
A
U
G
G
C
C
A
G
C
G
C
C
G
A
U
C
C
C
G
C
C
G
G
G
G
A
U
C
G
G
U
G
G
C
A
160
A
G
G
140
A
A
A
G
C
C
C
G
G
C
G
G
U
G
G
A
220
A
A
U
G
G
C
G
G
C
U
G
U
G
C
C
G
A
C
G
G
U
A
200
A
A
C
C
G
G
100
A
A G G
C
A
G
G
80
C
C
C
U
A
A
G
A
A
U
G
G
G
C
C
C
A
C
G
A
U
A
A
A
G
U
C
C
G
G
G
C
A
G
G
C
U
G
C
U
U
G
U
A
G
A
U
G
A
A
G
G
A
G
G
A
G
G
C
U
U
C
G
G
G
C
A
A
CA
U
A
C
U
G
A
C
A
G
A
C
U
G
U
C
G
G
G
A
C
G
G
C
A
G
G
C
G
C
U
U
C
G
U
G
G
G
G
C
C
CC
G
O
ON H
O
NH
2
N
N
N
N
O
N
H
O
Guanine
Uracil
C
C
G
G
A
A
A
U
A
G
G
C
C
C
A
A
G
G
U
U
C
A
G
U
G
C
U
A
A
C
G
U
G
C
G
C
C
280
A260
G
U
G
G
G
U
A
300
C
A
A
G
C
G
U
G
C
C
G
G G
U
A
G
U
U
G
A
C
330
U
A
C
C
AG
U
CG
A
A GG
U
CA
G
U
U
U
C
GAC
C
U
377
360
1
C
U
A
U
U
C
G
G
C
C C
A
A
G
A
C
A
G
C
A
C
20
60
G
C
G
U
A
U
U
GG
G
C
U
U
40
A
C
FIGURE 8–26 Secondary structure of RNAs. (a) Bulge, internal loop,
and hairpin loop. (b) The paired regions generally have an A-form
right-handed helix, as shown for a hairpin.
FIGURE 8–27 Base-paired helical structures in an RNA. Shown
here is the possible secondary structure of the M1 RNA component
of the enzyme RNase P of E. coli, with many hairpins. RNase P,
which also contains a protein component (not shown), functions in
the processing of transfer RNAs (see Fig. 26–23). The two brackets
indicate additional complementary sequences that may be paired in
the three-dimensional structure. The blue dots indicate non-Watson-
Crick GUU base pairs (boxed inset). Note that GUU base pairs are
allowed only when presynthesized strands of RNA fold up or anneal
with each other. There are no RNA polymerases (the enzymes that
synthesize RNAs on a DNA template) that insert a U opposite a
template G, or vice versa, during RNA synthesis.
SUMMARY 8.2 Nucleic Acid Structure
■ Many lines of evidence show that DNA bears
genetic information. In particular, the Avery-
MacLeod-McCarty experiment showed that DNA
isolated from one bacterial strain can enter and
transform the cells of another strain, endowing
it with some of the inheritable characteristics
of the donor. The Hershey-Chase experiment
showed that the DNA of a bacterial virus, but
not its protein coat, carries the genetic
message for replication of the virus in a host cell.
■ Putting together much published data, Watson
and Crick postulated that native DNA consists
of two antiparallel chains in a right-handed
double-helical arrangement. Complementary
base pairs, AUT and GqC, are formed by
hydrogen bonding within the helix. The base
Chapter 8 Nucleotides and Nucleic Acids290
FIGURE 8–28 Three-dimensional structure in RNA. (a) Three-
dimensional structure of phenylalanine tRNA of yeast (PDB ID 1TRA).
Some unusual base-pairing patterns found in this tRNA are shown.
Note also the involvement of the oxygen of a ribose phosphodiester
bond in one hydrogen-bonding arrangement, and a ribose 2H11032-hydroxyl
group in another (both in red). (b) A hammerhead ribozyme (so named
because the secondary structure at the active site looks like the head
of a hammer), derived from certain plant viruses (derived from PDB
ID 1MME). Ribozymes, or RNA enzymes, catalyze a variety of reac-
tions, primarily in RNA metabolism and protein synthesis. The com-
plex three-dimensional structures of these RNAs reflect the complexity
inherent in catalysis, as described for protein enzymes in Chapter 6.
(c) A segment of mRNA known as an intron, from the ciliated proto-
zoan Tetrahymena thermophila (derived from PDB ID 1GRZ). This
intron (a ribozyme) catalyzes its own excision from between exons in
an mRNA strand (discussed in Chapter 26).
pairs are stacked perpendicular to the long axis
of the double helix, 3.4 ? apart, with 10.5 base
pairs per turn.
■ DNA can exist in several structural forms. Two
variations of the Watson-Crick form, or B-DNA,
are A- and Z-DNA. Some sequence-dependent
structural variations cause bends in the DNA
molecule. DNA strands with appropriate se-
quences can form hairpin/cruciform structures
or triplex or tetraplex DNA.
■ Messenger RNA transfers genetic information
from DNA to ribosomes for protein synthesis.
Transfer RNA and ribosomal RNA are also
involved in protein synthesis. RNA can be
structurally complex; single RNA strands can
be folded into hairpins, double-stranded re-
gions, or complex loops.
8.3 Nucleic Acid Chemistry
To understand how nucleic acids function, we must un-
derstand their chemical properties as well as their struc-
tures. The role of DNA as a repository of genetic infor-
mation depends in part on its inherent stability. The
chemical transformations that do occur are generally very
slow in the absence of an enzyme catalyst. The long-term
storage of information without alteration is so important
to a cell, however, that even very slow reactions that
alter DNA structure can be physiologically significant.
Processes such as carcinogenesis and aging may be
intimately linked to slowly accumulating, irreversible al-
terations of DNA. Other, nondestructive alterations also
occur and are essential to function, such as the strand
separation that must precede DNA replication or tran-
scription. In addition to providing insights into physio-
logical processes, our understanding of nucleic acid
chemistry has given us a powerful array of technologies
that have applications in molecular biology, medicine, and
forensic science. We now examine the chemical proper-
ties of DNA and some of these technologies.
Double-Helical DNA and RNA Can Be Denatured
Solutions of carefully isolated, native DNA are highly
viscous at pH 7.0 and room temperature (25 H11034C). When
such a solution is subjected to extremes of pH or to tem-
peratures above 80 H11034C, its viscosity decreases sharply,
indicating that the DNA has undergone a physical
change. Just as heat and extremes of pH denature glob-
ular proteins, they also cause denaturation, or melting,
of double-helical DNA. Disruption of the hydrogen
bonds between paired bases and of base stacking causes
unwinding of the double helix to form two single strands,
completely separate from each other along the entire
length or part of the length (partial denaturation) of the
molecule. No covalent bonds in the DNA are broken
(Fig. 8–29).
Renaturation of a DNA molecule is a rapid one-step
process, as long as a double-helical segment of a dozen
or more residues still unites the two strands. When the
temperature or pH is returned to the range in which
most organisms live, the unwound segments of the two
strands spontaneously rewind, or anneal, to yield the
intact duplex (Fig. 8–29). However, if the two strands
are completely separated, renaturation occurs in two
steps. In the first, relatively slow step, the two strands
“find” each other by random collisions and form a short
segment of complementary double helix. The second
step is much faster: the remaining unpaired bases suc-
cessively come into register as base pairs, and the two
strands “zipper” themselves together to form the dou-
ble helix.
The close interaction between stacked bases in a
nucleic acid has the effect of decreasing its absorption
of UV light relative to that of a solution with the same
concentration of free nucleotides, and the absorption is
decreased further when two complementary nucleic
acids strands are paired. This is called the hypochromic
effect. Denaturation of a double-stranded nucleic acid
produces the opposite result: an increase in absorption
8.3 Nucleic Acid Chemistry 291
FIGURE 8–29 Reversible denaturation and annealing (renaturation)
of DNA.
called the hyperchromic effect. The transition from
double-stranded DNA to the single-stranded, denatured
form can thus be detected by monitoring the absorption
of UV light.
Viral or bacterial DNA molecules in solution dena-
ture when they are heated slowly (Fig. 8–30). Each
species of DNA has a characteristic denaturation tem-
perature, or melting point (t
m
): the higher its content
of GqC base pairs, the higher the melting point of the
DNA. This is because GqC base pairs, with three hy-
drogen bonds, require more heat energy to dissociate
than AUT base pairs. Careful determination of the melt-
ing point of a DNA specimen, under fixed conditions of
pH and ionic strength, can yield an estimate of its base
composition. If denaturation conditions are carefully
controlled, regions that are rich in AUT base pairs will
specifically denature while most of the DNA remains
double-stranded. Such denatured regions (called bub-
bles) can be visualized with electron microscopy (Fig.
8–31). Strand separation of DNA must occur in vivo dur-
ing processes such as DNA replication and transcrip-
tion. As we shall see, the DNA sites where these
processes are initiated are often rich in AUT base pairs.
Duplexes of two RNA strands or of one RNA strand
and one DNA strand (RNA-DNA hybrids) can also be
denatured. Notably, RNA duplexes are more stable than
DNA duplexes. At neutral pH, denaturation of a double-
helical RNA often requires temperatures 20 H11034C or more
higher than those required for denaturation of a DNA
molecule with a comparable sequence. The stability of
an RNA-DNA hybrid is generally intermediate between
that of RNA and that of DNA. The physical basis for
these differences in thermal stability is not known.
Nucleic Acids from Different Species
Can Form Hybrids
The ability of two complementary DNA strands to pair
with one another can be used to detect similar DNA se-
quences in two different species or within the genome
of a single species. If duplex DNAs isolated from human
cells and from mouse cells are completely denatured by
heating, then mixed and kept at 65 H11034C for many hours,
much of the DNA will anneal. Most of the mouse DNA
strands anneal with complementary mouse DNA strands
to form mouse duplex DNA; similarly, most human
DNA strands anneal with complementary human DNA
strands. However, some strands of the mouse DNA will
associate with human DNA strands to yield hybrid
Chapter 8 Nucleotides and Nucleic Acids292
100
G
H11001
C (% of total nucleotides)
80
0
70 80 90
t
m
(°C)
11060 100
60
40
20
100
Denaturation (%)
50
0
75 80 85
Temperature (°C)
t
m t
m
FIGURE 8–30 Heat denaturation of DNA. (a) The denaturation, or
melting, curves of two DNA specimens. The temperature at the mid-
point of the transition (t
m
) is the melting point; it depends on pH and
ionic strength and on the size and base composition of the DNA.
(b) Relationship between t
m
and the GqC content of a DNA.
(a)
(b)
FIGURE 8–31 Partially denatured DNA. This DNA was partially de-
natured, then fixed to prevent renaturation during sample preparation.
The shadowing method used to visualize the DNA in this electron mi-
crograph increases its diameter approximately fivefold and obliterates
most details of the helix. However, length measurements can be ob-
tained, and single-stranded regions are readily distinguishable from
double-stranded regions. The arrows point to some single-stranded
bubbles where denaturation has occurred. The regions that denature
are highly reproducible and are rich in AUT base pairs.
duplexes, in which segments of a mouse DNA strand
form base-paired regions with segments of a human DNA
strand (Fig. 8–32). This reflects a common evolutionary
heritage; different organisms generally have some pro-
teins and RNAs with similar functions and, often, simi-
lar structures. In many cases, the DNAs encoding these
proteins and RNAs have similar sequences. The closer
the evolutionary relationship between two species, the
more extensively their DNAs will hybridize. For exam-
ple, human DNA hybridizes much more extensively with
mouse DNA than with DNA from yeast.
The hybridization of DNA strands from different
sources forms the basis for a powerful set of techniques
essential to the practice of modern molecular genetics.
A specific DNA sequence or gene can be detected in the
presence of many other sequences, if one already has
an appropriate complementary DNA strand (usually la-
beled in some way) to hybridize with it (Chapter 9). The
complementary DNA can be from a different species or
from the same species, or it can be synthesized chemi-
cally in the laboratory using techniques described later
in this chapter. Hybridization techniques can be varied
to detect a specific RNA rather than DNA. The isolation
and identification of specific genes and RNAs rely on
these hybridization techniques. Applications of this
technology make possible the identification of an indi-
vidual on the basis of a single hair left at the scene of a
crime or the prediction of the onset of a disease decades
before symptoms appear (see Box 9–1).
Nucleotides and Nucleic Acids Undergo
Nonenzymatic Transformations
Purines and pyrimidines, along with the nucleotides of
which they are a part, undergo a number of spontaneous
alterations in their covalent structure. The rate of these
reactions is generally very slow, but they are physio-
logically significant because of the cell’s very low toler-
ance for alterations in its genetic information. Alter-
ations in DNA structure that produce permanent
changes in the genetic information encoded therein are
called mutations, and much evidence suggests an inti-
mate link between the accumulation of mutations in an
individual organism and the processes of aging and
carcinogenesis.
Several nucleotide bases undergo spontaneous loss
of their exocyclic amino groups (deamination) (Fig.
8–33a). For example, under typical cellular conditions,
deamination of cytosine (in DNA) to uracil occurs in
about one of every 10
7
cytidine residues in 24 hours.
This corresponds to about 100 spontaneous events per
day, on average, in a mammalian cell. Deamination of
adenine and guanine occurs at about 1/100th this rate.
The slow cytosine deamination reaction seems in-
nocuous enough, but is almost certainly the reason why
DNA contains thymine rather than uracil. The product
of cytosine deamination (uracil) is readily recognized as
foreign in DNA and is removed by a repair system
(Chapter 25). If DNA normally contained uracil, recog-
nition of uracils resulting from cytosine deamination
would be more difficult, and unrepaired uracils would
lead to permanent sequence changes as they were
paired with adenines during replication. Cytosine deam-
ination would gradually lead to a decrease in GqC base
pairs and an increase in AUU base pairs in the DNA of
all cells. Over the millennia, cytosine deamination could
eliminate GqC base pairs and the genetic code that de-
pends on them. Establishing thymine as one of the four
bases in DNA may well have been one of the crucial
turning points in evolution, making the long-term stor-
age of genetic information possible.
Another important reaction in deoxyribonu-
cleotides is the hydrolysis of the N-H9252-glycosyl bond be-
tween the base and the pentose (Fig. 8–33b). This oc-
curs at a higher rate for purines than for pyrimidines.
As many as one in 10
5
purines (10,000 per mammalian
cell) are lost from DNA every 24 hours under typical
8.3 Nucleic Acid Chemistry 293
FIGURE 8–32 DNA hybridization. Two DNA samples to be compared
are completely denatured by heating. When the two solutions are
mixed and slowly cooled, DNA strands of each sample associate with
their normal complementary partner and anneal to form duplexes. If
the two DNAs have significant sequence similarity, they also tend to
form partial duplexes or hybrids with each other: the greater the se-
quence similarity between the two DNAs, the greater the number of
hybrids formed. Hybrid formation can be measured in several ways.
One of the DNAs is usually labeled with a radioactive isotope to sim-
plify the measurements.
cellular conditions. Depurination of ribonucleotides and
RNA is much slower and generally is not considered
physiologically significant. In the test tube, loss of
purines can be accelerated by dilute acid. Incubation of
DNA at pH 3 causes selective removal of the purine
bases, resulting in a derivative called apurinic acid.
Other reactions are promoted by radiation. UV light
induces the condensation of two ethylene groups to
form a cyclobutane ring. In the cell, the same reaction
between adjacent pyrimidine bases in nucleic acids
forms cyclobutane pyrimidine dimers. This happens
most frequently between adjacent thymidine residues
on the same DNA strand (Fig. 8–34). A second type of
pyrimidine dimer, called a 6-4 photoproduct, is also
formed during UV irradiation. Ionizing radiation (x rays
and gamma rays) can cause ring opening and fragmen-
tation of bases as well as breaks in the covalent back-
bone of nucleic acids.
Virtually all forms of life are exposed to energy-rich
radiation capable of causing chemical changes in DNA.
Near-UV radiation (with wavelengths of 200 to 400 nm),
which makes up a significant portion of the solar spec-
trum, is known to cause pyrimidine dimer formation and
other chemical changes in the DNA of bacteria and of
human skin cells. We are subject to a constant field of
ionizing radiation in the form of cosmic rays, which can
penetrate deep into the earth, as well as radiation emit-
ted from radioactive elements, such as radium, pluto-
nium, uranium, radon,
14
C, and
3
H. X rays used in med-
ical and dental examinations and in radiation therapy of
cancer and other diseases are another form of ionizing
radiation. It is estimated that UV and ionizing radiations
are responsible for about 10% of all DNA damage caused
by environmental agents.
DNA also may be damaged by reactive chemicals in-
troduced into the environment as products of industrial
activity. Such products may not be injurious per se but
may be metabolized by cells into forms that are. Two
prominent classes of such agents (Fig. 8–35) are (1)
deaminating agents, particularly nitrous acid (HNO
2
) or
compounds that can be metabolized to nitrous acid or
nitrites, and (2) alkylating agents.
Nitrous acid, formed from organic precursors such
as nitrosamines and from nitrite and nitrate salts, is a
potent accelerator of the deamination of bases. Bisulfite
has similar effects. Both agents are used as preserva-
tives in processed foods to prevent the growth of toxic
bacteria. They do not appear to increase cancer risks
Chapter 8 Nucleotides and Nucleic Acids294
3
(a) Deamination
3
2
2
2
2
CH
N
HN
Hypoxanthine
Uracil
N
Cytosine
O
CH
NO
N
Xanthine
NO
N
O
HN
NH
NH
N
N
N
NH
Thymine5-Methylcytosine
O
HN
O
O
N
N
N
N
Adenine
Guanine
O N
H
N
N
O
N
N
N
H N
O
HNHN
FIGURE 8–33 Some well-characterized nonenzymatic reactions of
nucleotides. (a) Deamination reactions. Only the base is shown.
(b) Depurination, in which a purine is lost by hydrolysis of the N-H9252-
glycosyl bond. The deoxyribose remaining after depurination is readily
converted from the H9252-furanose to the aldehyde form (see Fig. 8–3). Fur-
ther nonenzymatic reactions are illustrated in Figures 8–34 and 8–35.
significantly when used in this way, perhaps because
they are used in small amounts and make only a minor
contribution to the overall levels of DNA damage. (The
potential health risk from food spoilage if these preser-
vatives were not used is much greater.)
Alkylating agents can alter certain bases of DNA.
For example, the highly reactive chemical dimethylsul-
fate (Fig. 8–35b) can methylate a guanine to yield O
6
-
methylguanine, which cannot base-pair with cytosine.
Many similar reactions are brought about by alkylating
agents normally present in cells, such as S-adenosyl-
methionine.
Possibly the most important source of mutagenic al-
terations in DNA is oxidative damage. Excited-oxygen
species such as hydrogen peroxide, hydroxyl radicals,
and superoxide radicals arise during irradiation or as a
byproduct of aerobic metabolism. Of these species, the
hydroxyl radicals are responsible for most oxidative
DNA damage. Cells have an elaborate defense system
to destroy reactive oxygen species, including enzymes
such as catalase and superoxide dismutase that convert
reactive oxygen species to harmless products. A frac-
tion of these oxidants inevitably escape cellular de-
fenses, however, and damage to DNA occurs through
any of a large, complex group of reactions ranging from
oxidation of deoxyribose and base moieties to strand
breaks. Accurate estimates for the extent of this dam-
age are not yet available, but every day the DNA of each
human cell is subjected to thousands of damaging
oxidative reactions.
This is merely a sampling of the best-understood
reactions that damage DNA. Many carcinogenic com-
pounds in food, water, or air exert their cancer-causing
effects by modifying bases in DNA. Nevertheless, the in-
tegrity of DNA as a polymer is better maintained than
that of either RNA or protein, because DNA is the only
macromolecule that has the benefit of biochemical repair
systems. These repair processes (described in Chapter
25) greatly lessen the impact of damage to DNA.
8.3 Nucleic Acid Chemistry 295
OH
C
N
H
OH
C
O
C
C
H
N
O
C
N
H
CH
3
C
O
C
C
H
N
UV lightUV light
Adjacent
thymines
Cyclobutane thymine dimer 6-4 Photoproduct
O
C
N
HCH
3
C
O
C
C
H
N
O
C
N
H CH
3
C
O
C
C
H
N
O
C
N
H CH
3
C
O
C
C
H
N
O
C
H CH
3
C
O
C
C
H
N
4
6
CH
3
5
665
(a)
N
P P
P
FIGURE 8–34 Formation of pyrimidine dimers induced by UV
light. (a) One type of reaction (on the left) results in the formation
of a cyclobutyl ring involving C-5 and C-6 of adjacent pyrimidine
residues. An alternative reaction (on the right) results in a 6-4
photoproduct, with a linkage between C-6 of one pyrimidine and
C-4 of its neighbor. (b) Formation of a cyclobutane pyrimidine
dimer introduces a bend or kink into the DNA.
Some Bases of DNA Are Methylated
Certain nucleotide bases in DNA molecules are enzy-
matically methylated. Adenine and cytosine are methy-
lated more often than guanine and thymine. Methyla-
tion is generally confined to certain sequences or
regions of a DNA molecule. In some cases the function
of methylation is well understood; in others the func-
tion remains unclear. All known DNA methylases use S-
adenosylmethionine as a methyl group donor. E. coli
has two prominent methylation systems. One serves as
part of a defense mechanism that helps the cell to dis-
tinguish its DNA from foreign DNA by marking its
own DNA with methyl groups and destroying (foreign)
DNA without the methyl groups (this is known as a
restriction-modification system; see Chapter 9). The
other system methylates adenosine residues within the
sequence (5H11032)GATC(3H11032) to N
6
-methyladenosine (Fig.
8–5a). This is mediated by the Dam (DNA adenine
methylation) methylase, a component of a system that
repairs mismatched base pairs formed occasionally dur-
ing DNA replication (see Fig. 25–20).
In eukaryotic cells, about 5% of cytidine residues in
DNA are methylated to 5-methylcytidine (Fig. 8–5a).
Methylation is most common at CpG sequences, pro-
ducing methyl-CpG symmetrically on both strands of the
DNA. The extent of methylation of CpG sequences
varies by molecular region in large eukaryotic DNA mol-
ecules. Methylation suppresses the migration of seg-
ments of DNA called transposons, described in Chapter
25. These methylations of cytosine also have structural
significance. The presence of 5-methylcytosine in an al-
ternating CpG sequence markedly increases the ten-
dency for that segment of DNA to assume the Z form.
The Sequences of Long DNA Strands
Can Be Determined
In its capacity as a repository of information, a DNA mol-
ecule’s most important property is its nucleotide se-
quence. Until the late 1970s, determining the sequence
of a nucleic acid containing even five or ten nucleotides
was difficult and very laborious. The development of two
new techniques in 1977, one by Alan Maxam and Walter
Gilbert and the other by Frederick Sanger, has made pos-
sible the sequencing of ever larger DNA molecules with
an ease unimagined just a few decades ago. The tech-
niques depend on an improved understanding of nu-
cleotide chemistry and DNA metabolism, and on elec-
trophoretic methods for separating DNA strands differing
in size by only one nucleotide. Electrophoresis of DNA is
similar to that of proteins (see Fig. 3–19). Polyacrylamide
is often used as the gel matrix in work with short DNA
molecules (up to a few hundred nucleotides); agarose is
generally used for longer pieces of DNA.
In both Sanger and Maxam-Gilbert sequencing, the
general principle is to reduce the DNA to four sets of la-
beled fragments. The reaction producing each set is
base-specific, so the lengths of the fragments correspond
to positions in the DNA sequence where a certain base
occurs. For example, for an oligonucleotide with the se-
quence pAATCGACT, labeled at the 5H11032 end (the left end),
a reaction that breaks the DNA after each C residue will
generate two labeled fragments: a four-nucleotide and a
seven-nucleotide fragment; a reaction that breaks the
DNA after each G will produce only one labeled, five-
nucleotide fragment. Because the fragments are radio-
actively labeled at their 5H11032 ends, only the fragment to the
5H11032 side of the break is visualized. The fragment sizes cor-
respond to the relative positions of C and G residues in
the sequence. When the sets of fragments corresponding
to each of the four bases are electrophoretically sepa-
rated side by side, they produce a ladder of bands from
which the sequence can be read directly (Fig. 8–36). We
illustrate only the Sanger method, because it has proven
to be technically easier and is in more widespread use.
It requires the enzymatic synthesis of a DNA strand com-
plementary to the strand under analysis, using a radio-
actively labeled “primer” and dideoxynucleotides.
Chapter 8 Nucleotides and Nucleic Acids296
FIGURE 8–35 Chemical agents that cause
DNA damage. (a) Precursors of nitrous acid,
which promotes deamination reactions.
(b) Alkylating agents.
8.3 Nucleic Acid Chemistry 297
(a)
P
P
dATP
dGTP
3H11032
P
P
P
OH
A
P
P P P
P
P PP
ATCGGC
OH
A
OH
G
P
C
P
C
P
G
P
T
5H11032
Primer
strand
Template
strand
PP
TC
FIGURE 8–36 DNA sequencing by the Sanger method. This method
makes use of the mechanism of DNA synthesis by DNA polymerases
(Chapter 25). (a) DNA polymerases require both a primer (a short
oligonucleotide strand), to which nucleotides are added, and a template
strand to guide selection of each new nucleotide. In cells, the 3H11032-hy-
droxyl group of the primer reacts with an incoming deoxynucleoside
triphosphate (dNTP) to form a new phosphodiester bond. (b) The Sanger
sequencing procedure uses dideoxynucleoside triphosphate (ddNTP)
analogs to interrupt DNA synthesis. (The Sanger method is also known
as the dideoxy method.) When a ddNTP is inserted in place of a dNTP,
strand elongation is halted after the analog is added, because it lacks
the 3H11032-hydroxyl group needed for the next step.
(c) The DNA to be sequenced is used as the tem-
plate strand, and a short primer, radioactively
or fluorescently labeled, is an-
nealed to it. By addition of small
amounts of a single ddNTP, for
example ddCTP, to an otherwise
normal reaction system, the synthesized
strands will be prematurely terminated at some lo-
cations where dC normally occurs. Given the excess of
dCTP over ddCTP, the chance that the analog will be incorporated
whenever a dC is to be added is small. However, ddCTP is present in
sufficient amounts to ensure that each new strand has a high proba-
bility of acquiring at least one ddC at some point during synthesis. The
result is a solution containing a mixture of labeled fragments, each end-
ing with a C residue. Each C residue in the sequence generates a set
of fragments of a particular length, such that the different-sized frag-
ments, separated by electrophoresis, reveal the location of C residues.
This procedure is repeated separately for each of the four ddNTPs, and
the sequence can be read directly from an autoradiogram of the gel.
Because shorter DNA fragments migrate faster, the fragments near the
bottom of the gel represent the nucleotide positions closest to the primer
(the 5H11032 end), and the sequence is read (in the 5H11032 n 3H11032 direction) from
bottom to top. Note that the sequence obtained is that of the strand
complementary to the strand being analyzed.
DNA sequencing is readily automated by a varia-
tion of Sanger’s sequencing method in which the
dideoxynucleotides used for each reaction are labeled
with a differently colored fluorescent tag (Fig. 8–37).
This technology allows DNA sequences containing thou-
sands of nucleotides to be determined in a few hours.
Entire genomes of many organisms have now been se-
quenced (see Table 1–4), and many very large DNA-
sequencing projects are in progress. Perhaps the most
ambitious of these is the Human Genome Project, in
which researchers have sequenced all 3.2 billion base
pairs of the DNA in a human cell (Chapter 9). Dideoxy
Sequencing of DNA
The Chemical Synthesis of DNA Has Been Automated
Another technology that has paved the way for many
biochemical advances is the chemical synthesis of
oligonucleotides with any chosen sequence. The chem-
ical methods for synthesizing nucleic acids were devel-
oped primarily by H. Gobind Khorana and his colleagues
in the 1970s. Refinement and automation of these meth-
ods have made possible the rapid and accurate synthe-
sis of DNA strands. The synthesis is carried out with the
growing strand attached to a solid support (Fig. 8–38),
using principles similar to those used by Merrifield in
peptide synthesis (see Fig. 3–29). The efficiency of each
addition step is very high, allowing the routine labora-
tory synthesis of polymers containing 70 or 80 nu-
cleotides and, in some laboratories, much longer
strands. The availability of relatively inexpensive DNA
polymers with predesigned sequences is having a pow-
erful impact on all areas of biochemistry (Chapter 9).
Chapter 8 Nucleotides and Nucleic Acids298
FIGURE 8–37 Strategy for automating DNA sequencing reactions.
Each dideoxynucleotide used in the Sanger method can be linked to
a fluorescent molecule that gives all the fragments terminating in that
nucleotide a particular color. All four labeled ddNTPs are added to a
single tube. The resulting colored DNA fragments are then separated
by size in a single electrophoretic gel contained in a capillary tube (a
refinement of gel electrophoresis that allows for faster separations). All
fragments of a given length migrate through the capillary gel in a single
peak, and the color associated with each peak is detected using a laser
beam. The DNA sequence is read by determining the sequence of
colors in the peaks as they pass the detector. This information is fed
directly to a computer, which determines the sequence.
Nucleoside
attached to
silica support
1
42Repeat steps to until all residues are added
CH
2
H
O
H
HH
Base
1
O
OH H
3H11032
5H11032
DMT
Nucleoside
protected
at 5H11032 hydroxyl
R
Si
O
H
O
H
HH
Base
1
O
H
CH
2
H
DMT
O
CH
2
H
O
H
H
Base
1
O
H
R
Si
H
O
H
OP
H
H
Base
2
O
R
Si
CH
2
H
O
HH
HH
Base
1
O
CH
2
H
O
O
O
H
2
NC (CH
2
)
DMT
O
H
OP
H
HH
Base
2
O
R
Si
CH
2
H
O
HH
HH
Base
1
O
CH
2
H
O
O
2
NC (CH
2
)
DMT
Oxidation to
form triester
4
DMT
Protecting
group removed
2
2
H
Next nucleotide
added
3
N
H11001
CH
2
CH
2
H
NCH(CH
3
)
2
CH
(CH
3
)
H
OP
H
HH
Base
2
O
Nucleotide
activated
at 3H11032 position
CH
2
H
O
O
(CH
3
)
(CH
3
)
2
NC (CH
2
)
Cyanoethyl
protecting group
Diisopropylamine byproduct
DMT
Diisopropylamino activating group
Remove protecting groups from bases
Remove cyanoethyl groups from phosphates
Cleave chain from silica support7
5
6
5H11032 3H11032
Oligonucleotide chain
8.3 Nucleic Acid Chemistry 299
FIGURE 8–38 Chemical synthesis of DNA. Automated DNA synthe-
sis is conceptually similar to the synthesis of polypeptides on a solid
support. The oligonucleotide is built up on the solid support (silica),
one nucleotide at a time, in a repeated series of chemical reactions
with suitably protected nucleotide precursors. 1 The first nucleoside
(which will be the 3H11032 end) is attached to the silica support at the 3H11032
hydroxyl (through a linking group, R) and is protected at the 5H11032 hy-
droxyl with an acid-labile dimethoxytrityl group (DMT). The reactive
groups on all bases are also chemically protected. 2 The protecting
DMT group is removed by washing the column with acid (the DMT
group is colored, so this reaction can be followed spectrophotomet-
rically). 3 The next nucleotide is activated with a diisopropylamino
group and reacted with the bound nucleotide to form a 5H11032,3H11032 linkage,
which in step 4 is oxidized with iodine to produce a phosphotri-
ester linkage. (One of the phosphate oxygens carries a cyanoethyl pro-
tecting group.) Reactions 2 through 4 are repeated until all nu-
cleotides are added. At each step, excess nucleotide is removed before
addition of the next nucleotide. In steps 5 and 6 the remaining
protecting groups on the bases and the phosphates are removed, and
in 7 the oligonucleotide is separated from the solid support and pu-
rified. The chemical synthesis of RNA is somewhat more complicated
because of the need to protect the 2H11032 hydroxyl of ribose without ad-
versely affecting the reactivity of the 3H11032 hydroxyl.
SUMMARY 8.3 Nucleic Acid Chemistry
■ Native DNA undergoes reversible unwinding
and separation of strands (melting) on heating
or at extremes of pH. DNAs rich in GqC pairs
have higher melting points than DNAs rich in
AUT pairs.
■ Denatured single-stranded DNAs from two
species can form a hybrid duplex, the degree
of hybridization depending on the extent of
sequence similarity. Hybridization is the basis
for important techniques used to study and
isolate specific genes and RNAs.
■ DNA is a relatively stable polymer. Spontaneous
reactions such as deamination of certain bases,
hydrolysis of base-sugar N-glycosyl bonds,
radiation-induced formation of pyrimidine
dimers, and oxidative damage occur at very low
rates, yet are important because of cells’ very
low tolerance for changes in genetic material.
■ DNA sequences can be determined and DNA
polymers synthesized with simple, automated
protocols involving chemical and enzymatic
methods.
8.4 Other Functions of Nucleotides
In addition to their roles as the subunits of nucleic acids,
nucleotides have a variety of other functions in every
cell: as energy carriers, components of enzyme cofac-
tors, and chemical messengers.
Nucleotides Carry Chemical Energy in Cells
The phosphate group covalently linked at the 5H11032 hy-
droxyl of a ribonucleotide may have one or two addi-
tional phosphates attached. The resulting molecules are
referred to as nucleoside mono-, di-, and triphosphates
(Fig. 8–39). Starting from the ribose, the three phos-
phates are generally labeled H9251, H9252, and H9253. Hydrolysis of
nucleoside triphosphates provides the chemical energy
to drive a wide variety of cellular reactions. Adenosine
5H11032-triphosphate, ATP, is by far the most widely used for
this purpose, but UTP, GTP, and CTP are also used in
some reactions. Nucleoside triphosphates also serve as
the activated precursors of DNA and RNA synthesis, as
described in Chapters 25 and 26.
The energy released by hydrolysis of ATP and the
other nucleoside triphosphates is accounted for by the
structure of the triphosphate group. The bond between
the ribose and the H9251 phosphate is an ester linkage. The
H9251,H9252 and H9252,H9253 linkages are phosphoanhydrides (Fig.
8–40). Hydrolysis of the ester linkage yields about 14
kJ/mol under standard conditions, whereas hydrolysis
of each anhydride bond yields about 30 kJ/mol. ATP hy-
drolysis often plays an important thermodynamic role
in biosynthesis. When coupled to a reaction with a pos-
itive free-energy change, ATP hydrolysis shifts the equi-
librium of the overall process to favor product forma-
Chapter 8 Nucleotides and Nucleic Acids300
Abbreviations of ribonucleoside
5H11032-phosphates
Base Mono- Di- Tri-
Adenine
Guanine
Cytosine
Thymine
AMP
GMP
CMP
UMP
ADP
GDP
CDP
UDP
ATP
GTP
CTP
UTP
Abbreviations of deoxyribonucleoside
Base Mono- Di- Tri-
Adenine
Guanine
Cytosine
Uracil
dAMP
dGMP
dCMP
dTMP
dADP
dGDP
dCDP
dTDP
dATP
dGTP
dCTP
dTTP
5H11032-phosphates
O
H11002
OCH
2
H
O
H11002
P
O
H11002
O
H11002
O
P
O
OOP
OO
H
H
H
H
Base
O
OH
H9251H9252H9253
NMP
NDP
NTP
FIGURE 8–39 Nucleoside phosphates. General structure of the nucleoside
5H11032-mono-, di-, and triphosphates (NMPs, NDPs, and NTPs) and their standard
abbreviations. In the deoxyribonucleoside phosphates (dNMPs, dNDPs, and
dNTPs), the pentose is 2H11032-deoxy-D-ribose.
FIGURE 8–40 The phosphate ester and phosphoanhydride bonds of
ATP. Hydrolysis of an anhydride bond yields more energy than hy-
drolysis of the ester. A carboxylic acid anhydride and carboxylic acid
ester are shown for comparison.
tion (recall the relationship between equilibrium con-
stant and free-energy change described by Eqn 6–3 on
p. 195).
Adenine Nucleotides Are Components of Many
Enzyme Cofactors
A variety of enzyme cofactors serving a wide range of
chemical functions include adenosine as part of their
structure (Fig. 8–41). They are unrelated structurally
except for the presence of adenosine. In none of these
cofactors does the adenosine portion participate directly
in the primary function, but removal of adenosine gen-
erally results in a drastic reduction of cofactor activi-
ties. For example, removal of the adenine nucleotide
(3H11032-phosphoadenosine diphosphate) from acetoacetyl-
CoA, the coenzyme A derivative of acetoacetate, re-
duces its reactivity as a substrate for H9252-ketoacyl-CoA
transferase (an enzyme of lipid metabolism) by a factor
of 10
6
. Although this requirement for adenosine has not
been investigated in detail, it must involve the binding
energy between enzyme and substrate (or cofactor) that
is used both in catalysis and in stabilizing the initial
enzyme-substrate complex (Chapter 6). In the case of
H9252-ketoacyl-CoA transferase, the nucleotide moiety of
coenzyme A appears to be a binding “handle” that helps
to pull the substrate (acetoacetyl-CoA) into the active
site. Similar roles may be found for the nucleoside por-
tion of other nucleotide cofactors.
Why is adenosine, rather than some other large mol-
ecule, used in these structures? The answer here may
involve a form of evolutionary economy. Adenosine is
8.4 Other Functions of Nucleotides 301
H11002
O
H
O
H11002
P
O
H11002
O
O
O
P
H
H
H
OOH
CH
2
O
OCH
3
C
N
CH
2
NH
2
N
N
N
NC CC
N
CH
2
C
NCH
2
CH
2
H
HH
H
CH
3
HS
O
O
OH
CH
3
5H11032
P
O
O
O
H11002
O
H11002
3H11032-Phosphoadenosine diphosphate
(3H11032-P-ADP)
Pantothenic acidb-Mercaptoethylamine
Coenzyme A
CH
2
CHOH
CHOH
CHOH
CH
2
O
O
P
H11001
O
O
O P O
H11002
CH
2
H
O
H
H
N
NH
2
N
N
N
H
OH
2H11032
4H11032 1H11032
3H11032
CH
2
O
H
ON
O
N
N
CH
3
NH
2
Riboflavin
Nicotinamide adenine dinucleotide (NAD
H11545
)
H
O
H
H
N
N
N
N
H
HOH
CH
2
O
NH
2
Flavin adenine dinucleotide (FAD)
H
O
H
H
N
H
HOH
CH
2
O
Nicotinamide
OP
H11002
O
O
O
O PO
H11002
O
O
FIGURE 8–41 Some coenzymes containing adenosine. The
adenosine portion is shaded in light red. Coenzyme A (CoA)
functions in acyl group transfer reactions; the acyl group (such as
the acetyl or acetoacetyl group) is attached to the CoA through a
thioester linkage to the H9252-mercaptoethylamine moiety. NAD
H11001
func-
tions in hydride transfers, and FAD, the active form of vitamin B
2
(riboflavin), in electron transfers. Another coenzyme incorporating
adenosine is 5H11032-deoxyadenosylcobalamin, the active form of vita-
min B
12
(see Box 17-2), which participates in intramolecular
group transfers between adjacent carbons.
certainly not unique in the amount of potential binding
energy it can contribute. The importance of adenosine
probably lies not so much in some special chemical char-
acteristic as in the evolutionary advantage of using one
compound for multiple roles. Once ATP became the uni-
versal source of chemical energy, systems developed to
synthesize ATP in greater abundance than the other nu-
cleotides; because it is abundant, it becomes the logical
choice for incorporation into a wide variety of struc-
tures. The economy extends to protein structure. A sin-
gle protein domain that binds adenosine can be used in
a wide variety of enzymes. Such a domain, called a
nucleotide-binding fold, is found in many enzymes
that bind ATP and nucleotide cofactors.
Some Nucleotides Are Regulatory Molecules
Cells respond to their environment by taking cues from
hormones or other external chemical signals. The in-
teraction of these extracellular chemical signals (“first
messengers”) with receptors on the cell surface often
leads to the production of second messengers inside
the cell, which in turn leads to adaptive changes in
the cell interior (Chapter 12). Often, the second mes-
senger is a nucleotide (Fig. 8–42). One of the most
common is adenosine 3H11541,5H11541-cyclic monophosphate
(cyclic AMP, or cAMP), formed from ATP in a reac-
tion catalyzed by adenylyl cyclase, an enzyme associ-
ated with the inner face of the plasma membrane. Cyclic
AMP serves regulatory functions in virtually every cell
outside the plant kingdom. Guanosine 3H11032,5H11032-cyclic mono-
phosphate (cGMP) occurs in many cells and also has
regulatory functions.
Another regulatory nucleotide, ppGpp (Fig. 8–42),
is produced in bacteria in response to a slowdown in
protein synthesis during amino acid starvation. This nu-
cleotide inhibits the synthesis of the rRNA and tRNA
molecules (see Fig. 28–24) needed for protein synthe-
sis, preventing the unnecessary production of nucleic
acids.
SUMMARY 8.4 Other Functions of Nucleotides
■ ATP is the central carrier of chemical energy in
cells. The presence of an adenosine moiety in a
variety of enzyme cofactors may be related to
binding-energy requirements.
■ Cyclic AMP, formed from ATP in a reaction
catalyzed by adenylyl cyclase, is a common
second messenger produced in response to
hormones and other chemical signals.
Chapter 8 Nucleotides and Nucleic Acids302
FIGURE 8–42 Three regulatory nucleotides.
Key Terms
gene 273
ribosomal RNA (rRNA) 273
messenger RNA (mRNA) 273
transfer RNA (tRNA) 273
nucleotide 273
nucleoside 273
pyrimidine 273
purine 273
deoxyribonucleotides 274
ribonucleotide 274
phosphodiester linkage 277
Terms in bold are defined in the glossary.
5H11032 end 277
3H11032 end 277
oligonucleotide 278
polynucleotide 278
base pair 279
major groove 282
minor groove 282
B-form DNA 284
A-form DNA 284
Z-form DNA 284
palindrome 285
hairpin 285
cruciform 285
triplex DNA 286
G tetraplex 287
H-DNA 287
monocistronic mRNA 287
polycistronic mRNA 288
mutation 293
second messenger 302
adenosine 3H11541,5H11541-cyclic monophos-
phate (cyclic AMP, cAMP) 302
Chapter 8 Problems 303
Further Reading
General
Chang, K.Y. & Varani, G. (1997) Nucleic acids structure and
recognition. Nat. Struct. Biol. 4 (Suppl.), 854–858.
Describes the application of NMR to determination of nucleic
acid structure.
Friedberg, E.C., Walker, G.C., & Siede, W. (1995) DNA Repair
and Mutagenesis, W. H. Freeman and Company, New York.
A good source for more information on the chemistry of
nucleotides and nucleic acids.
Hecht, S.M. (ed.) (1996) Bioorganic Chemistry: Nucleic Acids,
Oxford University Press, Oxford.
A very useful set of articles.
Kornberg, A. & Baker, T.A. (1991) DNA Replication, 2nd edn,
W. H. Freeman and Company, New York.
The best place to start to learn more about DNA structure.
Sinden, R.R. (1994) DNA Structure and Function, Academic
Press, Inc., San Diego.
Good discussion of many topics covered in this chapter.
Historical
Judson, H.F. (1996) The Eighth Day of Creation: Makers of the
Revolution in Biology, expanded edn, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
Olby, R.C. (1994) The Path to the Double Helix: The Discovery
of DNA, Dover Publications, Inc., New York.
Sayre, A. (1978) Rosalind Franklin and DNA, W. W. Norton &
Co., Inc., New York.
Watson, J.D. (1968) The Double Helix: A Personal Account of
the Discovery of the Structure of DNA, Atheneum, New York.
[Paperback edition, Touchstone Books, 2001.]
Variations in DNA Structure
Frank-Kamenetskii, M.D. & Mirkin, S.M. (1995) Triplex DNA
structures. Annu. Rev. Biochem. 64, 65–95.
Herbert, A. & Rich, A. (1996) The biology of left-handed Z-DNA.
J. Biol. Chem. 271, 11,595–11,598.
Htun, H. & Dahlberg, J.E. (1989) Topology and formation of
triple-stranded H-DNA. Science 243, 1571–1576.
Keniry, M.A. (2000) Quadruplex structures in nucleic acids.
Biopolymers 56, 123–146.
Good summary of the structural properties of quadruplexes.
Moore, P.B. (1999) Structural motifs in RNA. Annu. Rev.
Biochem. 68, 287–300.
Shafer, R.H. (1998) Stability and structure of model DNA
triplexes and quadruplexes and their interactions with small
ligands. Prog. Nucleic Acid Res. Mol. Biol. 59, 55–94.
Wells, R.D. (1988) Unusual DNA structures. J. Biol. Chem. 263,
1095–1098.
Minireview; a concise summary.
Nucleic Acid Chemistry
Collins, A.R. (1999) Oxidative DNA damage, antioxidants, and
cancer. Bioessays 21, 238–246.
Marnett, L.J. & Plastaras, J.P. (2001) Endogenous DNA
damage and mutation. Trends Genet. 17, 214–221.
ATP As Energy Carrier
Jencks, W.P. (1987) Economics of enzyme catalysis. Cold Spring
Harb. Symp. Quant. Biol. 52, 65–73.
A relatively short article, full of insights.
1. Nucleotide Structure Which positions in a purine
ring of a purine nucleotide in DNA have the potential to form
hydrogen bonds but are not involved in Watson-Crick base
pairing?
2. Base Sequence of Complementary DNA Strands
One strand of a double-helical DNA has the sequence
(5H11032)GCGCAATATTTCTCAAAATATTGCGC(3H11032). Write the base
sequence of the complementary strand. What special type of
sequence is contained in this DNA segment? Does the dou-
ble-stranded DNA have the potential to form any alternative
structures?
3. DNA of the Human Body Calculate the weight in
grams of a double-helical DNA molecule stretching from the
earth to the moon (~320,000 km). The DNA double helix
weighs about 1 H11003 10
H1100218
g per 1,000 nucleotide pairs; each
base pair extends 3.4 ?. For an interesting comparison, your
body contains about 0.5 g of DNA!
4. DNA Bending Assume that a poly(A) tract five base
pairs long produces a 20H11034 bend in a DNA strand. Calculate
the total (net) bend produced in a DNA if the center base
pairs (the third of five) of two successive (dA)
5
tracts are lo-
cated (a) 10 base pairs apart; (b) 15 base pairs apart. As-
sume 10 base pairs per turn in the DNA double helix.
5. Distinction between DNA Structure and RNA
Structure Hairpins may form at palindromic sequences in
single strands of either RNA or DNA. How is the helical struc-
ture of a long and fully base-paired (except at the end) hair-
pin in RNA different from that of a similar hairpin in DNA?
6. Nucleotide Chemistry The cells of many eukaryotic
organisms have highly specialized systems that specifically
repair G–T mismatches in DNA. The mismatch is repaired to
form a GqC (not AUT) base pair. This G–T mismatch repair
mechanism occurs in addition to a more general system that
repairs virtually all mismatches. Can you suggest why cells
might require a specialized system to repair G–T mismatches?
7. Nucleic Acid Structure Explain why the absorption
of UV light by double-stranded DNA increases (hyperchromic
effect) when the DNA is denatured.
Problems
Chapter 8 Nucleotides and Nucleic Acids304
8. Determination of Protein Concentration in a So-
lution Containing Proteins and Nucleic Acids The con-
centration of protein or nucleic acid in a solution containing
both can be estimated by using their different light absorp-
tion properties: proteins absorb most strongly at 280 nm and
nucleic acids at 260 nm. Their respective concentrations in a
mixture can be estimated by measuring the absorbance (A)
of the solution at 280 nm and 260 nm and using the table be-
low, which gives R
280/260
, the ratio of absorbances at 280 and
260 nm; the percentage of total mass that is nucleic acid; and
a factor, F, that corrects the A
280
reading and gives a more
accurate protein estimate. The protein concentration (in
mg/ml) H11005 F H11003 A
280
(assuming the cuvette is 1 cm wide). Cal-
culate the protein concentration in a solution of A
280
H11005 0.69
and A
260
H11005 0.94.
9. Base Pairing in DNA In samples of DNA isolated from
two unidentified species of bacteria, X and Y, adenine makes
up 32% and 17%, respectively, of the total bases. What rela-
tive proportions of adenine, guanine, thymine, and cytosine
would you expect to find in the two DNA samples? What as-
sumptions have you made? One of these species was isolated
from a hot spring (64 H11034C). Suggest which species is the ther-
mophilic bacterium. What is the basis for your answer?
10. Solubility of the Components of DNA Draw the fol-
lowing structures and rate their relative solubilities in water
(most soluble to least soluble): deoxyribose, guanine, phos-
phate. How are these solubilities consistent with the three-
dimensional structure of double-stranded DNA?
11. DNA Sequencing The following DNA fragment was
sequenced by the Sanger method. The red asterisk indicates
a fluorescent label.
A sample of the DNA was reacted with DNA polymerase and
each of the nucleotide mixtures (in an appropriate buffer)
listed below. Dideoxynucleotides (ddNTPs) were added in
relatively small amounts.
1. dATP, dTTP, dCTP, dGTP, ddTTP
2. dATP, dTTP, dCTP, dGTP, ddGTP
3. dATP, dCTP, dGTP, ddTTP
4. dATP, dTTP, dCTP, dGTP
The resulting DNA was separated by electrophoresis on an
agarose gel, and the fluorescent bands on the gel were
located. The band pattern resulting from nucleotide mixture
1 is shown below. Assuming that all mixtures were run on the
same gel, what did the remaining lanes of the gel look like?
12. Snake Venom Phosphodiesterase An exonuclease is
an enzyme that sequentially cleaves nucleotides from the end
of a polynucleotide strand. Snake venom phosphodiesterase,
which hydrolyzes nucleotides from the 3H11032 end of any oligonu-
cleotide with a free 3H11032-hydroxyl group, cleaves between the
3H11032 hydroxyl of the ribose or deoxyribose and the phosphoryl
group of the next nucleotide. It acts on single-stranded DNA
or RNA and has no base specificity. This enzyme was used in
sequence determination experiments before the development
of modern nucleic acid sequencing techniques. What are the
products of partial digestion by snake venom phosphodi-
esterase of an oligonucleotide with the following sequence?
(5H11032)GCGCCAUUGC(3H11032)–OH
13. Preserving DNA in Bacterial Endospores Bacter-
ial endospores form when the environment is no longer con-
ducive to active cell metabolism. The soil bacterium Bacillus
subtilis, for example, begins the process of sporulation when
one or more nutrients are depleted. The end product is a
Proportion of
R
280/260
nucleic acid (%) F
1.75 0.00 1.116
1.63 0.25 1.081
1.52 0.50 1.054
1.40 0.75 1.023
1.36 1.00 0.994
1.30 1.25 0.970
1.25 1.50 0.944
1.16 2.00 0.899
1.09 2.50 0.852
1.03 3.00 0.814
0.979 3.50 0.776
0.939 4.00 0.743
0.874 5.00 0.682
0.846 5.50 0.656
0.822 6.00 0.632
0.804 6.50 0.607
0.784 7.00 0.585
0.767 7.50 0.565
0.753 8.00 0.545
0.730 9.00 0.508
0.705 10.00 0.478
0.671 12.00 0.422
0.644 14.00 0.377
0.615 17.00 0.322
0.595 20.00 0.278
Chapter 8 Problems 305
small, metabolically dormant structure that can survive al-
most indefinitely with no detectable metabolism. Spores have
mechanisms to prevent accumulation of potentially lethal mu-
tations in their DNA over periods of dormancy that can ex-
ceed 1,000 years. B. subtilis spores are much more resistant
than the organism’s growing cells to heat, UV radiation, and
oxidizing agents, all of which promote mutations.
(a) One factor that prevents potential DNA damage in
spores is their greatly decreased water content. How would
this affect some types of mutations?
(b) Endospores have a category of proteins called small
acid-soluble proteins (SASPs) that bind to their DNA, pre-
venting formation of cyclobutane-type dimers. What causes
cyclobutane dimers, and why do bacterial endospores need
mechanisms to prevent their formation?
Biochemistry on the Internet
14. The Structure of DNA Elucidation of the three-
dimensional structure of DNA helped researchers understand
how this molecule conveys information that can be faithfully
replicated from one generation to the next. To see the second-
ary structure of double-stranded DNA, go to the Protein Data
Bank website (www.rcsb.org/pdb). Use the PDB identifiers
listed below to retrieve the data pages for the two forms of DNA.
Open the structures using RasMol or Chime, and use the dif-
ferent viewing options to complete the following exercises.
(a) Obtain the file for 141D, a highly conserved, re-
peated DNA sequence from the end of the HIV-1 (the virus
that causes AIDS) genome. Display the molecule as a stick
or ball-and-stick structure. Identify the sugar–phosphate
backbone for each strand of the DNA duplex. Locate and iden-
tify individual bases. Which is the 5H11032 end of this molecule?
Locate the major and minor grooves. Is this a right- or left-
handed helix?
(b) Obtain the file for 145D, a DNA with the Z confor-
mation. Display the molecule as a stick or ball-and-stick struc-
ture. Identify the sugar–phosphate backbone for each strand
of the DNA duplex. Is this a right- or left-handed helix?
(c) To fully appreciate the secondary structure of DNA,
select “Stereo” in the Options menu in the viewer. You will
see two images of the DNA molecule. Sit with your nose ap-
proximately 10 inches from the monitor and focus on the tip
of your nose. In the background you should see three images
of the DNA helix. Shift your focus from the tip of your nose
to the middle image, which should appear three-dimensional.
(Note that only one of the two authors can make this work.)
For additional tips, see the Study Guide or the textbook web-
site (www.whfreeman.com/lehninger).
chapter
W
e now turn to a technology that is fundamental
to the advance of modern biological sciences, defin-
ing present and future biochemical frontiers and illus-
trating many important principles of biochemistry.
Elucidation of the laws governing enzymatic catalysis,
macromolecular structure, cellular metabolism, and in-
formation pathways allows research to be directed at in-
creasingly complex biochemical processes. Cell division,
immunity, embryogenesis, vision, taste, oncogenesis,
cognition—all are orchestrated in an elaborate sym-
phony of molecular and macromolecular interactions
that we are now beginning to understand with increasing
clarity. The real implications of the biochemical journey
begun in the nineteenth century are found in the ever-
increasing power to analyze and alter living systems.
To understand a complex biological process, a bio-
chemist isolates and studies the individual components
in vitro, then pieces together the parts to get a coher-
ent picture of the overall process. A major source of mo-
lecular insights is the cell’s own information archive, its
DNA. The sheer size of chromosomes, however, pres-
ents an enormous challenge: how does one find and
study a particular gene among the tens of thousands of
genes nested in the billions of base pairs of a mammalian
genome? Solutions began to emerge in the 1970s.
Decades of advances by thousands of scientists
working in genetics, biochemistry, cell biology, and phys-
ical chemistry came together in the laboratories of Paul
Berg, Herbert Boyer, and Stanley Cohen to yield tech-
niques for locating, isolating, preparing, and studying
small segments of DNA derived from much larger chro-
mosomes. Techniques for DNA cloning paved the way
to the modern fields of genomics and proteomics, the
study of genes and proteins on the scale of whole cells
and organisms. These new methods are transforming ba-
sic research, agriculture, medicine, ecology, forensics,
and many other fields, while occasionally presenting so-
ciety with difficult choices and ethical dilemmas.
We begin this chapter with an outline of the funda-
mental biochemical principles of the now-classic disci-
pline of DNA cloning. Next, after laying the groundwork
for a discussion of genomics, we illustrate the range of
applications and the potential of these technologies,
with a broad emphasis on modern advances in genomics
and proteomics.
9.1 DNA Cloning: The Basics
A clone is an identical copy. This term originally applied
to cells of a single type, isolated and allowed to repro-
duce to create a population of identical cells. DNA
cloning involves separating a specific gene or DNA seg-
ment from a larger chromosome, attaching it to a small
molecule of carrier DNA, and then replicating this mod-
ified DNA thousands or millions of times through both
an increase in cell number and the creation of multiple
DNA-BASED INFORMATION
TECHNOLOGIES
9.1 DNA Cloning: The Basics 306
9.2 From Genes to Genomes 317
9.3 From Genomes to Proteomes 325
9.4 Genome Alterations and New Products
of Biotechnology 330
Of all the natural systems, living matter is the one which,
in the face of great transformations, preserves inscribed in
its organization the largest amount of its own past history.
—Emile Zuckerkandl and Linus Pauling, article in Journal
of Theoretical Biology, 1965
9
306
8885d_c09_306-342 2/7/04 8:14 AM Page 306 mac76 mac76:385_reb:
copies of the cloned DNA in each cell. The
result is selective amplification of a par-
ticular gene or DNA segment. Cloning of
DNA from any organism entails five gen-
eral procedures:
1. Cutting DNA at precise locations.
Sequence-specific endonucleases (re-
striction endonucleases) provide the
necessary molecular scissors.
2. Selecting a small molecule of DNA
capable of self-replication. These
DNAs are called cloning vectors (a vector is a
delivery agent). They are typically plasmids or
viral DNAs.
3. Joining two DNA fragments covalently. The
enzyme DNA ligase links the cloning vector and
DNA to be cloned. Composite DNA molecules
comprising covalently linked segments from two or
more sources are called recombinant DNAs.
4. Moving recombinant DNA from the test tube to
a host cell that will provide the enzymatic machin-
ery for DNA replication.
5. Selecting or identifying host cells that contain
recombinant DNA.
The methods used to accomplish these and related tasks
are collectively referred to as recombinant DNA tech-
nology or, more informally, genetic engineering.
Much of our initial discussion will focus on DNA
cloning in the bacterium Escherichia coli, the first or-
ganism used for recombinant DNA work and still the
most common host cell. E. coli has many advantages:
its DNA metabolism (like many other of its biochemical
processes) is well understood; many naturally occurring
cloning vectors associated with E. coli, such as plasmids
and bacteriophages (bacterial viruses; also called
phages), are well characterized; and techniques are
available for moving DNA expeditiously from one bac-
terial cell to another. We also address DNA cloning in
other organisms, a topic discussed more fully later in
the chapter.
Restriction Endonucleases and DNA Ligase
Yield Recombinant DNA
Particularly important to recombinant DNA technology
is a set of enzymes (Table 9–1) made available through
decades of research on nucleic acid metabolism. Two
classes of enzymes lie at the heart of the general ap-
proach to generating and propagating a recombinant
DNA molecule (Fig. 9–1). First, restriction endonu-
cleases (also called restriction enzymes) recognize and
cleave DNA at specific DNA sequences (recognition se-
quences or restriction sites) to generate a set of smaller
fragments. Second, the DNA fragment to be cloned can
be joined to a suitable cloning vector by using DNA lig-
ases to link the DNA molecules together. The recombi-
nant vector is then introduced into a host cell, which
amplifies the fragment in the course of many genera-
tions of cell division.
Restriction endonucleases are found in a wide range
of bacterial species. Werner Arber discovered in the
early 1960s that their biological function is to recognize
and cleave foreign DNA (the DNA of an infecting virus,
for example); such DNA is said to be restricted. In the
host cell’s DNA, the sequence that would be recognized
9.1 DNA Cloning: The Basics 307
Paul Berg Herbert Boyer Stanley N. Cohen
TABLE 9–1 Some Enzymes Used in Recombinant DNA Technology
Enzyme(s) Function
Type II restriction endonucleases Cleave DNAs at specific base sequences
DNA ligase Joins two DNA molecules or fragments
DNA polymerase I (E. coli) Fills gaps in duplexes by stepwise addition of nucleotides to 3H11032 ends
Reverse transcriptase Makes a DNA copy of an RNA molecule
Polynucleotide kinase Adds a phosphate to the 5H11032-OH end of a polynucleotide to label it or permit ligation
Terminal transferase Adds homopolymer tails to the 3H11032-OH ends of a linear duplex
Exonuclease III Removes nucleotide residues from the 3H11032 ends of a DNA strand
Bacteriophage H9261 exonuclease Removes nucleotides from the 5H11032 ends of a duplex to expose single-stranded 3H11032 ends
Alkaline phosphatase Removes terminal phosphates from either the 5H11032 or 3H11032 end (or both)
8885d_c09_306-342 2/7/04 8:14 AM Page 307 mac76 mac76:385_reb:
by its own restriction endonuclease is protected from
digestion by methylation of the DNA, catalyzed by a spe-
cific DNA methylase. The restriction endonuclease and
the corresponding methylase are sometimes referred to
as a restriction-modification system.
There are three types of restriction endonucleases,
designated I, II, and III. Types I and III are generally large,
multisubunit complexes containing both the endonucle-
Chapter 9 DNA-Based Information Technologies308
Cloning vector
is cleaved with
restriction
endonuclease.
Cloning
vector
(plasmid)
DNA is introduced
into the host cell.
Recombinant
vector
Eukaryotic
chromosome
DNA ligase
DNA fragment of interest
is obtained by cleaving
chromosome with a
restriction endonuclease.
Fragments are ligated
to the prepared cloning
vector.
1 2
3
4
Propagation (cloning)
produces many copies
of recombinant DNA.
5
FIGURE 9–1 Schematic illustration of DNA cloning. A cloning vec-
tor and eukaryotic chromosomes are separately cleaved with the same
restriction endonuclease. The fragments to be cloned are then ligated
to the cloning vector. The resulting recombinant DNA (only one re-
combinant vector is shown here) is introduced into a host cell where
it can be propagated (cloned). Note that this drawing is not to scale:
the size of the E. coli chromosome relative to that of a typical cloning
vector (such as a plasmid) is much greater than depicted here.
ase and methylase activities. Type I restriction endonu-
cleases cleave DNA at random sites that can be more
than 1,000 base pairs (bp) from the recognition se-
quence. Type III restriction endonucleases cleave the
DNA about 25 bp from the recognition sequence. Both
types move along the DNA in a reaction that requires
the energy of ATP. Type II restriction endonucleases,
first isolated by Hamilton Smith in 1970, are simpler, re-
quire no ATP, and cleave the DNA within the recogni-
tion sequence itself. The extraordinary utility of this
group of restriction endonucleases was demonstrated by
Daniel Nathans, who first used them to develop novel
methods for mapping and analyzing genes and genomes.
Thousands of restriction endonucleases have been
discovered in different bacterial species, and more than
100 different DNA sequences are recognized by one or
more of these enzymes. The recognition sequences are
usually 4 to 6 bp long and palindromic (see Fig. 8–20).
Table 9–2 lists sequences recognized by a few type II
restriction endonucleases. In some cases, the interac-
tion between a restriction endonuclease and its target
sequence has been elucidated in exquisite molecular de-
tail; for example, Figure 9–2 shows the complex of the
type II restriction endonuclease EcoRV and its target
sequence.
Some restriction endonucleases make staggered
cuts on the two DNA strands, leaving two to four nu-
cleotides of one strand unpaired at each resulting end.
These unpaired strands are referred to as sticky ends
(Fig. 9–3a), because they can base-pair with each other
or with complementary sticky ends of other DNA frag-
ments. Other restriction endonucleases cleave both
strands of DNA at the opposing phosphodiester bonds,
leaving no unpaired bases on the ends, often called
blunt ends (Fig. 9–3b).
The average size of the DNA fragments produced by
cleaving genomic DNA with a restriction endonuclease
depends on the frequency with which a particular re-
striction site occurs in the DNA molecule; this in turn
depends largely on the size of the recognition sequence.
In a DNA molecule with a random sequence in which all
four nucleotides were equally abundant, a 6 bp sequence
recognized by a restriction endonuclease such as BamHI
would occur on average once every 4
6
(4,096) bp, as-
suming the DNA had a 50% GmC content. Enzymes that
recognize a 4 bp sequence would produce smaller DNA
fragments from a random-sequence DNA molecule; a
recognition sequence of this size would be expected to
occur about once every 4
4
(256) bp. In natural DNA mol-
ecules, particular recognition sequences tend to occur
less frequently than this because nucleotide sequences
in DNA are not random and the four nucleotides are not
equally abundant. In laboratory experiments, the aver-
age size of the fragments produced by restriction en-
donuclease cleavage of a large DNA can be increased by
simply terminating the reaction before completion; the
result is called a partial digest. Fragment size can also
8885d_c09_306-342 2/7/04 8:14 AM Page 308 mac76 mac76:385_reb:
be increased by using a special class of endonucleases
called homing endonucleases (see Fig. 26–34). These
recognize and cleave much longer DNA sequences (14
to 20 bp).
Once a DNA molecule has been cleaved into frag-
ments, a particular fragment of known size can be en-
riched by agarose or acrylamide gel electrophoresis or
by HPLC (pp. 92, 90). For a typical mammalian genome,
however, cleavage by a restriction endonuclease usually
yields too many different DNA fragments to permit iso-
lation of a particular fragment by electrophoresis or
HPLC. A common intermediate step in the cloning of a
specific gene or DNA segment is the construction of a
DNA library (as described in Section 9.2).
After the target DNA fragment is isolated, DNA lig-
ase can be used to join it to a similarly digested cloning
vector—that is, a vector digested by the same restric-
tion endonuclease; a fragment generated by EcoRI, for
example, generally will not link to a fragment generated
by BamHI. As described in more detail in Chapter 25
(see Fig. 25–16), DNA ligase catalyzes the formation of
new phosphodiester bonds in a reaction that uses ATP
9.1 DNA Cloning: The Basics 309
g
*
BamHI (5H11032) G G A T C C (3H11032)
CCT AGG
* h
g
*
CIaI(5H11032) A T C G A T (3H11032)
T A G C T A
* h
g
*
EcoRI (5H11032) G A A T T C (3H11032)
C T T A A G
* h
g
EcoRV (5H11032) G A T A T C (3H11032)
C T A T A G
h
g
*
HaelII (5H11032) G G C C (3H11032)
C C G G
*h
g
HindIII (5H11032) A A G C T T (3H11032)
T T C G A A
h
g
Notl(5H11032) G C G G C C G C (3H11032)
C G C C G G C G
h
*
g
Pstl(5H11032) C T G C A G (3H11032)
G A C G T C
h *
g
Pvull (5H11032) C A G C T G (3H11032)
G T C G A C
h
g
Tth111l (5H11032) G A C N N N G T C (3H11032)
C T G N N N C A G
h
Recognition Sequences for Some Type II Restriction EndonucleasesTABLE 9–2
Arrows indicate the phosphodiester bonds cleaved by each restriction endonuclease. Asterisks indicate bases that are methylated by the corresponding
methylase (where known). N denotes any base. Note that the name of each enzyme consists of a three-letter abbreviation (in italics) of the bacterial
species from which it is derived, sometimes followed by a strain designation and Roman numerals to distinguish different restriction endonucleases
isolated from the same bacterial species. Thus BamHI is the first (I) restriction endonuclease characterized from Bacillus amyloliquefaciens, strain H.
FIGURE 9–2 Interaction of EcoRV restriction
endonuclease with its target sequence. (a) The
dimeric EcoRV endonuclease (its two subunits
in light blue and gray) is bound to the products
of DNA cleavage at the sequence recognized
by the enzyme. The DNA backbone is shown
in two shades of blue to distinguish the
segments separated by cleavage (PDB ID
1RVC). (b) In this view, showing just the
DNA, the DNA segment has been turned
180H11034. The enzyme creates blunt ends; the
cleavage points appear staggered on the two
DNA strands because the DNA is kinked.
Bound magnesium ions (orange) play a role
in catalysis of the cleavage reaction.
Restriction Endonucleases (b)
(a)
8885d_c09_306-342 2/7/04 8:14 AM Page 309 mac76 mac76:385_reb:
Chapter 9 DNA-Based Information Technologies310
G G T G A A T T C A G C T T C G C A T T A G C A G C T G T A G C
C C A
Cleavage
site
Cleavage
site
Recognition
sequences
Chromosomal DNA
G G T G
C C A C T T A A
EcoRI
restriction
endonuclease
PvuII
restriction
endonuclease
G
A A T T C A G C T T C G C A T T A G C A G
G A C A T C G
C T G T A G C
Sticky ends Blunt ends
DNA
ligase
Plasmid
cloning vector
cleaved with
EcoRI and PvuII
(a) (b)
CTTAAGTCGAA CGGTATCGTCGACATCG
TCGAAGCGTAATC GTC
A A T T C C T G C A G A A G C T T C C G G A T C C C C G G G
G
PstI
Synthetic polylinker
DNA
ligase
Plasmid cloning vector
cleaved with EcoRI
(c)
HindIII BamHI
SmaI
GACGTCTTCGAAGGCCTAGGGGCCCTTAA
C
T
T
A
A
G
A
A
T
T
CG
C
T
T
A
A
G
A
A
T
T
CG
A
A
T
T
C
G T
T
A
A
C
G
P
o
ly
link
e
r
P
s
t
I
H
i
n
d
II
I
Ba
m
H
I
S
m
a
I
E
c
o
R
I
FIGURE 9–3 Cleavage of DNA mole-
cules by restriction endonucleases.
Restriction endonucleases recognize
and cleave only specific sequences,
leaving either (a) sticky ends (with
protruding single strands) or (b) blunt
ends. Fragments can be ligated to
other DNAs, such as the cleaved
cloning vector (a plasmid) shown
here. This reaction is facilitated by the
annealing of complementary sticky
ends. Ligation is less efficient for
DNA fragments with blunt ends than
for those with complementary sticky
ends, and DNA fragments with
different (noncomplementary) sticky
ends generally are not ligated.
(c) A synthetic DNA fragment with
recognition sequences for several
restriction endonucleases can be
inserted into a plasmid that has been
cleaved by a restriction endonuclease.
The insert is called a linker; an insert
with multiple restriction sites is called
a polylinker.
or a similar cofactor. The base-pairing of complemen-
tary sticky ends greatly facilitates the ligation reaction
(Fig. 9–3a). Blunt ends can also be ligated, albeit less
efficiently. Researchers can create new DNA sequences
by inserting synthetic DNA fragments (called linkers)
between the ends that are being ligated. Inserted DNA
fragments with multiple recognition sequences for re-
striction endonucleases (often useful later as points for
inserting additional DNA by cleavage and ligation) are
called polylinkers (Fig. 9–3c).
The effectiveness of sticky ends in selectively join-
ing two DNA fragments was apparent in the earliest
recombinant DNA experiments. Before restriction endo-
nucleases were widely available, some workers found
they could generate sticky ends by the combined action
of the bacteriophage H9261 exonuclease and terminal trans-
ferase (Table 9–1). The fragments to be joined were given
complementary homopolymeric tails. Peter Lobban and
Dale Kaiser used this method in 1971 in the first ex-
periments to join naturally occurring DNA fragments.
8885d_c09_306-342 2/7/04 8:14 AM Page 310 mac76 mac76:385_reb:
Similar methods were used soon after in the laboratory
of Paul Berg to join DNA segments from simian virus 40
(SV40) to DNA derived from bacteriophage H9261, thereby
creating the first recombinant DNA molecule with DNA
segments from different species.
Cloning Vectors Allow Amplification
of Inserted DNA Segments
The principles that govern the delivery of recombinant
DNA in clonable form to a host cell, and its subsequent
amplification in the host, are well illustrated by consid-
ering three popular cloning vectors commonly used in
experiments with E. coli—plasmids, bacteriophages,
and bacterial artificial chromosomes—and a vector used
to clone large DNA segments in yeast.
Plasmids Plasmids are circular DNA molecules that
replicate separately from the host chromosome. Natu-
rally occurring bacterial plasmids range in size from
5,000 to 400,000 bp. They can be introduced into bac-
terial cells by a process called transformation. The
cells (generally E. coli) and plasmid DNA are incubated
together at 0 H11034C in a calcium chloride solution, then sub-
jected to a shock by rapidly shifting the temperature to
37 to 43 H11034C. For reasons not well understood, some of
the cells treated in this way take up the plasmid DNA.
Some species of bacteria are naturally competent for
DNA uptake and do not require the calcium chloride
treatment. In an alternative method, cells incubated
with the plasmid DNA are subjected to a high-voltage
pulse. This approach, called electroporation, tran-
siently renders the bacterial membrane permeable to
large molecules.
Regardless of the approach, few cells actually take
up the plasmid DNA, so a method is needed to select
those that do. The usual strategy is to use a plasmid that
includes a gene that the host cell requires for growth
under specific conditions, such as a gene that confers
resistance to an antibiotic. Only cells transformed by the
recombinant plasmid can grow in the presence of that
antibiotic, making any cell that contains the plasmid “se-
lectable” under those growth conditions. Such a gene is
called a selectable marker.
Investigators have developed many different plas-
mid vectors suitable for cloning by modifying naturally
occurring plasmids. The E. coli plasmid pBR322 offers
a good example of the features useful in a cloning vec-
tor (Fig. 9–4):
1. pBR322 has an origin of replication, ori, a
sequence where replication is initiated by cellular
enzymes (Chapter 25). This sequence is required
to propagate the plasmid and maintain it at a level
of 10 to 20 copies per cell.
2. The plasmid contains two genes that confer
resistance to different antibiotics (tet
R
, amp
R
),
allowing the identification of cells that contain the
intact plasmid or a recombinant version of the
plasmid (Fig. 9–5).
3. Several unique recognition sequences in pBR322
(PstI, EcoRI, BamHI, SalI, PvuII) are targets for
different restriction endonucleases, providing sites
where the plasmid can later be cut to insert for-
eign DNA.
4. The small size of the plasmid (4,361 bp) facilitates
its entry into cells and the biochemical manipula-
tion of the DNA.
Transformation of typical bacterial cells with purified
DNA (never a very efficient process) becomes less suc-
cessful as plasmid size increases, and it is difficult to
clone DNA segments longer than about 15,000 bp when
plasmids are used as the vector.
Bacteriophages Bacteriophage H9261 has a very efficient
mechanism for delivering its 48,502 bp of DNA into a
bacterium, and it can be used as a vector to clone some-
what larger DNA segments (Fig. 9–6). Two key features
contribute to its utility:
1. About one-third of the H9261 genome is nonessential
and can be replaced with foreign DNA.
2. DNA is packaged into infectious phage particles
only if it is between 40,000 and 53,000 bp long, a
constraint that can be used to ensure packaging of
recombinant DNA only.
9.1 DNA Cloning: The Basics 311
Ampicillin
resistance
(amp
R
)
Tetracycline
resistance
(tet
R
)
Origin of
replication
(ori)
PvuII
SalI
BamHI
EcoRI
PstI
pBR322
(4,361bp)
FIGURE 9–4 The constructed E. coli plasmid pBR322. Note the lo-
cation of some important restriction sites—for PstI, EcoRI, BamHI, SalI,
and PvuII; ampicillin- and tetracycline-resistance genes; and the repli-
cation origin (ori). Constructed in 1977, this was one of the early plas-
mids designed expressly for cloning in E. coli.
8885d_c09_306-342 2/7/04 8:14 AM Page 311 mac76 mac76:385_reb:
Researchers have developed bacteriophage H9261 vec-
tors that can be readily cleaved into three pieces, two
of which contain essential genes but which together are
only about 30,000 bp long. The third piece, “filler” DNA,
is discarded when the vector is to be used for cloning,
and additional DNA is inserted between the two essen-
tial segments to generate ligated DNA molecules long
enough to produce viable phage particles. In effect, the
packaging mechanism selects for recombinant viral
DNAs.
Bacteriophage H9261 vectors permit the cloning of DNA
fragments of up to 23,000 bp. Once the bacteriophage
H9261 fragments are ligated to foreign DNA fragments of suit-
able size, the resulting recombinant DNAs can be pack-
PstI restriction
endonuclease
pBR322
plasmids
amp
R
tet
R
Foreign DNA
DNA
ligase
Agar containing
tetracycline
(control)
Colonies with
recombinant
plasmids
Host
DNA
transformation
of E. coli cells
2
Foreign DNA is ligated to cleaved
pBR322. Where ligation is successful,
the ampicillin-resistance element is
disrupted. The tetracycline-resistance
element remains intact.
1
pBR322 is cleaved at the ampicillin-
resistance element by PstI.
3
E. coli cells are transformed, then
grown on agar plates containing
tetracycline to select for those that
have taken up plasmid.
4
Individual colonies are transferred
to matching positions on additional
plates. One plate contains tetracycline,
the other tetracycline and ampicillin.
5
Cells that grow on tetracycline but not on tetracycline +
ampicillin contain recombinant plasmids with disrupted
ampicillin resistance, hence the foreign DNA. Cells with pBR322
without foreign DNA retain ampicillin resistance and grow on
both plates.
Agar
containing
tetracycline
Agar containing
ampicillin +
tetracycline
All colonies
have plasmids
selection of
transformed cells
colonies transferred
for testing
FIGURE 9–5 Use of pBR322 to clone and identify foreign DNA in
E. coli. Plasmid Cloning
restriction
endonuclease
Filler DNA (not needed
for packaging)
Lack essential DNA
and/or are too small
to be packagedRecombinant DNAs
DNA ligase
in vitro
packaging
λ bacteriophage
containing foreign DNA
Foreign DNA
fragments
FIGURE 9–6 Bacteriophage H9261 cloning vectors. Recombinant DNA
methods are used to modify the bacteriophage H9261 genome, removing
the genes not needed for phage production and replacing them with
“filler” DNA to make the phage DNA large enough for packaging into
phage particles. As shown here, the filler is replaced with foreign DNA
in cloning experiments. Recombinants are packaged into viable phage
particles in vitro only if they include an appropriately sized foreign
DNA fragment as well as both of the essential H9261 DNA end fragments.
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9.1 DNA Cloning: The Basics 313
FIGURE 9–7 (above right) Bacterial artificial chromosomes (BACs) as
cloning vectors. The vector is a relatively simple plasmid, with a repli-
cation origin (ori) that directs replication. The par genes, derived from
a type of plasmid called an F plasmid, assist in the even distribution
of plasmids to daughter cells at cell division. This increases the likeli-
hood of each daughter cell carrying one copy of the plasmid, even
when few copies are present. The low number of copies is useful in
cloning large segments of DNA because it limits the opportunities for
unwanted recombination reactions that can unpredictably alter large
cloned DNAs over time. The BAC includes selectable markers. A lacZ
gene (required for the production of the enzyme H9252-galactosidase) is sit-
uated in the cloning region such that it is inactivated by cloned DNA
inserts. Introduction of recombinant BACs into cells by electroporation
is promoted by the use of cells with an altered (more porous) cell wall.
Recombinant DNAs are screened for resistance to the antibiotic chlo-
ramphenicol (Cm
R
). Plates also contain a substrate for H9252-galactosidase
that yields a colored product. Colonies with active H9252-galactosidase and
hence no DNA insert in the BAC vector turn blue; colonies without
H9252-galactosidase activity—and thus with the desired DNA inserts—are
white.
Cm
R
F plasmid
par genes
BAC
vector
ori
Cloning sites
(include lacZ)
restriction
endonuclease
DNA
ligase
Large foreign DNA
fragment with appropriate
sticky ends
Colonies with
recombinant BACs
are white.
Agar containing
chloramphenicol
and substrate for
b-galactosidase
electroporation
Recombinant
BAC
selection of
chloramphenicol-
resistant cells
aged into phage particles by adding them to crude bac-
terial cell extracts that contain all the proteins needed
to assemble a complete phage. This is called in vitro
packaging (Fig. 9–6). All viable phage particles will
contain a foreign DNA fragment. The subsequent trans-
mission of the recombinant DNA into E. coli cells is
highly efficient.
Bacterial Artificial Chromosomes (BACs) Bacterial artificial
chromosomes are simply plasmids designed for the
cloning of very long segments (typically 100,000 to
300,000 bp) of DNA (Fig. 9–7). They generally include
selectable markers such as resistance to the antibiotic
chloramphenicol (Cm
R
), as well as a very stable origin
of replication (ori) that maintains the plasmid at one or
two copies per cell. DNA fragments of several hundred
thousand base pairs are cloned into the BAC vector. The
large circular DNAs are then introduced into host bac-
teria by electroporation. These procedures use host
bacteria with mutations that compromise the structure
of their cell wall, permitting the uptake of the large DNA
molecules.
Yeast Artificial Chromosomes (YACs) E. coli cells are by no
means the only hosts for genetic engineering. Yeasts are
particularly convenient eukaryotic organisms for this
work. As with E. coli, yeast genetics is a well-developed
discipline. The genome of the most commonly used
yeast, Saccharomyces cerevisiae, contains only
14 H11003 10
6
bp (a simple genome by eukaryotic standards,
less than four times the size of the E. coli chromosome),
and its entire sequence is known. Yeast is also very easy
to maintain and grow on a large scale in the laboratory.
Plasmid vectors have been constructed for yeast, em-
ploying the same principles that govern the use of E.
coli vectors described above. Convenient methods are
now available for moving DNA into and out of yeast cells,
facilitating the study of many aspects of eukaryotic cell
biochemistry. Some recombinant plasmids incorporate
multiple replication origins and other elements that al-
low them to be used in more than one species (for ex-
ample, yeast or E. coli). Plasmids that can be propa-
gated in cells of two or more different species are called
shuttle vectors.
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Research work with large genomes and the associ-
ated need for high-capacity cloning vectors led to the
development of yeast artificial chromosomes
(YACS; Fig. 9–8). YAC vectors contain all the elements
needed to maintain a eukaryotic chromosome in the
yeast nucleus: a yeast origin of replication, two selec-
table markers, and specialized sequences (derived from
the telomeres and centromere, regions of the chromo-
some discussed in Chapter 24) needed for stability and
proper segregation of the chromosomes at cell division.
Before being used in cloning, the vector is propagated
as a circular bacterial plasmid. Cleavage with a restric-
tion endonuclease (BamH1 in Fig. 9–8) removes a
length of DNA between two telomere sequences (TEL),
leaving the telomeres at the ends of the linearized DNA.
Cleavage at another internal site (EcoRI in Fig. 9–8) di-
vides the vector into two DNA segments, referred to as
vector arms, each with a different selectable marker.
The genomic DNA is prepared by partial digestion
with restriction endonucleases (EcoRI in Fig. 9–8) to
obtain a suitable fragment size. Genomic fragments are
then separated by pulsed field gel electrophoresis,
a variation of gel electrophoresis (see Fig. 3–19) that
allows the separation of very large DNA segments. The
DNA fragments of appropriate size (up to about
2 H11003 10
6
bp) are mixed with the prepared vector arms
and ligated. The ligation mixture is then used to trans-
form treated yeast cells with very large DNA molecules.
Culture on a medium that requires the presence of both
selectable marker genes ensures the growth of only
those yeast cells that contain an artificial chromosome
with a large insert sandwiched between the two vector
arms (Fig. 9–8). The stability of YAC clones increases
with size (up to a point). Those with inserts of more
than 150,000 bp are nearly as stable as normal cellular
chromosomes, whereas those with inserts of less than
100,000 bp are gradually lost during mitosis (so gener-
ally there are no yeast cell clones carrying only the two
vector ends ligated together or with only short inserts).
YACs that lack a telomere at either end are rapidly
degraded.
Specific DNA Sequences Are Detectable
by Hybridization
DNA hybridization, a process outlined in Chapter 8 (see
Fig. 8–32), is the most common sequence-based process
for detecting a particular gene or segment of nucleic
acid. There are many variations of the basic method,
most making use of a labeled (such as radioactive) DNA
or RNA fragment, known as a probe, complementary to
the DNA being sought. In one classic approach to de-
tect a particular DNA sequence within a DNA library (a
collection of DNA clones), nitrocellulose paper is
pressed onto an agar plate containing many individual
bacterial colonies from the library, each colony with a
different recombinant DNA. Some cells from each
colony adhere to the paper, forming a replica of the
plate. The paper is treated with alkali to disrupt the cells
and denature the DNA within, which remains bound to
the region of the paper around the colony from which
it came. Added radioactive DNA probe anneals only to
its complementary DNA. After any unannealed probe
DNA is washed away, the hybridized DNA can be de-
tected by autoradiography (Fig. 9–9).
Chapter 9 DNA-Based Information Technologies314
TEL Xori
EcoRI digestion
creates two arms
BamHI digestion creates
linear chromosome with
telomeric ends
EcoRI
EcoRI
BamHIBamHI
CEN
ori CEN
CEN
ori
Selectable
marker X
Selectable
marker Y
TEL TEL
Y TEL
TEL XY
Right arm has
selectable marker Y
YAC
Left arm has
selectable marker X
TEL
Fragments of genomic
DNA generated by light
digestion with EcoRI
Ligate
Transform
Enzymatic
digestion
of cell wall
Yeast
cell
Yeast
spheroplast
Yeast with
YAC clone
Select for
X and Y
FIGURE 9–8 Construction of a yeast artificial chromosome (YAC). A
YAC vector includes an origin of replication (ori), a centromere (CEN),
two telomeres (TEL), and selectable markers (X and Y). Digestion with
BamH1 and EcoRI generates two separate DNA arms, each with a
telomeric end and one selectable marker. A large segment of DNA
(e.g., up to 2 H11003 10
6
bp from the human genome) is ligated to the two
arms to create a yeast artificial chromosome. The YAC transforms yeast
cells (prepared by removal of the cell wall to form spheroplasts), and
the cells are selected for X and Y; the surviving cells propagate the
DNA insert.
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A common limiting step in detecting and cloning a
gene is the generation of a complementary strand of
nucleic acid to use as a probe. The origin of a probe de-
pends on what is known about the gene under investi-
gation. Sometimes a homologous gene cloned from
another species makes a suitable probe. Or, if the pro-
tein product of a gene has been purified, probes can be
designed and synthesized by working backward from the
amino acid sequence, deducing the DNA sequence that
would code for it (Fig. 9–10). Now, researchers typically
obtain the necessary DNA sequence information from
sequence databases that detail the structure of millions
of genes from a wide range of organisms.
Expression of Cloned Genes Produces
Large Quantities of Protein
Frequently it is the product of the cloned gene, rather
than the gene itself, that is of primary interest—partic-
ularly when the protein has commercial, therapeutic, or
research value. With an increased understanding of the
fundamentals of DNA, RNA, and protein metabolism and
their regulation in E. coli, investigators can now ma-
nipulate cells to express cloned genes in order to study
their protein products.
Most eukaryotic genes lack the DNA sequence ele-
ments—such as promoters, sequences that instruct RNA
polymerase where to bind—required for their expression
in E. coli cells, so bacterial regulatory sequences for
transcription and translation must be inserted at ap-
propriate positions relative to the eukaryotic gene in the
vector DNA. (Promoters, regulatory sequences, and
other aspects of the regulation of gene expression are
discussed in Chapter 28.) In some cases cloned genes
are so efficiently expressed that their protein product
represents 10% or more of the cellular protein; they are
said to be overexpressed. At these concentrations some
foreign proteins can kill an E. coli cell, so gene expres-
sion must be limited to the few hours before the planned
harvest of the cells.
Cloning vectors with the transcription and transla-
tion signals needed for the regulated expression of a
cloned gene are often called expression vectors. The
rate of expression of the cloned gene is controlled by
replacing the gene’s own promoter and regulatory se-
quences with more efficient and convenient versions
supplied by the vector. Generally, a well-characterized
promoter and its regulatory elements are positioned
near several unique restriction sites for cloning, so that
genes inserted at the restriction sites will be expressed
from the regulated promoter element (Fig. 9–11). Some
of these vectors incorporate other features, such as a
bacterial ribosome binding site to enhance translation
of the mRNA derived from the gene, or a transcription
termination sequence.
Genes can similarly be cloned and expressed in eu-
karyotic cells, with various species of yeast as the usual
hosts. A eukaryotic host can sometimes promote post-
translational modifications (changes in protein structure
made after synthesis on the ribosomes) that might be
required for the function of a cloned eukaryotic protein.
9.1 DNA Cloning: The Basics 315
Press nitrocellulose paper onto
the agar plate. Some cells from
each colony stick to the paper.
Agar plate with
transformed
bacterial colonies
Nitrocellulose paper
DNA bound to paper
Probe annealed to
colonies of interest
Treat with alkali to disrupt
cells and expose denatured
DNA.
Incubate the paper with the
radiolabeled probe, then wash.
Radiolabeled DNA probe
Expose
x-ray film
to paper.
FIGURE 9–9 Use of hybridization to identify a clone with a par-
ticular DNA segment. The radioactive DNA probe hybridizes to
complementary DNA and is revealed by autoradiography. Once the
labeled colonies have been identified, the corresponding colonies
on the original agar plate can be used as a source of cloned DNA
for further study.
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Alterations in Cloned Genes Produce
Modified Proteins
Cloning techniques can be used not only to overproduce
proteins but to produce protein products subtly altered
from their native forms. Specific amino acids may be re-
placed individually by site-directed mutagenesis.
This powerful approach to studying protein structure
and function changes the amino acid sequence of a pro-
tein by altering the DNA sequence of the cloned gene.
If appropriate restriction sites flank the sequence to be
altered, researchers can simply remove a DNA segment
and replace it with a synthetic one that is identical to
the original except for the desired change (Fig. 9–12a).
When suitably located restriction sites are not present,
an approach called oligonucleotide-directed muta-
genesis (Fig. 9–12b) can create a specific DNA se-
quence change. A short synthetic DNA strand with a
specific base change is annealed to a single-stranded
copy of the cloned gene within a suitable vector. The
mismatch of a single base pair in 15 to 20 bp does not
prevent annealing if it is done at an appropriate tem-
perature. The annealed strand serves as a primer for the
synthesis of a strand complementary to the plasmid
vector. This slightly mismatched duplex recombinant
plasmid is then used to transform bacteria, where the
mismatch is repaired by cellular DNA repair enzymes
(Chapter 25). About half of the repair events will re-
move and replace the altered base and restore the gene
to its original sequence; the other half will remove and
Chapter 9 DNA-Based Information Technologies316
H
3
N
H11001
Gly Leu Pro Trp Glu Asp Met Trp Phe Val Arg COO
H11002
Known amino acid sequence
(5H11032) G G APossible codons
G G C
G G U
G G G
U U G
C
C
U U A
C
C
C C C
C C U
C C G
C C A U G G
G A G
G A A
G A U
G A C A U G U G G
U U U
U U C
G U C
G U U
G U G
G U A
A G G
C G A
C G C
A G A (3H11032)
C G U
C G GRegion of minimal degeneracy
Synthetic
probes
20 nucleotides long, 8 possible sequences
U G G G A G A A U G U G G U U G U
G
A C
U
C
U
AU
U
U
U
C
U
G
FIGURE 9–10 Probe to detect the gene for a protein of known amino
acid sequence. Because more than one DNA sequence can code for
any given amino acid sequence, the genetic code is said to be “de-
generate.” (As described in Chapter 27, an amino acid is coded for
by a set of three nucleotides called a codon. Most amino acids have
two or more codons; see Fig. 27–7.) Thus the correct DNA sequence
for a known amino acid sequence cannot be known in advance. The
probe is designed to be complementary to a region of the gene with
minimal degeneracy, that is, a region with the fewest possible codons
for the amino acids—two codons at most in the example shown here.
Oligonucleotides are synthesized with selectively randomized se-
quences, so that they contain either of the two possible nucleotides
at each position of potential degeneracy (shaded in pink). The oligonu-
cleotide shown here represents a mixture of eight different sequences:
one of the eight will complement the gene perfectly, and all eight will
match at least 17 of the 20 positions.
Bacterial promoter (P)
and operator (O)
sequences
Ribosome
binding
site
ori
P O
Gene encoding
repressor that
binds O and
regulates P
Polylinker with
unique sites for
several restriction
endonucleases
(i.e., cloning sites)
Transcription
termination
sequence
Selectable genetic
marker (e.g., antibiotic
resistance)
FIGURE 9–11 DNA sequences in a typical E. coli expression vector.
The gene to be expressed is inserted into one of the restriction sites
in the polylinker, near the promoter (P), with the end encoding the
amino terminus proximal to the promoter. The promoter allows effi-
cient transcription of the inserted gene, and the transcription termi-
nation sequence sometimes improves the amount and stability of the
mRNA produced. The operator (O) permits regulation by means of a
repressor that binds to it (Chapter 28). The ribosome binding site pro-
vides sequence signals needed for efficient translation of the mRNA
derived from the gene. The selectable marker allows the selection of
cells containing the recombinant DNA.
8885d_c09_306-342 2/7/04 8:14 AM Page 316 mac76 mac76:385_reb:
replace the normal base, retaining the desired muta-
tion. Transformants are screened (often by sequencing
their plasmid DNA) until a bacterial colony containing
a plasmid with the altered sequence is found.
Changes can also be introduced that involve more
than one base pair. Large parts of a gene can be deleted
by cutting out a segment with restriction endonucleases
and ligating the remaining portions to form a smaller
gene. Parts of two different genes can be ligated to cre-
ate new combinations. The product of such a fused gene
is called a fusion protein.
Researchers now have ingenious methods to bring
about virtually any genetic alteration in vitro. Reintro-
duction of the altered DNA into the cell permits inves-
tigation of the consequences of the alteration. Site-
directed mutagenesis has greatly facilitated research on
proteins by allowing investigators to make specific
changes in the primary structure of a protein and to ex-
amine the effects of these changes on the folding, three-
dimensional structure, and activity of the protein.
SUMMARY 9.1 DNA Cloning: The Basics
■ DNA cloning and genetic engineering involve
the cleavage of DNA and assembly of DNA
segments in new combinations—recombinant
DNA.
■ Cloning entails cutting DNA into fragments with
enzymes; selecting and possibly modifying a
fragment of interest; inserting the DNA
fragment into a suitable cloning vector;
transferring the vector with the DNA insert into
a host cell for replication; and identifying and
selecting cells that contain the DNA fragment.
■ Key enzymes in gene cloning include
restriction endonucleases (especially the type
II enzymes) and DNA ligase.
■ Cloning vectors include plasmids,
bacteriophages, and, for the longest DNA
inserts, bacterial artificial chromosomes (BACs)
and yeast artificial chromosomes (YACs).
■ Cells containing particular DNA sequences can
be identified by DNA hybridization methods.
■ Genetic engineering techniques manipulate
cells to express and/or alter cloned genes.
9.2 From Genes to Genomes
The modern science of genomics now permits the study
of DNA on a cellular scale, from individual genes to the
entire genetic complement of an organism—its genome.
Genomic databases are growing rapidly, as one se-
quencing milestone is superseded by the next. Biology
in the twenty-first century will move forward with the
aid of informational resources undreamed of only a few
years ago. We now turn to a consideration of some of
the technologies fueling these advances.
9.2 From Genes to Genomes 317
Recombinant
plasmid DNA
Gene
Synthetic DNA
fragment with
specific base-
pair change
restriction
endonucleases
DNA ligase
Plasmid contains
gene with desired
base-pair change.
(a)
Single strand
of recombinant
plasmid DNA
Gene
DNA polymerase,
dNTPs, DNA ligase
In E. coli cells, about half the
plasmids will have gene with
desired base-pair change.
(b)
Oligonucleotide
with sequence
change
transformation
of E. coli cells;
repair of DNA
A
G
C
A
G
C G
C
C
G
C
C
A
G
C
T
C
G
G
G
C
C
C
G
A
G
CG
C
C
FIGURE 9–12 Two approaches to site-directed mutagenesis. (a) A
synthetic DNA segment replaces a DNA fragment that has been re-
moved by cleavage with a restriction endonuclease. (b) A synthetic
oligonucleotide with a desired sequence change at one position is hy-
bridized to a single-stranded copy of the gene to be altered. This acts
as primer for synthesis of a duplex DNA (with one mismatch), which
is then used to transform cells. Cellular DNA repair systems will con-
vert about 50% of the mismatches to reflect the desired sequence
change.
8885d_c09_306-342 2/7/04 8:14 AM Page 317 mac76 mac76:385_reb:
DNA Libraries Provide Specialized Catalogs
of Genetic Information
A DNA library is a collection of DNA clones, gathered
together as a source of DNA for sequencing, gene dis-
covery, or gene function studies. The library can take a
variety of forms, depending on the source of the DNA.
Among the largest types of DNA library is a genomic
library, produced when the complete genome of a par-
ticular organism is cleaved into thousands of fragments,
and all the fragments are cloned by insertion into a
cloning vector.
The first step in preparing a genomic library is par-
tial digestion of the DNA by restriction endonucleases,
such that any given sequence will appear in fragments
of a range of sizes—a range that is compatible with the
cloning vector and ensures that virtually all sequences
are represented among the clones in the library. Frag-
ments that are too large or too small for cloning are re-
moved by centrifugation or electrophoresis. The cloning
vector, such as a BAC or YAC plasmid, is cleaved with
the same restriction endonuclease and ligated to the ge-
nomic DNA fragments. The ligated DNA mixture is then
used to transform bacterial or yeast cells to produce a
library of cell types, each type harboring a different re-
combinant DNA molecule. Ideally, all the DNA in the
genome under study will be represented in the library.
Each transformed bacterium or yeast cell grows into a
colony, or “clone,” of identical cells, each cell bearing
the same recombinant plasmid.
Using hybridization methods, researchers can order
individual clones in a library by identifying clones with
overlapping sequences. A set of overlapping clones rep-
resents a catalog for a long contiguous segment of a
genome, often referred to as a contig (Fig. 9–13). Pre-
viously studied sequences or entire genes can be located
within the library using hybridization methods to de-
termine which library clones harbor the known se-
quence. If the sequence has already been mapped on a
chromosome, investigators can determine the location
(in the genome) of the cloned DNA and any contig of
which it is a part. A well-characterized library may con-
tain thousands of long contigs, all assigned to and or-
dered on particular chromosomes to form a detailed
physical map. The known sequences within the library
(each called a sequence-tagged site, or STS) can pro-
vide landmarks for genomic sequencing projects.
As more and more genome sequences become avail-
able, the utility of genomic libraries is diminishing and
investigators are constructing more specialized libraries
designed to study gene function. An example is a library
that includes only those genes that are expressed—that
is, are transcribed into RNA—in a given organism or
even in certain cells or tissues. Such a library lacks the
noncoding DNA that makes up a large portion of many
eukaryotic genomes. The researcher first extracts
mRNA from an organism or from specific cells of an or-
ganism and then prepares complementary DNAs
(cDNAs) from the RNA in a multistep reaction cat-
alyzed by the enzyme reverse transcriptase (Fig. 9–14).
The resulting double-stranded DNA fragments are then
inserted into a suitable vector and cloned, creating a
population of clones called a cDNA library. The search
for a particular gene is made easier by focusing on a
cDNA library generated from the mRNAs of a cell known
to express that gene. For example, if we wished to clone
globin genes, we could first generate a cDNA library
from erythrocyte precursor cells, in which about half the
mRNAs code for globins. To aid in the mapping of large
genomes, cDNAs in a library can be partially sequenced
at random to produce a useful type of STS called an ex-
pressed sequence tag (EST). ESTs, ranging in size
from a few dozen to several hundred base pairs, can be
positioned within the larger genome map, providing
markers for expressed genes. Hundreds of thousands of
ESTs were included in the detailed physical maps used
as a guide to sequencing the human genome.
A cDNA library can be made even more specialized
by cloning a cDNA or cDNA fragment into a vector that
fuses the cDNA sequence with the sequence for a
marker, or reporter gene; the fused genes form a “re-
porter construct.” Two useful markers are the genes for
green fluorescent protein and epitope tags. A target
Chapter 9 DNA-Based Information Technologies318
A
Segment of chromosome from organism X
BAC clones
18
29
3
4
5
6
7
BC D E F G H I J K L M N O P Q
– – –– – –
FIGURE 9–13 Ordering of the clones in a DNA library. Shown here
is a segment of a chromosome from a hypothetical organism X, with
markers A through Q representing sequence-tagged sites (STSs—DNA
segments of known sequence, including known genes). Below the
chromosome is an array of ordered BAC clones, numbered 1 to 9. Or-
dering the clones on the genetic map is a many-stage process. The
presence or absence of an STS on an individual clone can be deter-
mined by hybridization—for example, by probing each clone with
PCR-amplified DNA from the STS. Once the STSs on each BAC clone
are identified, the clones (and the STSs themselves, if their location is
not yet known) can be ordered on the map. For example, compare
clones 3, 4, and 5. Marker E (blue) is found on all three clones; F (red)
on clones 4 and 5, but not on 3; and G (green) only on clone 5. This
indicates that the order of the sites is E, F, G. The clones partially over-
lap and their order must be 3, 4, 5. The resulting ordered series of
clones is called a contig.
8885d_c09_306-342 2/7/04 8:14 AM Page 318 mac76 mac76:385_reb:
gene fused with a gene for green fluorescent protein
(GFP) generates a fusion protein that is highly fluo-
rescent—it literally lights up (Fig. 9–15a). Just a few
molecules of this protein can be observed microscopi-
cally, allowing the study of its location and movements
in a cell. An epitope tag is a short protein sequence
that is bound tightly by a well-characterized monoclonal
antibody (Chapter 5). The tagged protein can be specif-
ically precipitated from a crude protein extract by in-
teraction with the antibody (Fig. 9–15b). If any other
proteins bind to the tagged protein, those will precipi-
tate as well, providing information about protein-protein
interactions in a cell. The diversity and utility of spe-
cialized DNA libraries are growing every year.
The Polymerase Chain Reaction Amplifies Specific
DNA Sequences
The Human Genome Project, along with the many as-
sociated efforts to sequence the genomes of organisms
of every type, is providing unprecedented access to gene
sequence information. This in turn is simplifying the
process of cloning individual genes for more detailed
biochemical analysis. If we know the sequence of at least
the flanking parts of a DNA segment to be cloned, we
can hugely amplify the number of copies of that DNA
segment, using the polymerase chain reaction
(PCR), a process conceived by Kary Mullis in 1983. The
amplified DNA can be cloned directly or used in a vari-
ety of analytical procedures.
The PCR procedure has an elegant simplicity. Two
synthetic oligonucleotides are prepared, complementary
to sequences on opposite strands of the target DNA at
positions just beyond the ends of the segment to be am-
plified. The oligonucleotides serve as replication primers
that can be extended by DNA polymerase. The 3H11032 ends
of the hybridized probes are oriented toward each other
and positioned to prime DNA synthesis across the
desired DNA segment (Fig. 9–16). (DNA polymerases
9.2 From Genes to Genomes 319
5H11032 A A A A A A A A
mRNA
5H11032
5H11032
mRNA-DNA hybrid
3H11032
A A A A A A A A
3H11032 T T T T T T T T
A A A A A A A A
3H11032
5H11032
Duplex DNA
3H11032
Reverse transcriptase and
dNTPs yield a complementary
DNA strand.
mRNA is degraded
with alkali.
DNA polymerase I and dNTPs
yield double-stranded DNA.
mRNA template is
annealed to synthetic
oligonucleotide (oligo dT) primer.
T T T T T T T T
T T T T T T T T
A A A A A A A A
T T T T T T T T
FIGURE 9–14 Construction of a cDNA library from mRNA. A cell’s
mRNA includes transcripts from thousands of genes, and the cDNAs
generated are correspondingly heterogeneous. The duplex DNA pro-
duced by this method is inserted into an appropriate cloning vector.
Reverse transcriptase can synthesize DNA on an RNA or a DNA tem-
plate (see Fig. 26–29).
FIGURE 9–15 Specialized DNA libraries. (a) Cloning of cDNA next
to a gene for green fluorescent protein (GFP) creates a reporter con-
struct. RNA transcription proceeds through the gene of interest (insert
DNA) and the reporter gene, and the mRNA transcript is then ex-
pressed as a fusion protein. The GFP part of the protein is visible
in the fluorescence microscope. The photograph shows a nematode
worm containing a GFP fusion protein expressed only in the four
“touch” neurons that run the length of its body. Reporter Con-
structs (b) If the cDNA is cloned next to a gene for an epitope tag,
the resulting fusion protein can be precipitated by antibodies to the
epitope. Any other proteins that interact with the tagged protein also
precipitate, helping to elucidate protein-protein interactions.
Transcription
Insert
cDNA
Insert
cDNA
Size
markers
Pure tagged
protein
Precipitate
tagged protein
with specific
antibody.
Make cell
extract.
Express tagged
protein in a cell.
Precipitate
Precipitate
Separate
precipitated
proteins.
Identify new proteins in
precipitate (e.g., with mass
spectrometry).
GFP
(a)
(b)
Epitope
tag
8885d_c09_306-342 2/7/04 8:14 AM Page 319 mac76 mac76:385_reb:
Chapter 9 DNA-Based Information Technologies320
(5H11032)GAATTC
CTTAAG(5H11032)
Heat to separate
strands.
Replication
EcoRI endonuclease
Anneal primers containing
noncomplementary regions
with cleavage site for
restriction endonuclease.
PCR
(5H11032)GAATTC GAATTC(3H11032)
CTTAAGCTTAAG
AATTC G
Clone by insertion
at an EcoRI site
in a cloning vector.
CTTAAG
(b)
2
1
CTTAAG(5H11032)
(5H11032)GAATTC
1 Heat to separate
strands.
2 Add synthetic oligo-
nucleotide primers; cool.
3H11032
5H11032
Region of target DNA
to be amplified
3 Add thermostable DNA
polymerase to catalyze
5H11032 → 3H11032 DNA synthesis.
3H11032
3H11032
5H11032
5H11032
5H11032
5H11032
Repeat steps 1 and 2 .
3H11032
5H11032
DNA synthesis (step 3 )
is catalyzed by the
thermostable DNA
polymerase (still present).
3H11032
Repeat steps 1
through 3 .
5H11032
3H11032
5H11032
After 25 cycles, the target sequence has
been amplified about 10
6
-fold.
(a)
5H11032
5H11032
FIGURE 9–16 Amplification of a DNA segment by the polymerase chain
reaction. (a) The PCR procedure has three steps. DNA strands are H220711
separated by heating, then H220712 annealed to an excess of short synthetic
DNA primers (blue) that flank the region to be amplified; H220713 new DNA
is synthesized by polymerization. The three steps are repeated for 25 or
30 cycles. The thermostable DNA polymerase TaqI (from Thermus
aquaticus, a bacterial species that grows in hot springs) is not denatured
by the heating steps. (b) DNA amplified by PCR can be cloned. The
primers can include noncomplementary ends that have a site for cleavage
by a restriction endonuclease. Although these parts of the primers do not
anneal to the target DNA, the PCR process incorporates them into the
DNA that is amplified. Cleavage of the amplified fragments at these sites
creates sticky ends, used in ligation of the amplified DNA to a cloning
vector. Polymerase Chain Reaction
8885d_c09_306-342 2/10/04 1:52 PM Page 320 mac34 mac34: kec_420:
synthesize DNA strands from deoxyribonucleotides,
using a DNA template, as described in Chapter 25.)
Isolated DNA containing the segment to be amplified is
heated briefly to denature it, and then cooled in the
presence of a large excess of the synthetic oligonucleo-
tide primers. The four deoxynucleoside triphosphates
are then added, and the primed DNA segment is repli-
cated selectively. The cycle of heating, cooling, and
replication is repeated 25 or 30 times over a few hours
in an automated process, amplifying the DNA segment
flanked by the primers until it can be readily analyzed
or cloned. PCR uses a heat-stable DNA polymerase, such
as the Taq polymerase (derived from a bacterium that
lives at 90 H11034C), which remains active after every heating
step and does not have to be replenished. Careful de-
sign of the primers used for PCR, such as including re-
striction endonuclease cleavage sites, can facilitate the
subsequent cloning of the amplified DNA (Fig. 9–16b).
This technology is highly sensitive: PCR can detect
and amplify as little as one DNA molecule in almost any
type of sample. Although DNA degrades over time (p.
293), PCR has allowed successful cloning of DNA from
samples more than 40,000 years old. Investigators have
used the technique to clone DNA fragments from the
mummified remains of humans and extinct animals such
as the woolly mammoth, creating the new fields of mo-
lecular archaeology and molecular paleontology. DNA
from burial sites has been amplified by PCR and used
to trace ancient human migrations. Epidemiologists can
use PCR-enhanced DNA samples from human remains
to trace the evolution of human pathogenic viruses.
Thus, in addition to its usefulness for cloning DNA, PCR
is a potent tool in forensic medicine (Box 9–1). It is also
being used for detection of viral infections before they
cause symptoms and for prenatal diagnosis of a wide ar-
ray of genetic diseases.
The PCR method is also important in advancing the
goal of whole genome sequencing. For example, the
mapping of expressed sequence tags to particular chro-
mosomes often involves amplification of the EST by
PCR, followed by hybridization of the amplified DNA to
clones in an ordered library. Investigators found many
other applications of PCR in the Human Genome Pro-
ject, to which we now turn.
Genome Sequences Provide the Ultimate
Genetic Libraries
The genome is the ultimate source of information about
an organism, and there is no genome we are more in-
terested in than our own. Less than 10 years after the
development of practical DNA sequencing methods, se-
rious discussions began about the prospects for se-
quencing the entire 3 billion base pairs of the human
genome. The international Human Genome Project got
underway with substantial funding in the late 1980s. The
effort eventually included significant contributions from
20 sequencing centers distributed among six nations:
the United States, Great Britain, Japan, France, China,
and Germany. General coordination was provided by the
Office of Genome Research at the National Institutes of
Health, led first by James Watson and after 1992 by
Francis Collins. At the outset, the task of sequencing a
3 H11003 10
9
bp genome seemed to be a titanic job, but it
gradually yielded to advances in technology. The com-
pleted sequence of the human genome was published
in April 2003, several years ahead of schedule.
This advance was the product of a carefully planned
international effort spanning 14 years. Research teams
first generated a detailed physical map of the human
genome, with clones derived from each chromosome or-
ganized into a series of long contigs (Fig. 9–17). Each
contig contained landmarks in the form of STSs at a dis-
tance of every 100,000 bp or less. The genome thus
mapped could be divided up between the international
sequencing centers, each center sequencing the
mapped BAC or YAC clones corresponding to its par-
ticular segments of the genome. Because many of the
9.2 From Genes to Genomes 321
DNA is digested into fragments;
fragments inserted into BACs.
Genomic DNA
Contigs are identified
and mapped.
BAC to be sequenced
is fragmented; fragments
sequenced at random.
Sequence overlaps
reveal final sequence.
G G G C T A C A T G A T
G G G C T A C A T G A T G G T C
C A T G A T G G T C
FIGURE 9–17 The Human Genome Project strategy. Clones isolated
from a genomic library were ordered into a detailed physical map,
then individual clones were sequenced by shotgun sequencing pro-
tocols. The strategy used by the commercial sequencing effort elimi-
nated the step of creating the physical map and sequenced the entire
genome by shotgun cloning.
8885d_c09_306-342 2/7/04 8:14 AM Page 321 mac76 mac76:385_reb:
clones were more than 100,000 bp long, and modern se-
quencing techniques can resolve only 600 to 750 bp of
sequence at a time, each clone had to be sequenced in
pieces. The sequencing strategy used a shotgun ap-
proach, in which researchers used powerful new auto-
mated sequencers to sequence random segments of a
given clone, then assembled the sequence of the entire
clone by computerized identification of overlaps. The
number of clone pieces sequenced was determined sta-
tistically so that the entire length of the clone was se-
quenced four to six times on average. The sequenced
DNA was then made available in a database covering the
entire genome. Construction of the physical map was a
time-consuming task, and its progress was followed in
annual reports in major journals throughout the 1990s—
by the end of which the map was largely in place.
Completion of the entire sequencing project was initially
projected for the year 2005, but circumstances and tech-
nology intervened to accelerate the process.
A competing commercial effort to sequence the
human genome was initiated by the newly established
Celera Corporation in 1997. Led by J. Craig Venter, the
Celera group made use of a different strategy called
“whole genome shotgun sequencing,” which eliminates
the step of assembling a physical map of the genome.
Instead, teams sequenced DNA segments from through-
Chapter 9 DNA-Based Information Technologies322
BOX 9–1 WORKING IN BIOCHEMISTRY
A Potent Weapon in Forensic Medicine
Traditionally, one of the most accurate methods for
placing an individual at the scene of a crime has been
a fingerprint. With the advent of recombinant DNA
technology, a more powerful tool is now available:
DNA fingerprinting (also called DNA typing or DNA
profiling).
DNA fingerprinting is based on sequence poly-
morphisms, slight sequence differences (usually sin-
gle base-pair changes) between individuals, 1 bp in
every 1,000 bp, on average. Each difference from the
prototype human genome sequence (the first one ob-
tained) occurs in some fraction of the human popula-
tion; every individual has some differences. Some of
the sequence changes affect recognition sites for re-
striction enzymes, resulting in variation in the size of
DNA fragments produced by digestion with a partic-
ular restriction enzyme. These variations are restric-
tion fragment length polymorphisms (RFLPs).
The detection of RFLPs relies on a specialized
hybridization procedure called Southern blotting
(Fig. 1). DNA fragments from digestion of genomic
DNA by restriction endonucleases are separated by
size electrophoretically, denatured by soaking the
agarose gel in alkali, and then blotted onto a nylon
membrane to reproduce the distribution of fragments
in the gel. The membrane is immersed in a solution
containing a radioactively labeled DNA probe. A probe
for a sequence that is repeated several times in the
human genome generally identifies a few of the thou-
sands of DNA fragments generated when the human
genome is digested with a restriction endonuclease.
Autoradiography reveals the fragments to which the
probe hybridizes, as in Figure 9–9.
The genomic DNA sequences used in these tests
are generally regions containing repetitive DNA
(short sequences repeated thousands of times in tan-
dem), which are common in the genomes of higher
eukaryotes (see Fig. 24–8). The number of repeated
units in these DNA regions varies among individuals
(except between identical twins). With a suitable
probe, the pattern of bands produced by DNA finger-
printing is distinctive for each individual. Combining
the use of several probes makes the test so selective
that it can positively identify a single individual in the
entire human population. However, the Southern blot
procedure requires relatively fresh DNA samples and
larger amounts of DNA than are generally present at
a crime scene. RFLP analysis sensitivity is augmented
by using PCR (see Fig. 9–16a) to amplify vanishingly
small amounts of DNA. This allows investigators to
obtain DNA fingerprints from a single hair follicle, a
drop of blood, a small semen sample from a rape vic-
tim, or samples that might be months or even many
years old.
These methods are proving decisive in court cases
worldwide. In the example in Figure 1, the DNA from
a semen sample obtained from a rape and murder vic-
tim was compared with DNA samples from the victim
and two suspects. Each sample was cleaved into frag-
ments and separated by gel electrophoresis. Radioac-
tive DNA probes were used to identify a small subset
of fragments that contained sequences complemen-
tary to the probe. The sizes of the identified fragments
varied from one individual to the next, as seen here
in the different patterns for the three individuals (vic-
tim and two suspects) tested. One suspect’s DNA ex-
hibits a banding pattern identical to that of a semen
sample taken from the victim. This test used a single
probe, but three or four different probes would be
used (in separate experiments) to achieve an unam-
biguous positive identification.
8885d_c09_306-342 2/7/04 8:14 AM Page 322 mac76 mac76:385_reb:
out the genome at random. The sequenced segments
were ordered by the computerized identification of
sequence overlaps (with some reference to the public
project’s detailed physical map). At the outset of the
Human Genome Project, shotgun sequencing on this
scale had been deemed impractical. However, advances
in computer software and sequencing automation had
made the approach feasible by 1997. The ensuing race
between the private and public sequencing efforts
substantially advanced the timeline for completion of
the project. Publication of the draft human genome se-
quence in 2001 was followed by two years of follow-up
work to eliminate nearly a thousand discontinuities and
9.2 From Genes to Genomes 323
Such results have been used to both convict and
acquit suspects and, in other cases, to establish pa-
ternity with an extraordinary degree of certainty. The
impact of these procedures on court cases will con-
tinue to grow as societies agree on the standards and
as formal methods become widely established in foren-
sic laboratories. Even decades-old murder mysteries
can be solved: in 1996, DNA fingerprinting helped to
confirm the identification of the bones of the last Russ-
ian czar and his family, who were assassinated in 1918.
Expose
x-ray
film
to
membrane.
Denature
DNA, and
transfer to
nylon
membrane.
Separate fragments by agarose
gel electrophoresis (unlabeled).
Chromosomal DNA
(e.g., Suspect 1)
Cleave with restriction
endonucleases.
DNA fragments
Radiolabeled
DNA probe
DNA markersDNA markers DNA markersDNA markers
Incubate
with
probe,
then
wash.
EvidenceVictimSuspect 2Suspect 1 EvidenceVictim Suspect 2Suspect 1
FIGURE 1 The Southern blot procedure, as applied to DNA
fingerprinting. This procedure was named after Jeremy Southern,
who developed the technique.
Francis S. Collins J. Craig Venter
8885d_c09_306-342 2/7/04 8:14 AM Page 323 mac76 mac76:385_reb:
to provide high-quality sequence data that are contigu-
ous throughout the genome.
The Human Genome Project marks the culmination
of twentieth-century biology and promises a vastly
changed scientific landscape for the new century. The
human genome is only part of the story, as the genomes
of many other species are also being (or have been) se-
quenced, including the yeasts Saccharomyces cere-
visiae (completed in 1996) and Schizosaccharomyces
pombe (2002), the nematode Caenorhabditis elegans
(1998), the fruit fly Drosophila melanogaster (2000),
the plant Arabidopsis thaliana (2000), the mouse Mus
musculus (2002), zebrafish, and dozens of bacterial and
archaebacterial species (Fig. 9–18). Most of the early
efforts have been focused on species commonly used in
laboratories. However, genome sequencing is destined
to branch out to many other species as experience grows
and technology improves. Broad efforts to map genes,
attempts to identify new proteins and disease genes, and
many other initiatives are currently under way.
The result is a database with the potential not only
to fuel rapid advances in biology but to change the way
that humans think about themselves. Early insights pro-
vided by the human genome sequence range from the
intriguing to the profound. We are not as complicated
as we thought. Decades-old estimates that humans pos-
sessed about 100,000 genes within the approximately
3.2 H11003 10
9
bp in the human genome have been sup-
planted by the discovery that we have only 30,000 to
35,000 genes. This is perhaps three times more genes
than a fruit fly (with 13,000) and twice as many as a
nematode worm (18,000). Although humans evolved
relatively recently, the human genome is very old. Of
1,278 protein families identified in one early screen, only
94 were unique to vertebrates. However, while we share
many protein domain types with plants, worms, and
flies, we use these domains in more complex arrange-
ments. Alternative modes of gene expression (Chapter
26) allow the production of more than one protein from
a single gene—a process that humans and other verte-
brates engage in more than do bacteria, worms, or any
other forms of life. This allows for greater complexity in
the proteins generated from our gene complement.
We now know that only 1.1% to 1.4% of our DNA
actually encodes proteins (Fig. 9–19). More than 50%
of our genome consists of short, repeated sequences,
the vast majority of which—about 45% of our genome
in all—come from transposons, short movable DNA se-
quences that are molecular parasites (Chapter 25).
Many of the transposons have been there a long time,
now altered so that they can no longer move to new
genomic locations. Others are still actively moving at
low frequencies, helping to make the genome an ever-
dynamic and evolving entity. At least a few transposons
have been co-opted by their host and appear to serve
useful cellular functions.
What does all this information tell us about how
much one human differs from another? Within the hu-
man population are millions of single-base differences,
called single nucleotide polymorphisms, or SNPs
(pronounced “snips”). Each human differs from the next
Chapter 9 DNA-Based Information Technologies324
1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004
Genome sequencing begins
H. influenzae
S. cerevisiae
E. coli
C. elegans
D. melanogaster
A. thaliana
H. sapiens
(draft)
H. sapiens
(completed)
S. pombe
M. musculus
FIGURE 9–18 Genomic sequencing timeline. Discussions in the mid-
1980s led to initiation of the project in 1989. Preparatory work, in-
cluding extensive mapping to provide genome landmarks, occupied
much of the 1990s. Separate projects were launched to sequence the
genomes of other organisms important to research. The first sequenc-
ing efforts to be completed included many bacterial species (such as
Haemophilus influenzae), yeast (S. cerevisiae), a nematode worm
(C. elegans), the fruit fly (D. melanogaster), and a plant (A. thaliana).
Completed sequences for mammalian genomes, including the human
genome, began to emerge in 2000. Each genome project has a web-
site that serves as a central repository for the latest data.
FIGURE 9–19 Snapshot of the human genome. The chart shows the
proportions of our genome made up of various types of sequences.
Translated into
protein
1.1%–1.4%
Transposons
45%
Other intergenic
DNA
20.7%
Large
duplications
5%
Transcribed
into RNA as
end product
25%
Simple repeats
(microsatellites)
3%
8885d_c09_306-342 2/7/04 8:14 AM Page 324 mac76 mac76:385_reb:
by about 1 bp in every 1,000 bp. From these small ge-
netic differences arises the human variety we are all
aware of—differences in hair color, eyesight, allergies
to medication, foot size, and even (to some unknown
degree) behavior. Some of the SNPs are linked to par-
ticular human populations and can provide important
information about human migrations that occurred
thousands of years ago and about our more distant evo-
lutionary past.
As spectacular as this advance is, the sequencing of
the human genome is easy compared with what comes
next—the effort to understand all the information in
each genome. The genome sequences being added
monthly to international databases are roadmaps, parts
of which are written in a language we do not yet un-
derstand. However, they have great utility in catalyzing
the discovery of new proteins and processes affecting
every aspect of biochemistry, as will become apparent
in chapters to come.
SUMMARY 9.2 From Genes to Genomes
■ The science of genomics broadly encompasses
the study of genomes and their gene content.
■ Genomic DNA segments can be organized in
libraries—such as genomic libraries and cDNA
libraries—with a wide range of designs and
purposes.
■ The polymerase chain reaction (PCR) can be
used to amplify selected DNA segments from a
DNA library or an entire genome.
■ In an international cooperative research effort,
the genomes of many organisms, including that
of humans, have been sequenced in their
entirety and are now available in public
databases.
9.3 From Genomes to Proteomes
A gene is not simply a DNA sequence; it is information
that is converted to a useful product—a protein or func-
tional RNA molecule—when and if needed by the cell.
The first and most obvious step in exploring a large se-
quenced genome is to catalog the products of the genes
within that genome. Genes that encode RNA as their fi-
nal product are somewhat harder to identify than are
protein-encoding genes, and even the latter can be very
difficult to spot in a vertebrate genome. The explosion
of DNA sequence information has also revealed a sober-
ing truth. Despite many years of biochemical advances,
there are still thousands of proteins in every eukaryotic
cell (and quite a few in bacteria) that we know nothing
about. These proteins may have functions in processes
not yet discovered, or may contribute in unexpected
ways to processes we think we understand. In addition,
the genomic sequences tell us nothing about the three-
dimensional structure of proteins or how proteins are
modified after they are synthesized. The proteins, with
their myriad critical functions in every cell, are now be-
coming the focus of new strategies for whole cell bio-
chemistry.
The complement of proteins expressed by a genome
is called its proteome, a term that first appeared in the
research literature in 1995. This concept rapidly evolved
into a separate field of investigation, called proteomics.
The problem addressed by proteomics research is
straightforward, although the solution is not. Each
genome presents us with thousands of genes encoding
proteins, and ideally we want to know the structure and
function of all those proteins. Given that many proteins
offer surprises even after years of study, the investiga-
tion of an entire proteome is a daunting enterprise.
Simply discovering the function of new proteins requires
intensive work. Biochemists can now apply shortcuts
in the form of a broad array of new and updated tech-
nologies.
Protein function can be described on three levels.
Phenotypic function describes the effects of a protein
on the entire organism. For example, the loss of the pro-
tein may lead to slower growth of the organism, an al-
tered development pattern, or even death. Cellular
function is a description of the network of interactions
engaged in by a protein at the cellular level. Interactions
with other proteins in the cell can help define the kinds
of metabolic processes in which the protein participates.
Finally, molecular function refers to the precise bio-
chemical activity of a protein, including details such as
the reactions an enzyme catalyzes or the ligands a re-
ceptor binds.
For several genomes, such as those of the yeast Sac-
charomyces cerevisiae and the plant Arabidopsis, a
massive effort is underway to inactivate each gene by
genetic engineering and to investigate the effect on the
organism. If the growth patterns or other properties of
the organism change (or if it does not grow at all), this
provides information on the phenotypic function of the
protein product of the gene.
There are three other main paths to investigating
protein function: (1) sequence and structural compar-
isons with genes and proteins of known function, (2)
determination of when and where a gene is expressed,
and (3) investigation of the interactions of the protein
with other proteins. We discuss each of these ap-
proaches in turn.
Sequence or Structural Relationships Provide
Information on Protein Function
One of the important reasons to sequence many genomes
is to provide a database that can be used to assign gene
functions by genome comparisons, an enterprise referred
9.3 From Genomes to Proteomes 325
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to as comparative genomics. Sometimes a newly dis-
covered gene is related by sequence homologies to a
gene previously studied in another or the same species,
and its function can be entirely or partly defined by that
relationship. Such genes—of different species but pos-
sessing a clear sequence and functional relationship to
each other—are called orthologs. Genes similarly re-
lated to each other within a single species are called
paralogs (see Fig. 1–37). If the function of a gene has
been characterized for one species, this information can
be used to assign gene function to the ortholog found
in the second species. The identity is easiest to make
when comparing genomes from relatively closely related
species, such as mouse and human, although many
clearly orthologous genes have been identified in
species as distant as bacteria and humans. Sometimes
even the order of genes on a chromosome is conserved
over large segments of the genomes of closely related
species (Fig. 9–20). Conserved gene order, called syn-
teny, provides additional evidence for an orthologous
relationship between genes at identical locations within
the related segments.
Alternatively, certain sequences associated with
particular structural motifs (Chapter 4) may be identi-
fied within a protein. The presence of a structural mo-
tif may suggest that it, say, catalyzes ATP hydrolysis,
binds to DNA, or forms a complex with zinc ions, help-
ing to define molecular function. These relationships are
determined with the aid of increasingly sophisticated
computer programs, limited only by the current infor-
mation on gene and protein structure and our capacity
to associate sequences with particular structural motifs.
To further the assignment of function based on
structural relationships, a large-scale structural pro-
teomics project has been initiated. The goal is to crys-
tallize and determine the structure of as many proteins
and protein domains as possible, in many cases with lit-
tle or no existing information about protein function.
The project has been assisted by the automation of some
of the tedious steps of protein crystallization (see Box
4–4). As these structures are revealed, they will be made
available in the structural databases described in Chap-
ter 4. The effort should help define the extent of varia-
tion in structural motifs. When a newly discovered pro-
tein is found to have structural folds that are clearly
related to motifs with known functions in the databases,
this information can suggest a molecular function for
the protein.
Cellular Expression Patterns Can Reveal the Cellular
Function of a Gene
In every newly sequenced genome, researchers find
genes that encode proteins with no evident structural
relationships to known genes or proteins. In these cases,
other approaches must be used to generate information
about gene function. Determining which tissues a gene
is expressed in, or what circumstances trigger the ap-
pearance of the gene product, can provide valuable
clues. Many different approaches have been developed
to study these patterns.
Two-Dimensional Gel Electrophoresis As shown in Figure
3–22, two-dimensional gel electrophoresis allows the
separation and display of up to 1,000 different proteins
on a single gel. Mass spectrometry (see Box 3–2) can
then be used to partially sequence individual protein
spots and assign each to a gene. The appearance and
nonappearance (or disappearance) of particular protein
spots in samples from different tissues, from similar tis-
sues at different stages of development, or from tissues
treated in ways that simulate a variety of biological con-
ditions can help define cellular function.
DNA Microarrays Major refinements of the technology
underlying DNA libraries, PCR, and hybridization have
come together in the development of DNA microar-
rays (sometimes called DNA chips), which allow the
rapid and simultaneous screening of many thousands of
genes. DNA segments from known genes, a few dozen
to hundreds of nucleotides long, are amplified by PCR
and placed on a solid surface, using robotic devices that
accurately deposit nanoliter quantities of DNA solution.
Many thousands of such spots are deposited in a pre-
designed array on a surface area of just a few square
centimeters. An alternative strategy is to synthesize
DNA directly on the solid surface, using photolithogra-
phy (Fig. 9–21). Once the chip is constructed, it can be
probed with mRNAs or cDNAs from a particular cell type
Chapter 9 DNA-Based Information Technologies326
Human 9 Mouse 2
EPB72
PSMB7
DNM1
LMX1B
CDK9
STXBP1
AK1
LCN2
Epb7.2
Psmb7
Dnm
Lmx1b
Cdk9
Stxbp1
Ak1
Lcn2
FIGURE 9–20 Synteny in the mouse and human genomes. Large seg-
ments of the mouse and human genomes have closely related genes
aligned in the same order on chromosomes, a relationship called syn-
teny. This diagram shows segments of human chromosome 9 and
mouse chromosome 2. The genes in these segments exhibit a very
high degree of homology as well as the same gene order. The differ-
ent lettering schemes for the gene names reflect different naming con-
ventions in the two organisms.
8885d_c09_306-342 2/7/04 8:14 AM Page 326 mac76 mac76:385_reb:
or cell culture to identify the genes being expressed in
those cells.
A microarray can answer such questions as which
genes are expressed at a given stage in the development
of an organism. The total complement of mRNA is iso-
lated from cells at two different stages of development
and converted to cDNA, using reverse transcriptase and
fluorescently labeled deoxynucleotides. The fluorescent
cDNAs are then mixed and used as probes, each hy-
bridizing to complementary sequences on the microar-
ray. In Figure 9–22, for example, the labeled nucleotides
used to make the cDNA for each sample fluoresce in
two different colors. The cDNA from the two samples
is mixed and used to probe the microarray. Spots that
fluoresce green represent mRNAs more abundant at
the single-cell stage; those that fluoresce red represent
sequences more abundant later in development. The
mRNAs that are equally abundant at both stages of
development fluoresce yellow. By using a mixture of two
samples to measure relative rather than absolute abun-
dance of sequences, the method corrects for variations
in the amounts of DNA originally deposited in each spot
on the grid and other possible inconsistencies among
spots in the microarray. The spots that fluoresce pro-
vide a snapshot of all the genes being expressed in the
cells at the moment they were harvested—gene ex-
pression examined on a genome-wide scale. For a gene
of unknown function, the time and circumstances of its
expression can provide important clues about its role in
the cell.
An example of this technique is illustrated in Fig-
ure 9–23, showing the dramatic results this technique
can produce. Segments from each of the more than
6,000 genes in the completely sequenced yeast genome
were separately amplified by PCR, and each segment
was deposited in a defined pattern to create the illus-
trated microarray. In a sense, this array provides a snap-
shot of the entire yeast genome.
Protein Chips Proteins, too, can be immobilized on a
solid surface and used to help define the presence or
absence of other proteins in a sample. For example, re-
searchers prepare an array of antibodies to particular
proteins by immobilizing them as individual spots on a
solid surface. A sample of proteins is added, and if the
protein that binds any of the antibodies is present in the
sample, it can be detected by a solid-state form of the
ELISA assay (see Fig. 5–28). Many other types and ap-
plications of protein chips are being developed.
Detection of Protein-Protein Interactions Helps
to Define Cellular and Molecular Function
A key to defining the function of any protein is to de-
termine what it binds to. In the case of protein-protein
interactions, the association of a protein of unknown
function with one whose function is known can provide
a useful and compelling “guilt by association.” The tech-
niques used in this effort are quite varied.
Comparisons of Genome Composition Although not evi-
dence of direct association, the mere presence of com-
binations of genes in particular genomes can hint at
9.3 From Genomes to Proteomes 327
Light
Light
Solid
surface
Desired sequences
Solution
containing
activated
A (A*)
G* solution
C* solution
Light
Opaque
screen
over spots
2 and 4
C
G
T
A
C
C
T
G
G
G
T
A
G
C
C
G
12
34
G
*
G
*
G
*
G
*
G
*
G
*
G
*
A
A
G
G G
A
A
C*
C*
C*
C*
C*
C*
C*
A G
A
C
G
A
* A
*
A
*
A
* A
*
A
*
A
*
Opaque
screen over
spots 1, 2,
and 3
Opaque
screen over
spots 1 and 3
Many more cycles
FIGURE 9–21 Photolithography. This technique for preparing a DNA
microarray makes use of nucleotide precursors that are activated by
light, joining one nucleotide to the next in a photoreaction (as op-
posed to the chemical process illustrated in Fig. 8–38). A computer is
programmed with the oligonucleotide sequences to be synthesized at
each point on a solid surface. The surface is washed successively with
solutions containing one type of activated nucleotide (A*, G*, etc.).
As in the chemical synthesis of DNA, the activated nucleotides are
blocked so that only one can be added to a chain in each cycle. A
screen covering the surface is opened over the areas programmed to
receive a particular nucleotide, and a flash of light joins the nucleotide
to the polymers in the uncovered areas. This continues until the re-
quired sequences are built up on each spot on the surface. Many poly-
mers with the same sequence are generated on each spot, not just the
single polymer shown. Also, the surfaces have thousands of spots with
different sequences (see Fig. 9–22); this array shows just four spots, to
illustrate the strategy.
8885d_c09_306-342 2/7/04 8:14 AM Page 327 mac76 mac76:385_reb:
protein function. We can simply search the genomic
databases for particular genes, then determine what
other genes are present in the same genomes (Fig.
9–24). When two genes always appear together in a
genome, it suggests that the proteins they encode may
be functionally related. Such correlations are most use-
ful if the function of at least one of the proteins is known.
Purification of Protein Complexes With the construction of
cDNA libraries in which each gene is contiguous with
(fused to) an epitope tag, workers can immunoprecipi-
tate the protein product of a gene by using the antibody
that binds to the epitope (Fig. 9–15b). If the tagged pro-
tein is expressed in cells, other proteins that bind to
it may also be precipitated with it. Identification of the
associated proteins reveals some of the protein-protein
interactions of the tagged protein. There are many vari-
ations of this process. For example, a crude extract of
cells that express a similarly tagged protein is added to
a column containing immobilized antibody. The tagged
protein binds to the antibody, and proteins that inter-
act with the tagged protein are sometimes also retained
Chapter 9 DNA-Based Information Technologies328
mRNA
1
Isolate mRNAs from cells
at two stages of development;
each mRNA sample represents
all the genes expressed in
the cells at that stage.
cDNA
DNA
microarray
reverse
transcriptase
2
Convert mRNAs to cDNAs
by reverse transcriptase,
using fluorescently labeled
deoxyribonucleotide
triphosphates.
3
Add the cDNAs to a
microarray; fluorescent
cDNAs anneal to
complementary sequences
on the microarray.
4
Each fluorescent spot
represents a gene expressed
in the cells.
Removal of
unhybridized probe
FIGURE 9–22 DNA microarray. A microarray can be prepared from
any known DNA sequence, from any source, generated by chemical
synthesis or by PCR. The DNA is positioned on a solid surface (usu-
ally specially treated glass slides) with the aid of a robotic device ca-
pable of depositing very small (nanoliter) drops in precise patterns.
UV light cross-links the DNA to the glass slides. Once the DNA is at-
tached to the surface, the microarray can be probed with other fluo-
rescently labeled nucleic acids. Here, mRNA samples are collected
from cells at two different stages in the development of a frog. The
cDNA probes are made with nucleotides that fluoresce in different
colors for each sample; a mixture of the cDNAs is used to probe
the microarray. Green spots represent mRNAs more abundant at the
single-cell stage; red spots, sequences more abundant later in devel-
opment. The yellow spots indicate approximately equal abundance at
both stages. Synthesizing an Oligonucleotide Array
FIGURE 9–23 Enlarged image of a DNA microarray. Each glowing
spot in this microarray contains DNA from one of the 6,200 genes of
the yeast (S. cerevisiae) genome, with every gene represented in the
array. The microarray has been probed with fluorescently labeled nu-
cleic acid derived from the mRNAs obtained (1) when the cells were
growing normally in culture and (2) five hours after the cells began to
form spores. The green spots represent genes expressed at higher lev-
els during normal growth; the red spots, genes expressed at higher
levels during sporulation. The yellow spots represent genes that do
not change their levels of expression during sporulation. This image
is enlarged; the microarray actually measures only 1.8 H11003 1.8 cm.
Screening Oligonucleotide Array for Patterns of Gene Expression
8885d_c09_306-342 2/7/04 8:14 AM Page 328 mac76 mac76:385_reb:
on the column. The connection between the protein
and the tag is cleaved with a specific protease, and the
protein complexes are eluted from the column and
analyzed. Researchers can use these methods to define
complex networks of interactions within a cell.
A variety of useful protein tags are available. A com-
mon one is a histidine tag, often just a string of six His
residues. A poly-His sequence binds quite tightly to
metals such as nickel. If a protein is cloned so that its
sequence is contiguous with a His tag, it will have the
extra His residues at its carboxyl terminus. The protein
can then be purified by chromatography on columns
with immobilized nickel. These procedures are conven-
ient but require caution, because the additional amino
acid residues in an epitope or His tag can affect protein
activity.
Yeast Two-Hybrid Analysis A sophisticated genetic ap-
proach to defining protein-protein interactions is based
on the properties of the Gal4 protein (Gal4p), which ac-
tivates transcription of certain genes in yeast (see Fig.
28–28). Gal4p has two domains, one that binds to a spe-
cific DNA sequence and another that activates the RNA
polymerase that synthesizes mRNA from an adjacent
reporter gene. The domains are stable when separated,
but activation of the RNA polymerase requires interac-
tion with the activation domain, which in turn requires
positioning by the DNA-binding domain. Hence, the do-
mains must be brought together to function correctly
(Fig. 9–25a).
9.3 From Genomes to Proteomes 329
Species
Protein 1 2 3 4
P1
P2
P3
P4
P5
P6
P7
H11001
H11001
H11001
H11001H11001H11001
H11001
H11001H11001 H11001
H11001
H11001
H11001H11002
H11002H11002
H11002
H11001H11001 H11001H11002
H11002
H11002H11002
H11002H11002H11002
H11002
FIGURE 9–24 Use of comparative genomics to identify functionally
related genes. One use of comparative genomics is to prepare phylo-
genetic profiles in order to identify genes that always appear together
in a genome. This example shows a comparison of genes from four
organisms, but in practice, computer searches can look at dozens of
species. The designations P1, P2, and so forth refer to proteins en-
coded by each species. This technique does not require homologous
proteins. In this example, because proteins P3 and P6 always appear
together in a genome they may be functionally related. Further test-
ing would be needed to confirm this inference.
Reporter gene
Gal4p DNA-
binding domain
Gal4p
binding site
Gal4p
activation
domain
RNA
polymerase
Increased
transcription
Reporter gene
(a)
X
Y
Yeast strain 1
with Gal4p–binding
domain fusions
Yeast strain 2 with
Gal4p–activation
domain fusions
Mate to produce diploid cells.
Plate on medium requiring
interaction of the binding and
activation domains for cell
survival.
Survivors
form colonies.
Sequence fusion proteins to identify
which proteins are interacting.
(b)
FIGURE 9–25 The yeast two-hybrid system. (a) In this system for de-
tecting protein-protein interactions, the aim is to bring together the
DNA-binding domain and the activation domain of the yeast Gal4
protein through the interaction of two proteins, X and Y, to which each
domain is fused. This interaction is accompanied by the expression of
a reporter gene. (b) The two fusions are created in separate yeast
strains, which are then mated. The mated mixture is plated on a
medium on which the yeast cannot survive unless the reporter gene
is expressed. Thus, all surviving colonies have interacting protein fu-
sion pairs. Sequencing of the fusion proteins in the survivors reveals
which proteins are interacting. Yeast Two-Hybrid Systems
8885d_c09_306-342 2/7/04 8:14 AM Page 329 mac76 mac76:385_reb:
In this method, the protein-coding regions of genes
to be analyzed are fused to the coding sequences of ei-
ther the DNA-binding domain or the activation domain
of Gal4p, and the resulting genes express a series of
fusion proteins. If a protein fused to the DNA-binding
domain interacts with a protein fused to the activation
domain, transcription is activated. The reporter gene
transcribed by this activation is generally one that yields
a protein required for growth, or is an enzyme that cat-
alyzes a reaction with a colored product. Thus, when
grown on the proper medium, cells that contain such a
pair of interacting proteins are easily distinguished from
those that do not. Typically, many genes are fused to
the Gal4p DNA-binding domain gene in one yeast strain,
and many other genes are fused to the Gal4p activation
domain gene in another yeast strain, then the yeast
strains are mated and individual diploid cells grown into
colonies (Fig. 9–25b). This allows for large-scale screen-
ing for proteins that interact in the cell.
All these techniques provide important clues to pro-
tein function. However, they do not replace classical
biochemistry. They simply provide researchers with an
expedited entrée into important new biological prob-
lems. In the end, a detailed functional understanding
of any new protein requires traditional biochemical
analyses—such as were used for the many well-studied
proteins described in this text. When paired with the si-
multaneously evolving tools of biochemistry and molec-
ular biology, genomics and proteomics are speeding the
discovery not only of new proteins but of new biologi-
cal processes and mechanisms.
SUMMARY 9.3 From Genomes to Proteomes
■ A proteome is the complement of proteins
produced by a cell’s genome. The new field of
proteomics encompasses an effort to catalog
and determine the functions of all the proteins
in a cell.
■ One of the most effective ways to determine
the function of a new gene is by comparative
genomics, the search of databases for genes
with similar sequences. Paralogs and orthologs
are proteins (and their genes) with clear
functional and sequence relationships in the
same or in different species. In some cases, the
presence of a gene in combination with certain
other genes, observed as a pattern in several
genomes, can point toward a possible function.
■ Cellular proteomes can be displayed by two-
dimensional gel electrophoresis and explored
with the aid of mass spectrometry.
■ The cellular function of a protein can
sometimes be inferred by determining when
and where its gene is expressed. Researchers
use DNA microarrays (chips) and protein chips
to explore gene expression at the cellular level.
■ Several new techniques, including comparative
genomics, immunoprecipitation, and yeast two-
hybrid analysis, can identify protein-protein
interactions. These interactions provide
important clues to protein function.
9.4 Genome Alterations and New Products
of Biotechnology
We don’t need to look far to find practical applications
for the new biotechnologies or to find new opportunities
for breakthroughs in basic research. Herein lie both the
promise and the challenge of genomics. As our knowl-
edge of the genome increases, we will improve our un-
derstanding of every aspect of biological function. We
will enhance our capacity to engineer organisms and pro-
duce new pharmaceutical agents and, as a consequence,
will improve human nutrition and health. This promise
can be realized only if practical safeguards are in place
to ensure responsible application of these techniques.
A Bacterial Plant Parasite Aids Cloning in Plants
We not only can understand genomes, we can change
them. This is perhaps the ultimate manifestation of the
new technologies. The introduction of recombinant
DNA into plants has enormous implications for agricul-
ture, making possible the alteration of the nutritional
profile or yield of crops or their resistance to environ-
mental stresses, such as insect pests, diseases, cold,
salinity, and drought. Fertile plants of some species may
be generated from a single transformed cell, so that an
introduced gene passes to progeny through the seeds.
As yet, researchers have not found any naturally oc-
curring plant cell plasmids to facilitate cloning in plants,
so the biggest technical challenge is getting DNA into
plant cells. An important and adaptable ally in this ef-
fort is the soil bacterium Agrobacterium tumefaciens.
This bacterium can invade plants at the site of a wound,
transform nearby cells, and induce them to form a tu-
mor called a crown gall. Agrobacterium contains the
large (200,000 bp) Ti plasmid (Fig. 9–26a). When the
bacterium is in contact with a damaged plant cell, a
23,000 bp segment of the Ti plasmid called T DNA is
transferred from the plasmid and integrated at a ran-
dom position in one of the plant cell chromosomes (Fig.
9–26b). The transfer of T DNA from Agrobacterium to
the plant cell chromosome depends on two 25 bp re-
peats that flank the T DNA and on the products of the
virulence (vir) genes on the Ti plasmid (Fig. 9–26a).
The T DNA encodes enzymes that convert plant
metabolites to two classes of compounds that benefit
Chapter 9 DNA-Based Information Technologies330
8885d_c09_306-342 2/7/04 8:14 AM Page 330 mac76 mac76:385_reb:
the bacterium (Fig. 9–27). The first group consists of
plant growth hormones (auxins and cytokinins) that
stimulate growth of the transformed plant cells to form
the crown gall tumor. The second constitutes a series
of unusual amino acids called opines, which serve as a
food source for the bacterium. The opines are produced
in high concentrations in the tumor cells and secreted
to the surroundings, where they can be metabolized only
by Agrobacterium, using enzymes encoded elsewhere
on the Ti plasmid. The bacterium thereby diverts plant
resources by converting them to a form that benefits
only itself.
9.4 Genome Alterations and New Products of Biotechnology 331
vir genes
T DNA
25 bp repeats
(a)
Ti plasmid
(b)
Wounded plant
cell produces
acetosyringone.
Acetosyringone
activates vir genes.
Copy of T DNA is
transferred and
integrated into a
plant chromosome.
Agrobacterium cell
Plant cell
nucleus
Plant cell synthesizes
auxins, cytokinins,
opines; tumor
forms.
FIGURE 9–26 Transfer of DNA to plant cells by a bacterial parasite.
(a) The Ti (tumor-inducing) plasmid of Agrobacterium tumefaciens.
(b) Wounded plant cells produce and release the phenolic compound
acetosyringone. When Agrobacterium detects this compound, the
virulence (vir) genes on the Ti plasmid are expressed. The vir genes
encode enzymes needed to introduce the T DNA segment of the Ti
plasmid into the genome of nearby plant cells. A single-stranded copy
of the T DNA is synthesized and transferred to the plant cell, where
it is converted to duplex DNA and integrated into a plant cell chro-
mosome. The T DNA encodes enzymes that synthesize both plant
growth hormones and opines (see Fig. 9–27); the latter compounds
are metabolized (as a nutrient source) only by Agrobacterium. Ex-
pression of the T DNA genes by transformed plant cells thus leads to
both aberrant plant cell growth (tumor formation) and the diversion
of plant cell nutrients to the invading bacteria.
H
CH
2
Opines
C
Auxins
O
G
D
P
H
G
N
N
N
Isopentenyl adenine
(i
6
Ade)
N
H
N
HHOCH
2
CH
2
C
O
G
D
PC
D
H
G
CH
3
N
N
N
N
H
N
Indoleacetate
H
N
C
E
CH
3
CH
2
CH
3
Zeatin
Cytokinins
COO
H11002
A
H
2
)
2
Mannopine
O
O
CHO COO
H11002
HN
H
2
CHO
(C
OCH
2
A
C
O
H
2
N OOO
B
(CHOH)
4
D
G
N
D
H
2
H
2
N
H11001
OO
H11002
CH
3
O
A
H
2
)
3
Octopine
(CO
CH C
O
HN
H
C
A
C OC
O
OO
H11002
OO
H11002
O C
H11002
OOC
C
A
C NH OC
O
OO
H11002
O
H
2
)
3
(COONHOO
H
A
NH
(C CHH
2
)
2
Nopaline
G
N
D
H
2
H
2
N
H11001
FIGURE 9–27 Metabolites produced in Agrobacterium-infected
plant cells. Auxins and cytokinins are plant growth hormones. The most
common auxin, indoleacetate, is derived from tryptophan. Cytokinins
are adenine derivatives. Opines generally are derived from amino acid
precursors; at least 14 different opines are produced by enzymes en-
coded by the Ti plasmids of different Agrobacterium species.
8885d_c09_306-342 2/7/04 8:14 AM Page 331 mac76 mac76:385_reb:
This rare example of DNA transfer from a prokary-
ote to a eukaryotic cell is a natural genetic engineering
process—one that researchers can harness to transfer
recombinant DNA (instead of T DNA) to the plant
genome. A common cloning strategy employs an
Agrobacterium with two different recombinant plas-
mids. The first is a Ti plasmid from which the T DNA
segment has been removed in the laboratory (Fig.
9–28a). The second is an Agrobacterium–E. coli shut-
tle vector in which the 25 bp repeats of the T DNA flank
a foreign gene that the researcher wants to introduce
into the plant cell, along with a selectable marker such
as resistance to the antibiotic kanamycin (Fig. 9–28b).
The engineered Agrobacterium is used to infect a leaf,
but crown galls are not formed because the T DNA genes
for the auxin, cytokinin, and opine biosynthetic enzymes
are absent from both plasmids. Instead, the vir gene
products from the altered Ti plasmid direct the trans-
formation of the plant cells by the foreign gene—the
gene flanked by the T DNA 25 bp repeats in the second
plasmid. The transformed plant cells can be selected by
growth on agar plates that contain kanamycin, and ad-
dition of growth hormones induces the formation of new
plants that contain the foreign gene in every cell.
The successful transfer of recombinant DNA into
plants was vividly illustrated by an experiment in which
the luciferase gene from fireflies was introduced into the
cells of a tobacco plant (Fig. 9–29)—a favorite plant for
transformation experiments because its cells are par-
ticularly easy to transform with Agrobacterium. The
potential of this technology is not limited to the pro-
duction of glow-in-the-dark plants, of course. The same
approach has been used to produce crop plants that are
resistant to herbicides, plant viruses, and insect pests
(Fig. 9–30). Potential benefits include increased yields
and less need for environmentally harmful agricultural
chemicals.
Biotechnology can introduce new traits into a plant
much faster than traditional methods of plant breeding.
A prominent example is the development of soybeans
that are resistant to the general herbicide glyphosate (the
active ingredient in the product RoundUp). Glyphosate
breaks down rapidly in the environment (glyphosate-
sensitive plants can be planted in a treated area after as
little as 48 hours), and its use does not generally lead to
contamination of groundwater or carryover from one year
to the next. A field of glyphosate-resistant soybeans can
be treated once with glyphosate during a summer grow-
ing season to eliminate essentially all weeds in the field,
while leaving the soybeans unaffected (Fig. 9–31). Po-
tential pitfalls of the technology, such as the evolution
of glyphosate-resistant weeds or the escape of difficult-
to-control recombinant plants, remain a concern of re-
searchers and the public.
Chapter 9 DNA-Based Information Technologies332
vir
Agrobacterium cell
Ti plasmid without
T DNA
(b)
(a)
Recombinant plasmid
with foreign gene and
kanamycin-resistance
gene between T DNA
25 bp repeats
25 bp repeats
Foreign gene
Kanamycin resistance
Bacteria invade at wound
sites (where leaf is cut).
Leaf segments are
transferred to agar dish.
Plants are regenerated
from leaf segments.
Agar plate with growth
hormones and kanamycin
These kanamycin-
resistant plants
contain the foreign gene.
FIGURE 9–28 A two-plasmid strategy to create a recombinant plant.
(a) One plasmid is a modified Ti plasmid that contains the vir genes
but lacks T DNA. (b) The other plasmid contains a segment of DNA
that bears both a foreign gene (the gene of interest, e.g., the gene for
the insecticidal protein described in Fig. 9–30) and an antibiotic-
resistance element (here, kanamycin resistance), flanked by the two
25 bp repeats of T DNA that are required for transfer of the plasmid
genes to the plant chromosome. This plasmid also contains the repli-
cation origin needed for propagation in Agrobacterium.
When bacteria invade at the site of a wound (the edge of the cut
leaf), the vir genes on the first plasmid mediate transfer into the plant
genome of the segment of the second plasmid that is flanked by the
25 bp repeats. Leaf segments are placed on an agar dish that contains
both kanamycin and appropriate levels of plant growth hormones, and
new plants are generated from segments with the transformed cells.
Nontransformed cells are killed by the kanamycin. The foreign gene
and the antibiotic-resistance element are normally transferred together,
so plant cells that grow in this medium generally contain the foreign
gene.
8885d_c09_306-342 2/7/04 8:14 AM Page 332 mac76 mac76:385_reb:
9.4 Genome Alterations and New Products of Biotechnology 333
FIGURE 9–29 A tobacco plant expressing the gene for firefly luciferase.
Light was produced after the plant was watered with a solution con-
taining luciferin, the substrate for the light-producing luciferase enzyme
(see Box 13–2). Don’t expect glow-in-the-dark ornamental plants at your
local plant nursery anytime soon. The light is actually quite weak; this
photograph required a 24-hour exposure. The real point—that this tech-
nology allows the introduction of new traits into plants—is nevertheless
elegantly made.
FIGURE 9–30 Tomato plants engineered to be resistant to insect lar-
vae. Two tomato plants were exposed to equal numbers of moth lar-
vae. The plant on the left has not been genetically altered. The plant
on the right expresses a gene for a protein toxin derived from the bac-
terium Bacillus thuringiensis. This protein, introduced by a protocol
similar to that depicted in Figure 9–28, is toxic to the larvae of some
moth species while being harmless to humans and other organisms.
Insect resistance has also been genetically engineered in cotton and
other plants.
FIGURE 9–31 Glyphosate-resistant soybean plants. The photographs
show two areas of a soybean field in Wisconsin. (a) Without glyphosate
treatment, this part of the field is overrun with weeds. (b) Glyphosate-
resistant soybean plants thrive in the glyphosate-treated section of the
field. Glyphosate breaks down rapidly in the environment. Agricul-
tural use of engineered plants such as these proceeds only after con-
siderable deliberation, balancing the extraordinary promise of the
technology with the need to select new traits with care. Both science
and society as a whole have a stake in ensuring that the use of the re-
sultant plants has no adverse impact on the environment or on hu-
man health.
(a)
(b)
PNH
H11002
O
Glyphosate
CH
2
CH
2
O
O
H11002
COO
H11002
Manipulation of Animal Cell Genomes Provides
Information on Chromosome Structure
and Gene Expression
The transformation of animal cells by foreign genetic
material offers an important mechanism for expanding
our knowledge of the structure and function of animal
genomes, as well as for the generation of animals with
new traits. This potential has stimulated intensive re-
search into more-sophisticated means of cloning animals.
Most work of this kind requires a source of cells into
which DNA can be introduced. Although intact tissues
are often difficult to maintain and manipulate in vitro,
many types of animal cells can be isolated and grown in
the laboratory if their growth requirements are carefully
met. Cells derived from a particular animal tissue and
8885d_c09_306-342 2/7/04 8:14 AM Page 333 mac76 mac76:385_reb:
grown under appropriate tissue culture conditions can
maintain their differentiated properties (for example, a
hepatocyte (liver cell) remains a hepatocyte) for weeks
or even months.
No suitable plasmidlike vector is available for intro-
ducing DNA into an animal cell, so transformation usu-
ally requires the integration of the DNA into a host-cell
chromosome. The efficient delivery of DNA to a cell nu-
cleus and integration of this DNA into a chromosome
without disrupting any critical genes remain the major
technical problems in the genetic engineering of animal
cells.
Available methods for carrying DNA into an animal
cell vary in efficiency and convenience. Some success
has been achieved with spontaneous uptake of DNA or
electroporation, techniques roughly comparable to the
common methods used to transform bacteria. They are
inefficient in animal cells, however, transforming only 1
in 100 to 10,000 cells. Microinjection—the injection
of DNA directly into a nucleus, using a very fine nee-
dle—has a high success rate for skilled practitioners,
but the total number of cells that can be treated is small,
because each must be injected individually.
The most efficient and widely used methods for
transforming animal cells rely on liposomes or viral vec-
tors. Liposomes are small vesicles consisting of a lipid
bilayer that encloses an aqueous compartment (see Fig.
11–4). Liposomes that enclose a recombinant DNA mol-
ecule can be fused with the membranes of target cells
to deliver DNA into the cell. The DNA sometimes
reaches the nucleus, where it can integrate into a chro-
mosome (mostly at random locations). Viral vectors
are even more efficient at delivering DNA. Animal
viruses have effective mechanisms for introducing their
nucleic acids into cells, and several types also have
mechanisms to integrate their DNA into a host-cell
chromosome. Some of these, such as retroviruses (see
Fig. 26–30) and adenoviruses, have been modified to
serve as viral vectors to introduce foreign DNA into
mammalian cells.
The work on retroviral vectors illustrates some of
the strategies being used (Fig. 9–32). When an engi-
neered retrovirus enters a cell, its RNA genome is tran-
scribed to DNA by reverse transcriptase and then inte-
grated into the host genome by the enzyme viral
integrase. Special regions of DNA are required for this
Chapter 9 DNA-Based Information Technologies334
Reverse transcriptase
converts RNA genome
to double-stranded DNA.
LTR
H9274
gag pol env LTR
Retroviral genome (single-stranded RNA)
Viral genes are replaced
with a foreign gene.
LTR
H9274
gag pol env LTR
DNA
Recombinant DNA is
introduced into cells
in tissue culture.
LTR
H9274
LTR
Recombinant defective
retroviral DNA
RNA copies of
recombinant viruses
are produced in cells
containing a helper
virus and packaged into
viral particles.
Reverse transcriptase
and integrase
Retroviral RNA
genome with foreign gene
Retroviral genome
with foreign gene
is integrated into
the target cell
chromosome.
Recombinant virus
particles infect a
target cell.
FIGURE 9–32 Use of retroviral vectors in mammalian cell cloning.
A typical retroviral genome (somewhat simplified here), engineered to
carry a foreign gene (pink), is added to a host-cell tissue culture. The
helper virus (not shown) lacks the packaging sequence, H9274 , so its RNA
transcripts cannot be packaged into viral particles, but it provides the
gag, pol, and env gene products needed to package the engineered
retrovirus into functional viral particles. This enables the foreign gene
in the recombinant retroviral genome to be introduced efficiently into
the target cells.
procedure: long terminal repeat (LTR) sequences to in-
tegrate retroviral DNA into the host chromosome and
the H9274 (psi) sequence to package the viral RNA in viral
particles (see Fig. 26–30).
The gag, pol, and env genes of the retroviral
genome, required for retroviral replication and assem-
bly of viral particles, can be replaced with foreign DNA.
To assemble viruses that contain the recombinant ge-
netic information, researchers must introduce the DNA
into cultured cells that are simultaneously infected with
a “helper virus” that has the genes to produce viral par-
ticles but lacks the H9274 sequence required for packaging.
Thus the recombinant DNA can be transcribed and its
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RNA packaged into viral particles. These particles can
act as vectors to introduce the recombinant RNA into
target cells. Viral reverse transcriptase and integrase en-
zymes (produced by the helper virus) are also packaged
in the viral particle and introduced into the target cells.
Once the engineered viral genome is inside a cell, these
enzymes create a DNA copy of the recombinant viral
RNA genome and integrate it into a host chromosome.
The integrated recombinant DNA then becomes a per-
manent part of the target cell’s chromosome and is repli-
cated with the chromosome at every cell division. The
cell itself is not endangered by the integrated viral DNA,
because the recombinant virus lacks the genes needed
to produce RNA copies of its genome and package them
into new viral particles. The use of recombinant retro-
viruses is often the best method for introducing DNA
into large numbers of mammalian cells.
Each type of virus has different attributes, so sev-
eral classes of animal viruses are being engineered as
vectors to transform mammalian cells. Adenoviruses, for
example, lack a mechanism for integrating DNA into a
chromosome. Recombinant DNA introduced via an ade-
noviral vector is therefore expressed for only a short
time and then destroyed. This can be useful if the ob-
jective is transient expression of a gene.
Transformation of animal cells by any of the above
techniques has its problems. Introduced DNA is gener-
ally integrated into chromosomes at random locations.
Even when the foreign DNA contains a sequence simi-
lar to a sequence in a host chromosome, allowing tar-
geting to that position, nonhomologous integrants still
outnumber the targeted ones by several orders of mag-
nitude. If these integration events disrupt essential
genes, they can sometimes alter cellular functions (most
cells are diploid or polyploid, however, so an integration
usually leaves at least one unaffected copy of any given
gene). A particularly poor outcome would involve an in-
tegration event that inadvertently activated a gene that
stimulated cell division, potentially creating a cancer
cell. Although such an event was once thought to be
rare, recent trials suggest it is a significant hazard (Box
9–2). Finally, the site of an integration can determine
the level of expression of the integrated gene, because
integrants are not transcribed equally well everywhere
in the genome.
Despite these challenges, the transformation of ani-
mal cells has been used extensively to study chromosome
structure and the function, regulation, and expression of
genes. The successful introduction of recombinant DNA
into an animal can be illustrated by an experiment that
permanently altered an easily observable inheritable
physical trait. Microinjection of DNA into the nuclei of
fertilized mouse eggs can produce efficient transforma-
tion (chromosomal integration). When the injected eggs
are introduced into a female mouse and allowed to de-
velop, the new gene is often expressed in some of the
newborn mice. Those in which the germ line has been
9.4 Genome Alterations and New Products of Biotechnology 335
FIGURE 9–33 Cloning in mice. The gene for human growth hormone
was introduced into the genome of the mouse on the right. Expres-
sion of the gene resulted in the unusually large size of this mouse.
altered can be identified by testing their offspring. By
careful breeding of these mice, researchers can establish
a transgenic mouse line in which all the mice are ho-
mozygous for the new gene or genes. This technology was
used to introduce into mice the gene for human growth
hormone, under the control of an inducible promoter.
When the mice were fed a diet that included the inducer,
some of the mice that developed from injected embryos
grew to an unusually large size (Fig. 9–33). Transgenic
mice have now been produced with a wide range of ge-
netic variations, including many relevant to human dis-
eases and their control, pointing the way to human gene
therapy (Box 9–2). A very similar approach is used to
generate mice in which a particular gene has been inac-
tivated (“knockout mice”), a way of establishing the func-
tion of the inactivated gene. Creating a Transgenic Mouse
New Technologies Promise to Expedite the Discovery
of New Pharmaceuticals
It is difficult to summarize all the ways in which
genomics and proteomics might affect the de-
velopment of pharmaceutical agents, but a few exam-
ples illustrate the potential. Hypertension, congestive
heart failure, hypercholesterolemia, and obesity are
treated by pharmaceutical drugs that alter human phys-
iology. Therapies are arrived at by identifying an enzyme
or receptor involved in the process and discovering an
inhibitor that interferes with its action. Proteomics will
play an increasing role in identifying such potential drug
targets. For example, the most potent vasoconstrictor
known is the peptide hormone urotensin II. First dis-
covered in fish spinal fluid, urotensin II is a small cyclic
peptide, with 11 amino acid residues in humans and 12
or 13 in some other organisms. The vasoconstriction it
induces can cause or exacerbate hypertension, conges-
tive heart failure, and coronary artery disease. Some of
the methods described in Section 9.3 for elucidating
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protein-protein interactions have been used to demon-
strate that urotensin II is bound by a G-protein-coupled
receptor called GPR14. As we shall see in Chapter 12,
G proteins play an important role in many signaling
pathways. However, GPR14 was an “orphan” receptor,
in that human genome sequencing had identified it as a
G-protein-coupled receptor, but with no known func-
tion. The association of urotensin II with GPR14 now
makes the latter protein a key target for drug therapies
aimed at interfering with the action of urotensin II.
336
BOX 9–2 BIOCHEMISTRY IN MEDICINE
The Human Genome and Human Gene Therapy
As biotechnology gained momentum in the 1980s, a
rational approach to the treatment of genetic diseases
became increasingly attractive. In principle, DNA can
be introduced into human cells to correct inherited
genetic deficiencies. Genetic correction may even be
targeted to a specific tissue by inoculating an individ-
ual with a genetically engineered, tissue-specific virus
carrying a payload of DNA to be incorporated into de-
ficient cells. The goal is entrancing, but the research
path is strewn with impediments.
Altering chromosomal DNA entails substantial
risk—a risk that cannot be quantified in the early
stages of discovery. Consequently, early efforts at
human gene therapy were directed at only a small
subset of genetic diseases. Panels of scientists and
ethicists developed a list of several conditions that
should be satisfied to justify the risk involved, includ-
ing the following. (1) The genetic defect must be a
well-characterized, single-gene disorder. (2) Both the
mutant and the normal gene must be cloned and se-
quenced. (3) In the absence of a technique for elimi-
nating the existing mutant gene, the functional gene
must function well in the presence of the mutant gene.
(4) Finally, and most important, the risks inherent in
a new technology must be outweighed by the seri-
ousness of the disease. Protocols for human clinical
trials were submitted by scientists in several nations
and reviewed for scientific rigor and ethical compli-
ance by carefully selected advisory panels in each
country; then human trials commenced.
Early targets of gene therapy included cancer and
genetic diseases affecting the immune system. Immu-
nity is mediated by leukocytes (white blood cells) of
several different types, all arising from undifferenti-
ated stem cells in the bone marrow. These cells divide
quickly and have special metabolic requirements. Dif-
ferentiation can become blocked in several ways, re-
sulting in a condition called severe combined immune
deficiency (SCID). One form of SCID results from ge-
netically inherited defects in the gene encoding
adenosine deaminase (ADA), an enzyme involved in
nucleotide biosynthesis (discussed in Chapter 22).
Another form of SCID arises from a defect in a cell-
surface receptor protein that binds chemical signals
called cytokines, which trigger differentiation. In both
cases, the progenitor stem cells cannot differentiate
into the mature immune system cells, such as T and B
lymphocytes (Chapter 5). Children with these rare hu-
man diseases are highly susceptible to bacterial and vi-
ral infections, and often suffer from a range of related
physiological and neurological problems. In the absence
of an effective therapy, the children must be confined
in a sterile environment. About 20% of these children
have a human leukocyte antigen (HLA)–identical sib-
ling who can serve as a bone marrow transplant donor,
a procedure that can cure the disease. The remaining
children need a different approach.
The first human gene therapy trial was carried out
at the National Institutes of Health in Bethesda, Mary-
land, in 1990. The patient was a four-year-old girl crip-
pled by ADA deficiency. Bone marrow cells from the
child were transformed with an engineered retrovirus
containing a functional ADA gene; when the alteration
of cells is done in this way—in the laboratory rather
than in the living patient—the procedure is said to be
done ex vivo. The treated cells were reintroduced into
the patient’s marrow. Four years later, the child was
leading a normal life, going to school, and even testi-
fying about her experiences before Congress. However,
her recovery cannot be uniquely attributed to gene
therapy. Before the gene therapy clinical trials began,
researchers had developed a new treatment for ADA
deficiency, in which synthetic ADA was administered
in a complex with polyethylene glycol (PEG). For many
ADA-SCID patients, injection of the ADA-PEG com-
plex allowed some immune system development, with
weight gain and reduced infection, although not full
immune reconstitution. The new gene therapy was
risky, and withdrawing the inoculation treatment from
patients in the gene therapy trial was judged unethi-
cal. So trial participants received both treatments at
once, making it unclear which treatment was primarily
responsible for the positive clinical outcome. Never-
theless, the clinical trial provided important informa-
tion: it was feasible to transfer genes ex vivo to large
numbers of leukocytes, and cells bearing the trans-
ferred gene were still detectable years after treatment,
suggesting that long-term correction was possible. In
addition, the risk associated with use of the retroviral
vectors appeared to be low.
Through the 1990s, hundreds of human gene ther-
apy clinical trials were carried out, targeting a variety
of genetic diseases, but the results in most cases were
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Glu–Thr–Pro–Asp–Cys–S–S–Cys–Val
–
–
–
–
Phe Tyr
Trp–Lys
Urotensin II
Another objective of medical research is to identify
new agents that can treat the diseases caused by hu-
man pathogens. This now means identifying enzymatic
targets in microbial pathogens that can be inactivated
with a new drug. The ideal microbial target enzyme
337
discouraging. One major impediment proved to be the
inefficiency of introducing new genes into cells. Trans-
formation failed in many cells, and the number of
transformed cells often proved insufficient to reverse
the disorder. In the ADA trials, achieving a sufficient
population of transformed cells was particularly diffi-
cult, because of the ongoing ADA-PEG therapy. Nor-
mally, stem cells with the correct ADA gene would
have a growth advantage over the untreated cells, ex-
panding their population and gradually predominating
in the bone marrow. However, the injections of ADA-
PEG in the same patients allowed the untransformed
(ADA-deficient) cells to live and develop, and the
transformed cells did not have the needed growth ad-
vantage to expand their population at the expense of
the others.
A gene therapy trial initiated in 1999 was success-
ful in correcting a form of SCID caused by defective cy-
tokine receptors (in particular a subunit called H9253c), as
reported in 2000 by physician researchers in France,
Italy, and Britain. These researchers introduced the
corrected gene for the H9253c cytokine–receptor subunit
into CD34
H11001
cells. (The stem cells that give rise to im-
mune system cells have a protein called CD34 on their
surface; these cells can be separated from other bone
marrow cells by antibodies to CD34.) The transformed
cells were placed back into the patients’ bone marrow.
In this trial, introduction of the corrected gene clearly
conferred a growth advantage over the untreated cells.
A functioning immune system was detected in four of
the first five patients within 6 to 12 weeks, and levels
of mature immune system T lymphocytes reached the
levels found in age-matched control subjects (who did
not have SCID) within 6 to 8 months. Immune system
function was restored, and nearly 4 years later (mid-
2003) most of the children are leading normal lives.
Similar results have been obtained with four additional
patients. This provided dramatic confirmation that
human gene therapy could cure a serious genetic
disease.
In early 2003 came a setback. One of the original
four patients who had received cells with the correct
cytokine receptor gene developed a severe form of
leukemia. During the gene therapy treatment, one of
the introduced retroviruses had by chance inserted it-
self into a chromosome of one CD34
H11001
cell, resulting
in abnormally high expression of a gene called LMO-
2. The affected cell differentiated into an immune sys-
tem T cell, and the elevated expression of LMO-2 led
to uncontrolled growth of the cell, giving rise to the
leukemia. As of mid-2003 the patient had responded
well to chemotherapy, but there may be more chap-
ters to write. The incident shows that early worries
about the risk associated with retroviral vectors were
well founded. After a review of the gene therapy trial
protocols, including consultations with ethicists and
parents of children affected by these diseases, further
gene therapy trials are still planned for children who
are not candidates for bone marrow transplants. The
reason is simple enough. The potential benefit to the
children with these debilitating conditions has been
judged to outweigh the demonstrated risk.
Human gene therapy is not limited to genetic dis-
eases. Cancer cells are being targeted by delivering
genes for proteins that might destroy the cell or
restore the normal control of cell division. Immune
system cells associated with tumors, called tumor-
infiltrating lymphocytes, can be genetically modified to
produce tumor necrosis factor (TNF; see Fig. 12–50).
When these lymphocytes are taken from a cancer pa-
tient, modified, and reintroduced, the engineered cells
target the tumor, and the TNF they produce causes tu-
mor shrinkage. AIDS may also be treatable with gene
therapy; DNA that encodes an RNA molecule comple-
mentary to a vital HIV mRNA could be introduced into
immune system cells (the targets of HIV). The RNA
transcribed from the introduced DNA would pair with
the HIV mRNA, preventing its translation and inter-
fering with the virus’s life cycle. Alternatively, a gene
could be introduced that encodes an inactive form of
one subunit of a multisubunit HIV enzyme; with one
nonfunctional subunit, the entire enzyme might be in-
activated.
Our growing understanding of the human genome
and the genetic basis for some diseases brings the
promise of early diagnosis and constructive interven-
tion. As the early results demonstrate, however, the
road to effective therapies will be a long one, with
many detours. We need to learn more about cellular
metabolism, more about how genes interact, and
more about how to manage the dangers. The prospect
of vanquishing life-destroying genetic defects and
other debilitating diseases provides the motivation to
press on.
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should be (1) essential to the pathogen cell’s survival,
(2) well-conserved among a wide range of pathogens,
and (3) absent or significantly different in humans. The
task of identifying metabolic processes that are critical
to microorganisms but absent in humans is made much
easier by comparative genomics, augmented by the
functional information available from genomics and
proteomics. ■
Recombinant DNA Technology Yields New Products
and Challenges
The products of recombinant DNA technology range
from proteins to engineered organisms. The technology
can produce large amounts of commercially useful pro-
teins, can design microorganisms for special tasks, and
can engineer plants or animals with traits that are use-
ful in agriculture or medicine. Some products of this
technology have been approved for consumer or profes-
sional use, and many more are in development. Genetic
engineering has been transformed over a few years from
a promising new technology to a multibillion-dollar in-
dustry, with much of the growth occurring in the phar-
maceutical industry. Some major classes of new products
are listed in Table 9–3.
Erythropoietin is typical of the newer products.
This protein hormone (M
r
51,000) stimulates
erythrocyte production. People with diseases that com-
promise kidney function often have a deficiency of this
protein, resulting in anemia. Erythropoietin produced
by recombinant DNA technology can be used to treat
these individuals, reducing the need for repeated blood
transfusions. ■
Other applications of this technology continue to
emerge. Enzymes produced by recombinant DNA tech-
nology are already used in the production of detergents,
sugars, and cheese. Engineered proteins are being used
as food additives to supplement nutrition, flavor, and
fragrance. Microorganisms are being engineered with al-
tered or entirely novel metabolic pathways to extract oil
and minerals from ground deposits, to digest oil spills,
and to detoxify hazardous waste dumps and sewage. En-
gineered plants with improved resistance to drought,
frost, pests, and disease are increasing crop yields and
reducing the need for agricultural chemicals. Complete
animals can be cloned by moving an entire nucleus and
all of its genetic material to a prepared egg from which
the nucleus has been removed.
The extraordinary promise of modern biotechnol-
ogy does not come without controversy. The cloning of
mammals challenges societal mores and may be accom-
panied by serious deficiencies in the health and
longevity of the cloned animal. If useful pharmaceutical
agents can be produced, so can toxins suitable for bio-
logical warfare. The potential for hazards posed by the
release of engineered plants and other organisms into
Chapter 9 DNA-Based Information Technologies338
TABLE 9–3 Some Recombinant DNA Products in Medicine
Product category Examples/uses
Anticoagulants Tissue plasminogen activator (TPA); activates plasmin, an enzyme involved in dissolving
clots; effective in treating heart attack patients.
Blood factors Factor VIII; promotes clotting; it is deficient in hemophiliacs; treatment with factor VIII
produced by recombinant DNA technology eliminates infection risks associated
with blood transfusions.
Colony-stimulating factors Immune system growth factors that stimulate leukocyte production; treatment of immune
deficiencies and infections.
Erythropoietin Stimulates erythrocyte production; treatment of anemia in patients with kidney disease.
Growth factors Stimulate differentiation and growth of various cell types; promote wound healing.
Human growth hormone Treatment of dwarfism.
Human insulin Treatment of diabetes.
Interferons Interfere with viral reproduction; used to treat some cancers.
Interleukins Activate and stimulate different classes of leukocytes; possible uses in treatment of
wounds, HIV infection, cancer, and immune deficiencies.
Monoclonal antibodies Extraordinary binding specificity is used in: diagnostic tests; targeted transport of drugs,
toxins, or radioactive compounds to tumors as a cancer therapy; many other
applications.
Superoxide dismutase Prevents tissue damage from reactive oxygen species when tissues briefly deprived of O
2
during surgery suddenly have blood flow restored.
Vaccines Proteins derived from viral coats are as effective in “priming” an immune system as is the
killed virus more traditionally used for vaccines, and are safer; first developed was
the vaccine for hepatitis B.
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the biosphere continues to be monitored carefully. The
full range of the long-term consequences of this tech-
nology for our species and for the global environment
is impossible to foresee, but will certainly demand our
increasing understanding of both cellular metabolism
and ecology.
SUMMARY 9.4 Genome Alterations and New
Products of Biotechnology
■ Advances in whole genome sequencing and
genetic engineering methods are enhancing our
ability to modify genomes in all species.
■ Cloning in plants, which makes use of the Ti
plasmid vector from Agrobacterium, allows the
introduction of new plant traits.
■ In animal cloning, researchers introduce foreign
DNA primarily with the use of viral vectors or
microinjection. These techniques can produce
transgenic animals and provide new methods
for human gene therapy.
■ The use of genomics and proteomics in basic and
pharmaceutical research is greatly advancing the
discovery of new drugs. Biotechnology is also
generating an ever-expanding range of other
products and technologies.
Chapter 9 Further Reading 339
Key Terms
cloning 306
vector 307
recombinant DNA 307
restriction endonucleases 307
DNA ligase 307
plasmid 311
bacterial artificial chromosome
(BAC) 313
yeast artificial chromosome
(YAC) 314
site-directed mutagenesis 316
fusion protein 317
genomics 317
genomic library 318
contig 318
sequence-tagged site (STS) 318
complementary DNA
(cDNA) 318
cDNA library 318
expressed sequence tag (EST) 318
epitope tag 319
polymerase chain reaction
(PCR) 319
DNA fingerprinting 322
restriction fragment length
polymorphisms (RFLPs) 322
Southern blot 322
single nucleotide polymorphisms
(SNPs) 324
proteome 325
proteomics 325
orthologs 326
synteny 326
DNA microarray 326
Ti plasmid 330
transgenic 335
Terms in bold are defined in the glossary.
Further Reading
General
Jackson, D.A., Symons, R.H., & Berg, P. (1972) Biochemical
method for inserting new genetic information into DNA of simian
virus 40: circular SV40 DNA molecules containing lambda phage
genes and the galactose operon of Escherichia coli. Proc. Natl.
Acad. Sci. USA 69, 2904–2909.
The first recombinant DNA experiment linking DNA from two
species.
Lobban, P.E. & Kaiser, A.D. (1973) Enzymatic end-to-end
joining of DNA molecules. J. Mol. Biol. 78, 453–471.
Report of the first recombinant DNA experiment.
Sambrook, J., Fritsch, E.F., & Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual, 2nd edn, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY.
Although supplanted by more recent manuals, this three-
volume set includes much useful background information on
the biological, chemical, and physical principles underlying both
classic and still-current techniques.
Gene Cloning
Arnheim, N. & Erlich, H. (1992) Polymerase chain reaction
strategy. Annu. Rev. Biochem. 61, 131–156.
Hofreiter, M., Serre, D., Poinar, H.N., Kuch, M., & Paabo, S.
(2001) Ancient DNA. Nat. Rev. Genet. 2, 353–359.
Successes and pitfalls in the retrieval of DNA from very old
samples.
Ivanov, P.L., Wadhams, M.J., Roby, R.K., Holland, M.M.,
Weedn, V.W., & Parsons, T.J. (1996) Mitochondrial DNA
sequence heteroplasmy in the Grand Duke of Russia Georgij
Romanov establishes the authenticity of the remains of Tsar
Nicholas II. Nat. Genet. 12, 417–420.
Lindahl, T. (1997) Facts and artifacts of ancient DNA.
Cell 90, 1–3.
Good description of how nucleic acid chemistry affects the
retrieval of DNA in archaeology.
Genomics
Adams, M.D., Kelley, J.M., Gocayne, J.D., Dubnick, M.,
Polymeropoulos, M.H., Xiao, H., Merril, C.R., Wu, A., Olde,
B., Moreno, R.F., et al. (1991) Complementary DNA sequencing:
expressed sequence tags and Human Genome Project. Science
252, 1651–1656.
The paper that introduced expressed sequence tags (ESTs).
Bamshad, M. & Wooding, S.P. (2003) Signatures of natural
selection in the human genome. Nat. Rev. Genet. 4, 99A–111A.
Use of the human genome to trace human evolution.
Brenner, S. (2004) Genes to genomics. Annu. Rev. Genet. 38,
in press.
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Chapter 9 DNA-Based Information Technologies340
Carroll, S.B. (2003) Genetics and the making of Homo sapiens.
Nature 422, 849–857.
Clark, M.S. (1999) Comparative genomics: the key to understand-
ing the Human Genome Project. Bioessays 21, 121–130.
Useful background on some reasons for the importance of
sequencing the genomes of many organisms.
Collins, F.S., Green, E.D., Guttmacher, A.E., & Guyer, M.S.
(2003) A vision for the future of genomics research. Nature 422,
835–847.
A wide-ranging overview of the enormous potential of genomics
research.
Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody,
M.C., Baldwin, J., Devon, K., Dewar, K., Doyle, M.,
FitzHugh, W., et al. (2001) Initial sequencing and analysis of the
human genome. Nature 409, 860–921.
Discussion of the draft genome sequence put together by the
international Human Genome Project. Many other useful arti-
cles are to be found in this issue.
Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R.J.,
Sutton, G.G., Smith, H.O., Yandell, M., Evans, C.A., Holt,
R.A., et al. (2001) The sequence of the human genome. Science
291, 1304–1351.
Description of the draft of the human genome sequence pro-
duced by Celera Corporation. Many other articles in the same
issue provide insight and additional information.
Proteomics
Brown, P.O. & Botstein, D. (1999) Exploring the new world of
the genome with DNA microarrays. Nat. Genet. 21, 33–37.
Eisenberg, D., Marcotte, E.M., Xenarios, I., & Yeates, T.O.
(2000) Protein function in the post-genomic era. Nature 405,
823–826.
Pandey, A. & Mann, M. (2000) Proteomics to study genes and
genomes. Nature 405, 837–846.
An especially good description of the various strategies and
methods used to identify proteins and their functions.
Zhu, H., Bilgin, M., & Snyder, M. (2003) Proteomics. Annu.
Rev. Biochem. 72, 783–812.
Applying Biotechnology
Foster, E.A., Jobling, M.A., Taylor, P.G., Donnelly, P.,
de Knijff, P., Mieremet, R., Zerjal, T., & Tyler-Smith, C.
(1999) The Thomas Jefferson paternity case. Nature 397, 32.
Last article of a series in an interesting case study of the uses
of biotechnology to address historical questions.
Hansen, G. & Wright M.S. (1999) Recent advances in the trans-
formation of plants. Trends Plant Sci. 4, 226–231.
Koopman, P., Gubbay, J., Vivian, N., Goodfellow, P., &
Lovell-Badge, R. (1991) Male development of chromosomally
female mice transgenic for Sry. Nature 351, 117–121.
Recombinant DNA technology shows that a single gene directs
development of chromosomally female mice into males.
Lapham, E.V., Kozma, C., & Weiss, J. (1996) Genetic discrimi-
nation: perspectives of consumers. Science 274, 621–624.
The upside and downside of knowing what is in your genome.
Mahowald, M.B., Verp, M.S., & Anderson, R.R. (1998) Genetic
counseling: clinical and ethical challenges. Annu. Rev. Genet. 32,
547–559.
Ohlstein, E.H., Ruffolo, R.R., Jr., & Elliott, J.D. (2000) Drug
discovery in the next millennium. Annu. Rev. Pharmacol. Toxi-
col. 40, 177–191.
Palmiter, R.D., Brinster, R.L., Hammer, R.E., Trumbauer,
M.E., Rosenfeld, M.G., Birnberg, N.C., & Evans, R.M. (1982)
Dramatic growth of mice that develop from eggs microinjected
with metallothionein-growth hormone fusion genes. Nature 300,
611–615.
A description of how to make giant mice.
Pfeifer, A. & Verma, I. M. (2001) Gene therapy: promises and
problems. Annu. Rev. Genomics Hum. Genet. 2, 177–211.
Thompson, J. & Donkersloot, J.A. (1992) N-(Carboxyalkyl)
amino acids: occurrence, synthesis, and functions. Annu. Rev.
Biochem. 61, 517–557.
A summary of the structure and biological functions of opines.
Wadhwa, P.D., Zielske, S.P., Roth, J.C., Ballas, C.B.,
Bowman, J.E., & Gerson, S.L. (2002) Cancer gene therapy:
scientific basis. Annu. Rev. Med. 53, 437–452.
1. Cloning When joining two or more DNA fragments, a
researcher can adjust the sequence at the junction in a vari-
ety of subtle ways, as seen in the following exercises.
(a) Draw the structure of each end of a linear DNA frag-
ment produced by an EcoRI restriction digest (include those
sequences remaining from the EcoRI recognition sequence).
(b) Draw the structure resulting from the reaction of
this end sequence with DNA polymerase I and the four de-
oxynucleoside triphosphates (see Fig. 8–36).
(c) Draw the sequence produced at the junction that
arises if two ends with the structure derived in (b) are lig-
ated (see Fig. 25–16).
(d) Draw the structure produced if the structure derived
in (a) is treated with a nuclease that degrades only single-
stranded DNA.
(e) Draw the sequence of the junction produced if an
end with structure (b) is ligated to an end with structure (d).
(f) Draw the structure of the end of a linear DNA frag-
ment that was produced by a PvuII restriction digest (include
those sequences remaining from the PvuII recognition
sequence).
(g) Draw the sequence of the junction produced if an
end with structure (b) is ligated to an end with structure (f).
(h) Suppose you can synthesize a short duplex DNA
fragment with any sequence you desire. With this synthetic
fragment and the procedures described in (a) through (g),
design a protocol that would remove an EcoRI restriction site
from a DNA molecule and incorporate a new BamHI restric-
tion site at approximately the same location. (See Fig. 9–3.)
(i) Design four different short synthetic double-
stranded DNA fragments that would permit ligation of struc-
ture (a) with a DNA fragment produced by a PstI restriction
Problems
8885d_c09_306-342 2/7/04 8:14 AM Page 340 mac76 mac76:385_reb:
Chapter 9 Problems 341
digest. In one of these fragments, design the sequence so that
the final junction contains the recognition sequences for both
EcoRI and PstI. In the second and third fragments, design the
sequence so that the junction contains only the EcoRI and
only the PstI recognition sequence, respectively. Design the
sequence of the fourth fragment so that neither the EcoRI
nor the PstI sequence appears in the junction.
2. Selecting for Recombinant Plasmids When cloning
a foreign DNA fragment into a plasmid, it is often useful to
insert the fragment at a site that interrupts a selectable
marker (such as the tetracycline-resistance gene of pBR322).
The loss of function of the interrupted gene can be used to
identify clones containing recombinant plasmids with foreign
DNA. With a bacteriophage H9261 vector it is not necessary to do
this, yet one can easily distinguish vectors that incorporate
large foreign DNA fragments from those that do not. How are
these recombinant vectors identified?
3. DNA Cloning The plasmid cloning vector pBR322 (see
Fig. 9–4) is cleaved with the restriction endonuclease PstI.
An isolated DNA fragment from a eukaryotic genome (also
produced by PstI cleavage) is added to the prepared vector
and ligated. The mixture of ligated DNAs is then used to trans-
form bacteria, and plasmid-containing bacteria are selected
by growth in the presence of tetracycline.
(a) In addition to the desired recombinant plasmid, what
other types of plasmids might be found among the trans-
formed bacteria that are tetracycline resistant? How can the
types be distinguished?
(b) The cloned DNA fragment is 1,000 bp long and has
an EcoRI site 250 bp from one end. Three different recom-
binant plasmids are cleaved with EcoRI and analyzed by gel
electrophoresis, giving the patterns shown. What does each
pattern say about the cloned DNA? Note that in pBR322, the
PstI and EcoRI restriction sites are about 750 bp apart. The
entire plasmid with no cloned insert is 4,361 bp. Size mark-
ers in lane 4 have the number of nucleotides noted.
4. Identifying the Gene for a Protein with a Known
Amino Acid Sequence Using Figure 27–7 to translate the
genetic code, design a DNA probe that would allow you to iden-
tify the gene for a protein with the following amino-terminal
amino acid sequence. The probe should be 18 to 20 nucleotides
long, a size that provides adequate specificity if there is suffi-
cient homology between the probe and the gene.
H
3
N
H11001
–Ala–Pro–Met–Thr–Trp–Tyr–Cys–Met–
Asp–Trp–Ile–Ala–Gly–Gly–Pro–Trp–Phe–Arg–
Lys–Asn–Thr–Lys–
5. Designing a Diagnostic Test for a Genetic Disease
Huntington’s disease (HD) is an inherited neurodegenerative
disorder, characterized by the gradual, irreversible impair-
ment of psychological, motor, and cognitive functions. Symp-
toms typically appear in middle age, but onset can occur at
almost any age. The course of the disease can last 15 to 20
years. The molecular basis of the disease is becoming better
understood. The genetic mutation underlying HD has been
traced to a gene encoding a protein (M
r
350,000) of unknown
function. In individuals who will not develop HD, a region of
the gene that encodes the amino terminus of the protein has
a sequence of CAG codons (for glutamine) that is repeated
6 to 39 times in succession. In individuals with adult-onset
HD, this codon is typically repeated 40 to 55 times. In indi-
viduals with childhood-onset HD, this codon is repeated more
than 70 times. The length of this simple trinucleotide repeat
indicates whether an individual will develop HD, and at ap-
proximately what age the first symptoms will occur.
A small portion of the amino-terminal coding sequence
of the 3,143-codon HD gene is given below. The nucleotide
sequence of the DNA is shown in black, the amino acid se-
quence corresponding to the gene is shown in blue, and the
CAG repeat is shaded. Using Figure 27–7 to translate the ge-
netic code, outline a PCR-based test for HD that could be car-
ried out using a blood sample. Assume the PCR primer must
be 25 nucleotides long. By convention, unless otherwise spec-
ified a DNA sequence encoding a protein is displayed with
the coding strand (the sequence identical to the mRNA tran-
scribed from the gene) on top such that it is read 5H11032 to 3H11032,
left to right.
Source: The Huntington’s Disease Collaborative Research Group. (1993) A novel gene
containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease
chromosomes. Cell 72, 971–983.
6. Using PCR to Detect Circular DNA Molecules In
a species of ciliated protist, a segment of genomic DNA is
sometimes deleted. The deletion is a genetically programmed
reaction associated with cellular mating. A researcher pro-
poses that the DNA is deleted in a type of recombination
called site-specific recombination, with the DNA on either end
of the segment joined together and the deleted DNA ending
up as a circular DNA reaction product.
proposed
reaction
ATGGCGACCCTGGAAAAGCTGATGAAGGCCTTCGAGTCCCTCAAGTCCTTC
M
307
1 ATLEKLMKAFESLKS
CAGCAGTTCCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAG
Q
358
18 QFQQQQQQQQQQQQQQ
F
CAGCAGCAGCAGCAGCAGCAGCAACAGCCGCCACCGCCGCCGCCGCCGCCG
Q
409
35 QQQQQQQQPPPPPP PP
CCGCCTCCTCAGCTTCCTCAGCCGCCGCCG
P
460
52 PPQLPQPPP
750
Electrophoresis
1234
3,000
1,000
250
Nucleotide
length
500
1,500
5,000
8885d_c09_306-342 2/7/04 8:14 AM Page 341 mac76 mac76:385_reb:
Chapter 9 DNA-Based Information Technologies342
Suggest how the researcher might use the polymerase chain
reaction (PCR) to detect the presence of the circular form of
the deleted DNA in an extract of the protist.
7. RFLP Analysis for Paternity Testing DNA finger-
printing and RFLP analysis are often used to test for pater-
nity. A child inherits chromosomes from both mother and
father, so DNA from a child displays restriction fragments de-
rived from each parent. In the gel shown here, which child,
if any, can be excluded as being the biological offspring of the
putative father? Explain your reasoning. Lane M is the sam-
ple from the mother, F from the putative father, and C1, C2,
and C3 from the children.
8. Mapping a Chromosome Segment A group of over-
lapping clones, designated A through F, is isolated from one
region of a chromosome. Each of the clones is separately
cleaved by a restriction enzyme and the pieces resolved by
agarose gel electrophoresis, with the results shown in the fig-
ure below. There are nine different restriction fragments in
this chromosomal region, with a subset appearing in each
clone. Using this information, deduce the order of the re-
striction fragments in the chromosome.
9. Cloning in Plants The strategy outlined in Figure
9–28 employs Agrobacterium cells that contain two separate
plasmids. Suggest why the sequences on the two plasmids are
not combined on one plasmid.
10. DNA Fingerprinting and RFLP Analysis DNA is ex-
tracted from the blood cells of two different humans, indi-
viduals 1 and 2. In separate experiments, the DNA from each
individual is cleaved by restriction endonucleases A, B, and
C, and the fragments separated by electrophoresis. A hypo-
thetical map of a 10,000 bp segment of a human chromosome
is shown (1 kbp H11005 1,000 bp). Individual 2 has point muta-
tions that eliminate restriction recognition sites B* and C*.
You probe the gel with a radioactive oligonucleotide comple-
mentary to the indicated sequence and expose a piece of x-
ray film to the gel. Indicate where you would expect to see
bands on the film. The lanes of the gel are marked in the ac-
companying diagram.
11. Use of Photolithography to Make a DNA Microar-
ray Figure 9–21 shows the first steps in the process of mak-
ing a DNA microarray, or DNA chip, using photolithography.
Describe the remaining steps needed to obtain the desired
sequences (a different four-nucleotide sequence on each of
the four spots) shown in the first panel of the figure. After
each step, give the resulting nucleotide sequence attached at
each spot.
12. Cloning in Mammals The retroviral vectors described
in Figure 9–32 make possible the efficient integration of for-
eign DNA into a mammalian genome. Explain how these vec-
tors, which lack genes for replication and viral packaging
(gag, pol, env), are assembled into infectious viral particles.
Suggest why it is important that these vectors lack the repli-
cation and packaging genes.
C
*
B
probe
B
*
AA
0kbp
B
1
C
2 345678
C
910
M 12 12 12
M 12 12 12
10
9
8
7
6
5
4
3
2
1
A B C
ABC
Electrophoresis
ABCDEF
Nine
restriction
fragments
1
2
3
4
5
6
7
8
9
Overlapping clones
Electrophoresis
M F C1 C3C2
8885d_c09_306-342 2/7/04 8:14 AM Page 342 mac76 mac76:385_reb:
chapter
B
iological lipids are a chemically diverse group of com-
pounds, the common and defining feature of which
is their insolubility in water. The biological functions of
the lipids are as diverse as their chemistry. Fats and oils
are the principal stored forms of energy in many or-
ganisms. Phospholipids and sterols are major structural
elements of biological membranes. Other lipids, al-
though present in relatively small quantities, play cru-
cial roles as enzyme cofactors, electron carriers, light-
absorbing pigments, hydrophobic anchors for proteins,
“chaperones” to help membrane proteins fold, emulsi-
fying agents in the digestive tract, hormones, and
intracellular messengers. This chapter introduces rep-
resentative lipids of each type, with emphasis on their
chemical structure and physical properties. We discuss
the energy-yielding oxidation of lipids in Chapter 17 and
their synthesis in Chapter 21.
10.1 Storage Lipids
The fats and oils used almost universally as stored forms
of energy in living organisms are derivatives of fatty
acids. The fatty acids are hydrocarbon derivatives, at
about the same low oxidation state (that is, as highly
reduced) as the hydrocarbons in fossil fuels. The cellu-
lar oxidation of fatty acids (to CO
2
and H
2
O), like the
controlled, rapid burning of fossil fuels in internal com-
bustion engines, is highly exergonic.
We introduce here the structures and nomenclature
of the fatty acids most commonly found in living or-
ganisms. Two types of fatty acid–containing compounds,
triacylglycerols and waxes, are described to illustrate
the diversity of structure and physical properties in this
family of compounds.
Fatty Acids Are Hydrocarbon Derivatives
Fatty acids are carboxylic acids with hydrocarbon chains
ranging from 4 to 36 carbons long (C
4
to C
36
). In some
fatty acids, this chain is unbranched and fully saturated
(contains no double bonds); in others the chain con-
tains one or more double bonds (Table 10–1). A few
contain three-carbon rings, hydroxyl groups, or methyl-
group branches. A simplified nomenclature for these
compounds specifies the chain length and number of
double bonds, separated by a colon; for example, the
16-carbon saturated palmitic acid is abbreviated 16:0,
and the 18-carbon oleic acid, with one double bond, is
18:1. The positions of any double bonds are specified by
superscript numbers following H9004 (delta); a 20-carbon
fatty acid with one double bond between C-9 and C-10
(C-1 being the carboxyl carbon) and another between
C-12 and C-13 is designated 20:2(H9004
9,12
). The most
commonly occurring fatty acids have even numbers
of carbon atoms in an unbranched chain of 12 to 24
carbons (Table 10–1). As we shall see in Chapter 21, the
even number of carbons results from the mode of
LIPIDS
10.1 Storage Lipids 343
10.2 Structural Lipids in Membranes 348
10.3 Lipids as Signals, Cofactors, and Pigments 357
10.4 Working with Lipids 363
The fatty substance, separated from the salifiable bases,
was dissolved in boiling alcohol. On cooling, it was
obtained crystallized and very pure, and in this state it
was examined. As it has not been hitherto described . . .
I purpose to call it margarine, from the Greek word
signifying pearl, because one of its characters is to have
the appearance of mother of pearl, which it communicates
to several of the combinations of which it forms with the
salifiable bases.
—Michel-Eugène Chevreul,
article in Philosophical Magazine, 1814
10
343
8885d_c10_343-368 1/12/04 1:06 PM Page 343 mac76 mac76:385_reb:
synthesis of these compounds, which involves conden-
sation of two-carbon (acetate) units.
There is also a common pattern in the location
of double bonds; in most monounsaturated fatty acids
the double bond is between C-9 and C-10 (H9004
9
), and
the other double bonds of polyunsaturated fatty acids
are generally H9004
12
and H9004
15
. (Arachidonic acid is an
exception to this generalization.) The double bonds
of polyunsaturated fatty acids are almost never con-
jugated (alternating single and double bonds, as in
OCHUCHOCHUCHO), but are separated by a meth-
ylene group: OCHUCHOCH
2
OCHUCHO. In nearly
all naturally occurring unsaturated fatty acids, the dou-
ble bonds are in the cis configuration. Trans fatty acids
are produced by fermentation in the rumen of dairy an-
imals and are obtained from dairy products and meat.
They are also produced during hydrogenation of fish or
vegetable oils. Because diets high in trans fatty acids
correlate with increased blood levels of LDL (bad cho-
lesterol) and decreased HDL (good cholesterol), it is
generally recommended that one avoid large amounts
of these fatty acids. Unfortunately, French fries, dough-
nuts, and cookies tend to be high in trans fatty acids.
The physical properties of the fatty acids, and of
compounds that contain them, are largely determined
by the length and degree of unsaturation of the hydro-
carbon chain. The nonpolar hydrocarbon chain accounts
for the poor solubility of fatty acids in water. Lauric acid
(12:0, M
r
200), for example, has a solubility in water of
0.063 mg/g—much less than that of glucose (M
r
180),
which is 1,100 mg/g. The longer the fatty acyl chain and
the fewer the double bonds, the lower is the solubility
Chapter 10 Lipids344
TABLE 10–1 Some Naturally Occurring Fatty Acids: Structure, Properties, and Nomenclature
Solubility at 30 H11034C
Carbon Common name Melting
(mg/g solvent)
skeleton Structure* Systematic name
?
(derivation) point (H11034C) Water Benzene
12:0 CH
3
(CH
2
)
10
COOH n-Dodecanoic acid Lauric acid 44.2 0.063 2,600
(Latin laurus,
“laurel plant”)
14:0 CH
3
(CH
2
)
12
COOH n-Tetradecanoic acid Myristic acid 53.9 0.024 874
(Latin Myristica,
nutmeg genus)
16:0 CH
3
(CH
2
)
14
COOH n-Hexadecanoic acid Palmitic acid 63.1 0.0083 348
(Latin palma,
“palm tree”)
18:0 CH
3
(CH
2
)
16
COOH n-Octadecanoic acid Stearic acid 69.6 0.0034 124
(Greek stear,
“hard fat”)
20:0 CH
3
(CH
2
)
18
COOH n-Eicosanoic acid Arachidic acid 76.5
(Latin Arachis,
legume genus)
24:0 CH
3
(CH
2
)
22
COOH n-Tetracosanoic acid Lignoceric acid 86.0
(Latin lignum,
“wood” H11001 cera,
“wax“)
16:1(H9004
9
)CH
3
(CH
2
)
5
CHUCH(CH
2
)
7
COOH cis-9-Hexadecenoic acid Palmitoleic acid 1–0.5
18:1(H9004
9
)CH
3
(CH
2
)
7
CHUCH(CH
2
)
7
COOH cis-9-Octadecenoic acid Oleic acid 13.4
(Latin oleum, “oil”)
18:2(H9004
9,12
)CH
3
(CH
2
)
4
CHUCHCH
2
CHU cis-,cis-9,12-Octadecadienoic Linoleic acid 1–5
CH(CH
2
)
7
COOH acid (Greek linon, “flax”)
18:3(H9004
9,12,15
)CH
3
CH
2
CHUCHCH
2
CHU cis-,cis-,cis-9,12,15- H9251-Linolenic acid H1100211
CHCH
2
CHUCH(CH
2
)
7
COOH Octadecatrienoic acid
20:4(H9004
5,8,11,14
)CH
3
(CH
2
)
4
CHUCHCH
2
CHU cis-,cis-,cis-,cis-5,8,11,14- Arachidonic acid H1100249.5
CHCH
2
CHUCHCH
2
CHU Icosatetraenoic acid
CH(CH
2
)
3
COOH
*All acids are shown in their nonionized form. At pH 7, all free fatty acids have an ionized carboxylate. Note that numbering of carbon atoms begins at the carboxyl carbon.
?
The prefix n- indicates the “normal” unbranched structure. For instance, “dodecanoic” simply indicates 12 carbon atoms, which could be arranged in a variety of branched
forms; “n-dodecanoic” specifies the linear, unbranched form. For unsaturated fatty acids, the configuration of each double bond is indicated; in biological fatty acids the
configuration is almost always cis.
8885d_c10_343-368 1/12/04 1:06 PM Page 344 mac76 mac76:385_reb:
in water. The carboxylic acid group is polar (and ion-
ized at neutral pH) and accounts for the slight solubil-
ity of short-chain fatty acids in water.
Melting points are also strongly influenced by the
length and degree of unsaturation of the hydrocarbon
chain. At room temperature (25 H11034C), the saturated fatty
acids from 12:0 to 24:0 have a waxy consistency, whereas
unsaturated fatty acids of these lengths are oily liquids.
This difference in melting points is due to different de-
grees of packing of the fatty acid molecules (Fig. 10–1).
In the fully saturated compounds, free rotation around
each carbon–carbon bond gives the hydrocarbon chain
great flexibility; the most stable conformation is the fully
extended form, in which the steric hindrance of neigh-
boring atoms is minimized. These molecules can pack to-
gether tightly in nearly crystalline arrays, with atoms all
along their lengths in van der Waals contact with the
atoms of neighboring molecules. In unsaturated fatty
acids, a cis double bond forces a kink in the hydrocar-
bon chain. Fatty acids with one or several such kinks
cannot pack together as tightly as fully saturated fatty
acids, and their interactions with each other are there-
fore weaker. Because it takes less thermal energy to
disorder these poorly ordered arrays of unsaturated
fatty acids, they have markedly lower melting points
than saturated fatty acids of the same chain length
(Table 10–1).
In vertebrates, free fatty acids (unesterified fatty
acids, with a free carboxylate group) circulate in the
blood bound noncovalently to a protein carrier, serum
albumin. However, fatty acids are present in blood
plasma mostly as carboxylic acid derivatives such as es-
ters or amides. Lacking the charged carboxylate group,
these fatty acid derivatives are generally even less sol-
uble in water than are the free fatty acids.
Triacylglycerols Are Fatty Acid Esters of Glycerol
The simplest lipids constructed from fatty acids are the
triacylglycerols, also referred to as triglycerides, fats,
or neutral fats. Triacylglycerols are composed of three
fatty acids each in ester linkage with a single glycerol
(Fig. 10–2). Those containing the same kind of fatty acid
10.1 Storage Lipids 345
(a) Carboxyl
Hydrocarbon
group
chain
C
H11002
OO (b)
C
H11002
O O
Saturated
fatty acids
(c) (d)
Mixture of saturated and
unsaturated fatty acids
FIGURE 10–1 The packing of fatty acids into stable aggregates. The
extent of packing depends on the degree of saturation. (a) Two rep-
resentations of the fully saturated acid stearic acid (stearate at pH 7)
in its usual extended conformation. Each line segment of the zigzag
represents a single bond between adjacent carbons. (b) The cis dou-
ble bond (shaded) in oleic acid (oleate) does not permit rotation and
introduces a rigid bend in the hydrocarbon tail. All other bonds in the
chain are free to rotate. (c) Fully saturated fatty acids in the extended
form pack into nearly crystalline arrays, stabilized by many hydro-
phobic interactions. (d) The presence of one or more cis double bonds
interferes with this tight packing and results in less stable aggregates.
C
O
O
CH
2
13
2
O
C
O
H
CH
2
O C
O
1-Stearoyl, 2-linoleoyl, 3-palmitoyl glycerol,
a mixed triacylglycerol
Glycerol
HO
CH
2
OH
H
CH
2
OHC
C
FIGURE 10–2 Glycerol and a triacylglycerol. The mixed triacylglyc-
erol shown here has three different fatty acids attached to the glyc-
erol backbone. When glycerol has two different fatty acids at C-1 and
C-3, the C-2 is a chiral center (p. 76).
8885d_c10_343-368 1/12/04 1:06 PM Page 345 mac76 mac76:385_reb:
in all three positions are called simple triacylglycerols
and are named after the fatty acid they contain. Simple
triacylglycerols of 16:0, 18:0, and 18:1, for example, are
tristearin, tripalmitin, and triolein, respectively. Most
naturally occurring triacylglycerols are mixed; they con-
tain two or more different fatty acids. To name these
compounds unambiguously, the name and position of
each fatty acid must be specified.
Because the polar hydroxyls of glycerol and the
polar carboxylates of the fatty acids are bound in ester
linkages, triacylglycerols are nonpolar, hydrophobic mol-
ecules, essentially insoluble in water. Lipids have lower
specific gravities than water, which explains why mix-
tures of oil and water (oil-and-vinegar salad dressing, for
example) have two phases: oil, with the lower specific
gravity, floats on the aqueous phase.
Triacylglycerols Provide Stored Energy and Insulation
In most eukaryotic cells, triacylglycerols form a sepa-
rate phase of microscopic, oily droplets in the aqueous
cytosol, serving as depots of metabolic fuel. In verte-
brates, specialized cells called adipocytes, or fat cells,
store large amounts of triacylglycerols as fat droplets
that nearly fill the cell (Fig. 10–3a). Triacylglycerols are
also stored as oils in the seeds of many types of plants,
providing energy and biosynthetic precursors during
seed germination (Fig. 10–3b). Adipocytes and germi-
nating seeds contain lipases, enzymes that catalyze the
hydrolysis of stored triacylglycerols, releasing fatty
acids for export to sites where they are required as fuel.
There are two significant advantages to using tria-
cylglycerols as stored fuels, rather than polysaccharides
such as glycogen and starch. First, because the carbon
atoms of fatty acids are more reduced than those of sug-
ars, oxidation of triacylglycerols yields more than twice
as much energy, gram for gram, as the oxidation of car-
bohydrates. Second, because triacylglycerols are hy-
drophobic and therefore unhydrated, the organism that
carries fat as fuel does not have to carry the extra weight
of water of hydration that is associated with stored poly-
saccharides (2 g per gram of polysaccharide). Humans
have fat tissue (composed primarily of adipocytes) un-
der the skin, in the abdominal cavity, and in the mam-
mary glands. Moderately obese people with 15 to 20 kg
of triacylglycerols deposited in their adipocytes could
meet their energy needs for months by drawing on their
fat stores. In contrast, the human body can store less
than a day’s energy supply in the form of glycogen. Car-
bohydrates such as glucose and glycogen do offer cer-
tain advantages as quick sources of metabolic energy,
one of which is their ready solubility in water.
In some animals, triacylglycerols stored under the
skin serve not only as energy stores but as insulation
against low temperatures. Seals, walruses, penguins, and
other warm-blooded polar animals are amply padded
with triacylglycerols. In hibernating animals (bears, for
example), the huge fat reserves accumulated before
hibernation serve the dual purposes of insulation and
energy storage (see Box 17–1). The low density of tri-
acylglycerols is the basis for another remarkable func-
tion of these compounds. In sperm whales, a store of
triacylglycerols and waxes allows the animals to match
the buoyancy of their bodies to that of their surround-
ings during deep dives in cold water (Box 10–1).
Many Foods Contain Triacylglycerols
Most natural fats, such as those in vegetable oils, dairy
products, and animal fat, are complex mixtures of sim-
ple and mixed triacylglycerols. These contain a variety
of fatty acids differing in chain length and degree of sat-
uration (Fig. 10–4). Vegetable oils such as corn (maize)
and olive oil are composed largely of triacylglycerols
with unsaturated fatty acids and thus are liquids at room
temperature. They are converted industrially into solid
Chapter 10 Lipids346
8 m(a) H9262
3 H9262m
(b)
H9262
FIGURE 10–3 Fat stores in cells. (a) Cross section of four guinea pig
adipocytes, showing huge fat droplets that virtually fill the cells. Also
visible are several capillaries in cross section. (b) Cross section of a
cotyledon cell from a seed of the plant Arabidopsis. The large dark
structures are protein bodies, which are surrounded by stored oils in
the light-colored oil bodies.
8885d_c10_343-368 1/12/04 1:06 PM Page 346 mac76 mac76:385_reb:
fats by catalytic hydrogenation, which reduces some
of their double bonds to single bonds and converts oth-
ers to trans double bonds. Triacylglycerols containing
only saturated fatty acids, such as tristearin, the major
component of beef fat, are white, greasy solids at room
temperature.
When lipid-rich foods are exposed too long to the
oxygen in air, they may spoil and become rancid. The
unpleasant taste and smell associated with rancidity re-
sult from the oxidative cleavage of the double bonds in
10.1 Storage Lipids 347
Fatty acids (% of total)
Natural fats at 25 °C
Olive oil,
liquid
Butter,
soft solid
Beef fat,
hard solid
C
16
and C
18
saturated
C
16
and C
18
unsaturated
C
4
to C
14
saturated
20
40
60
80
100
FIGURE 10–4 Fatty acid composition of three food fats. Olive oil,
butter, and beef fat consist of mixtures of triacylglycerols, differing in
their fatty acid composition. The melting points of these fats—and
hence their physical state at room temperature (25H11034C)—are a direct
function of their fatty acid composition. Olive oil has a high propor-
tion of long-chain (C
16
and C
18
) unsaturated fatty acids, which ac-
counts for its liquid state at 25H11034C. The higher proportion of long-chain
(C
16
and C
18
) saturated fatty acids in butter increases its melting point,
so butter is a soft solid at room temperature. Beef fat, with an even
higher proportion of long-chain saturated fatty acids, is a hard solid.
BOX 10–1 THE WORLD OF BIOCHEMISTRY
Sperm Whales: Fatheads of the Deep
Studies of sperm whales have uncovered another way
in which triacylglycerols are biologically useful. The
sperm whale’s head is very large, accounting for over
one-third of its total body weight. About 90% of the
weight of the head is made up of the spermaceti or-
gan, a blubbery mass that contains up to 3,600 kg
(about 4 tons) of spermaceti oil, a mixture of triacyl-
glycerols and waxes containing an abundance of un-
saturated fatty acids. This mixture is liquid at the
normal resting body temperature of the whale, about
37 H11034C, but it begins to crystallize at about 31 H11034C and
becomes solid when the temperature drops several
more degrees.
The probable biological function of spermaceti oil
has been deduced from research on the anatomy and
feeding behavior of the sperm whale. These mammals
feed almost exclusively on squid in very deep water.
In their feeding dives they descend 1,000 m or more;
the deepest recorded dive is 3,000 m (almost 2 miles).
At these depths, there are no competitors for the very
plentiful squid; the sperm whale rests quietly, waiting
for schools of squid to pass.
For a marine animal to remain at a given depth
without a constant swimming effort, it must have the
same density as the surrounding water. The sperm
whale undergoes changes in buoyancy to match the
density of its surroundings—from the tropical ocean
surface to great depths where the water is much
colder and thus denser. The key is the freezing point
of spermaceti oil. When the temperature of the oil is
lowered several degrees during a deep dive, it con-
geals or crystallizes and becomes denser. Thus the
buoyancy of the whale changes to match the density
of seawater. Various physiological mechanisms pro-
mote rapid cooling of the oil during a dive. During the
return to the surface, the congealed spermaceti oil
warms and melts, decreasing its density to match that
of the surface water. Thus we see in the sperm whale
a remarkable anatomical and biochemical adaptation.
The triacylglycerols and waxes synthesized by the
sperm whale contain fatty acids of the necessary chain
length and degree of unsaturation to give the sper-
maceti oil the proper melting point for the animal’s
diving habits.
Unfortunately for the sperm whale population,
spermaceti oil was at one time considered the finest
lamp oil and continues to be commercially valuable as
a lubricant. Several centuries of intensive hunting of
these mammals have driven sperm whales onto the
endangered species list.
Spermaceti
organ
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unsaturated fatty acids, which produces aldehydes and
carboxylic acids of shorter chain length and therefore
higher volatility.
Waxes Serve as Energy Stores and Water Repellents
Biological waxes are esters of long-chain (C
14
to C
36
)
saturated and unsaturated fatty acids with long-chain
(C
16
to C
30
) alcohols (Fig. 10–5). Their melting points
(60 to 100 H11034C) are generally higher than those of tria-
cylglycerols. In plankton, the free-floating microorgan-
isms at the bottom of the food chain for marine animals,
waxes are the chief storage form of metabolic fuel.
Waxes also serve a diversity of other functions re-
lated to their water-repellent properties and their firm
consistency. Certain skin glands of vertebrates secrete
waxes to protect hair and skin and keep it pliable, lu-
bricated, and waterproof. Birds, particularly waterfowl,
secrete waxes from their preen glands to keep their
feathers water-repellent. The shiny leaves of holly,
rhododendrons, poison ivy, and many tropical plants
are coated with a thick layer of waxes, which prevents
excessive evaporation of water and protects against
parasites.
Biological waxes find a variety of applications in the
pharmaceutical, cosmetic, and other industries. Lanolin
(from lamb’s wool), beeswax (Fig. 10–5), carnauba wax
(from a Brazilian palm tree), and wax extracted from
spermaceti oil (from whales; see Box 10–1) are widely
used in the manufacture of lotions, ointments, and
polishes.
SUMMARY 10.1 Storage Lipids
■ Lipids are water-insoluble cellular components
of diverse structure that can be extracted by
nonpolar solvents.
■ Almost all fatty acids, the hydrocarbon
components of many lipids, have an even
number of carbon atoms (usually 12 to 24); they
are either saturated or unsaturated, with double
bonds almost always in the cis configuration.
■ Triacylglycerols contain three fatty acid
molecules esterified to the three hydroxyl
groups of glycerol. Simple triacylglycerols
contain only one type of fatty acid; mixed
triacylglycerols, two or three types.
Triacylglycerols are primarily storage fats;
they are present in many foods.
10.2 Structural Lipids in Membranes
The central architectural feature of biological mem-
branes is a double layer of lipids, which acts as a bar-
rier to the passage of polar molecules and ions. Mem-
brane lipids are amphipathic: one end of the molecule
is hydrophobic, the other hydrophilic. Their hydropho-
bic interactions with each other and their hydrophilic
interactions with water direct their packing into sheets
called membrane bilayers. In this section we describe
five general types of membrane lipids: glycerophospho-
lipids, in which the hydrophobic regions are composed
of two fatty acids joined to glycerol; galactolipids and
sulfolipids, which also contain two fatty acids esterified
to glycerol, but lack the characteristic phosphate of phos-
pholipids; archaebacterial tetraether lipids, in which two
very long alkyl chains are ether-linked to glycerol at both
ends; sphingolipids, in which a single fatty acid is joined
to a fatty amine, sphingosine; and sterols, compounds
characterized by a rigid system of four fused hydrocar-
bon rings.
The hydrophilic moieties in these amphipathic com-
pounds may be as simple as a single OOH group at
one end of the sterol ring system, or they may be much
more complex. In glycerophospholipids and some sphin-
golipids, a polar head group is joined to the hydropho-
bic moiety by a phosphodiester linkage; these are the
phospholipids. Other sphingolipids lack phosphate but
have a simple sugar or complex oligosaccharide at their
polar ends; these are the glycolipids (Fig. 10–6). Within
these groups of membrane lipids, enormous diversity re-
sults from various combinations of fatty acid “tails” and
polar “heads.” The arrangement of these lipids in mem-
branes, and their structural and functional roles therein,
are considered in the next chapter.
Chapter 10 Lipids348
FIGURE 10–5 Biological wax. (a) Triacontanoylpalmitate, the major
component of beeswax, is an ester of palmitic acid with the alcohol
triacontanol. (b) A honeycomb, constructed of beeswax, is firm at
25H11034C and completely impervious to water. The term “wax” originates
in the Old English weax, meaning “the material of the honeycomb.”
(b)
CH
3
(CH
2
)
14
C
O
O CH
2
(CH
2
)
28
CH
3
Palmitic acid
(a)
1-Triacontanol
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Glycerophospholipids Are Derivatives of
Phosphatidic Acid
Glycerophospholipids, also called phosphoglycerides,
are membrane lipids in which two fatty acids are attached
in ester linkage to the first and second carbons of glyc-
erol, and a highly polar or charged group is attached
through a phosphodiester linkage to the third carbon.
Glycerol is prochiral; it has no asymmetric carbons, but
attachment of phosphate at one end converts it into a
chiral compound, which can be correctly named either
L-glycerol 3-phosphate, D-glycerol 1-phosphate, or sn-
glycerol 3-phosphate (Fig. 10–7). Glycerophospholipids
are named as derivatives of the parent compound, phos-
phatidic acid (Fig. 10–8), according to the polar alcohol
in the head group. Phosphatidylcholine and phosphati-
dylethanolamine have choline and ethanolamine in their
polar head groups, for example. In all these compounds,
the head group is joined to glycerol through a phos-
phodiester bond, in which the phosphate group bears
a negative charge at neutral pH. The polar alcohol
may be negatively charged (as in phosphatidylinositol
4,5-bisphosphate), neutral (phosphatidylserine), or pos-
itively charged (phosphatidylcholine, phosphatidylethan-
olamine). As we shall see in Chapter 11, these charges
contribute greatly to the surface properties of membranes.
The fatty acids in glycerophospholipids can be any
of a wide variety, so a given phospholipid (phospha-
tidylcholine, for example) may consist of a number of
molecular species, each with its unique complement of
fatty acids. The distribution of molecular species is spe-
cific for different organisms, different tissues of the
same organism, and different glycerophospholipids in
the same cell or tissue. In general, glycerophospholipids
contain a C
16
or C
18
saturated fatty acid at C-1 and a
C
18
to C
20
unsaturated fatty acid at C-2. With few ex-
ceptions, the biological significance of the variation in
fatty acids and head groups is not yet understood.
Some Phospholipids Have Ether-Linked Fatty Acids
Some animal tissues and some unicellular organisms are
rich in ether lipids, in which one of the two acyl chains
is attached to glycerol in ether, rather than ester, link-
age. The ether-linked chain may be saturated, as in the
alkyl ether lipids, or may contain a double bond between
C-1 and C-2, as in plasmalogens (Fig. 10–9). Vertebrate
heart tissue is uniquely enriched in ether lipids; about
half of the heart phospholipids are plasmalogens. The
membranes of halophilic bacteria, ciliated protists, and
certain invertebrates also contain high proportions of
10.2 Structural Lipids in Membranes 349
Storage
lipids
(neutral)
Membrane lipids (polar)
Phospholipids Glycolipids
Triacylglycerols Glycerophospholipids Sphingolipids Sphingolipids
Alcohol
Sphingosine
Archaebacterial ether lipids
Glycerol Glycerol
Fatty acid Fatty acid
Fatty acid Fatty acid Fatty acid Fatty acid
Fatty acid Diphytanyl
Fatty acid
Fatty acid PO
4
PO
4
PO
4
PO
4
Choline
Sphingosine
Glycerol
Glycerol
Diphytanyl
Glycerol
(SO
4
)
Glycerol
Galactolipids (sulfolipids)
Mono- or
oligosaccharide
Mono- or
disaccharide
( ether linkage)
FIGURE 10–6 Some common types of storage and membrane lipids.
All the lipid types shown here have either glycerol or sphingosine as
the backbone (pink screen), to which are attached one or more long-
chain alkyl groups (yellow) and a polar head group (blue). In triacyl-
glycerols, glycerophospholipids, galactolipids, and sulfolipids, the
alkyl groups are fatty acids in ester linkage. Sphingolipids contain a
single fatty acid, in amide linkage to the sphingosine backbone. The
membrane lipids of archaebacteria are variable; that shown here has
two very long, branched alkyl chains, each end in ether linkage with
a glycerol moiety. In phospholipids the polar head group is joined
through a phosphodiester, whereas glycolipids have a direct glycosidic
linkage between the head-group sugar and the backbone glycerol.
1
CH
2
OH
2
COHH
3
CH
2
O O
H11002
O
O
H11002
P
L-Glycerol 3-phosphate
(sn-glycerol 3-phosphate)
FIGURE 10–7 L-Glycerol 3-phosphate, the backbone of phospho-
lipids. Glycerol itself is not chiral, as it has a plane of symmetry through
C-2. However, glycerol can be converted to a chiral compound by
adding a substituent such as phosphate to either of the OCH
2
OH
groups; that is, glycerol is prochiral. One unambiguous nomenclature
for glycerol phosphate is the DL system (described on p. 77), in which
the isomers are named according to their stereochemical relationships
to glyceraldehyde isomers. By this system, the stereoisomer of glycerol
phosphate found in most lipids is correctly named either L-glycerol
3-phosphate or D-glycerol 1-phosphate. Another way to specify
stereoisomers is the stereospecific numbering (sn) system, in which
C-1 is, by definition, that group of the prochiral compound that oc-
cupies the pro-S position. The common form of glycerol phosphate in
phospholipids is, by this system, sn-glycerol 3-phosphate.
8885d_c10_349 1/16/04 8:18 AM Page 349 mac76 mac76:385_reb:
ether lipids. The functional significance of ether lipids in
these membranes is unknown; perhaps their resistance
to the phospholipases that cleave ester-linked fatty acids
from membrane lipids is important in some roles.
At least one ether lipid, platelet-activating
factor, is a potent molecular signal. It is released
from leukocytes called basophils and stimulates platelet
aggregation and the release of serotonin (a vasocon-
strictor) from platelets. It also exerts a variety of effects
on liver, smooth muscle, heart, uterine, and lung tissues
and plays an important role in inflammation and the
allergic response. ■
Chapter 10 Lipids350
Glycerophospholipid
Head-group
(general structure)
substituent
2
C
1
CH
2
O C
O
3
CH
2
O P
O
O
H11002
O X
H O
O
C
Saturated fatty acid
(e.g., palmitic acid)
Unsaturated fatty acid
(e.g., oleic acid)
Name of X Formula of X
Net charge
(at pH 7)
H H110021
Ethanolamine 0
Choline 0
Serine H110021
Glycerol H110021
myo-Inositol 4,5-
bisphosphate
H110024
Phosphatidyl- H110022
glycerol
CH
2
CH
2
N
H11001
H
3
CH
2
CH
2
N
H11001
(CH
3
)
3
CH
2
C
COO
H11002
H N
H11001
H
3
H
OH
HO
C
C
CH
2
HOH
H
2
O P
O
O
H11002
O CH
2
C
CH
2
O C
O
R
2
H O C
O
R
1
OH
H
H
H
OP
OP
HH
HO
2
HCHC
2
1
23
4
56
HC
OH
Name of
glycerophospholipid
Phosphatidic acid
Phosphatidylethanolamine
Phosphatidylcholine
Phosphatidylserine
Phosphatidylglycerol
Phosphatidylinositol
4,5-bisphosphate
Cardiolipin
FIGURE 10–8 Glycerophospholipids. The common glycerophospho-
lipids are diacylglycerols linked to head-group alcohols through a
phosphodiester bond. Phosphatidic acid, a phosphomonoester, is the
parent compound. Each derivative is named for the head-group alco-
hol (X), with the prefix “phosphatidyl-.” In cardiolipin, two phospha-
tidic acids share a single glycerol.
8885d_c10_343-368 1/12/04 1:06 PM Page 350 mac76 mac76:385_reb:
Chloroplasts Contain Galactolipids and Sulfolipids
The second group of membrane lipids are those that
predominate in plant cells: the galactolipids, in which
one or two galactose residues are connected by a gly-
cosidic linkage to C-3 of a 1,2-diacylglycerol (Fig. 10–10;
see also Fig. 10–6). Galactolipids are localized in the
thylakoid membranes (internal membranes) of chloro-
plasts; they make up 70% to 80% of the total membrane
lipids of a vascular plant. They are probably the most
abundant membrane lipids in the biosphere. Phosphate
is often the limiting plant nutrient in soil, and perhaps
the evolutionary pressure to conserve phosphate for
more critical roles favored plants that made phosphate-
free lipids. Plant membranes also contain sulfolipids, in
which a sulfonated glucose residue is joined to a di-
acylglycerol in glycosidic linkage. In sulfolipids, the sul-
fonate on the head group bears a fixed negative charge
like that of the phosphate group in phospholipids (Fig.
10–10).
10.2 Structural Lipids in Membranes 351
ether-linked alkene
2
C
1
CH
2
O C
H
C
H
H O C
O
3
CH
2
O
O P
H11002
O
O CH
2
CH
2
N
H11001
(CH
3
)
3
Plasmalogen
choline
O P
O
3
C
2
C
1
CH
2
O CH
2
CH
2
H O C
O
CH
3
H
2
H11002
O
O CH
2
CH
2
N
H11001
(CH
3
)
3
acetyl ester
Platelet-activating factor
ether-linked alkane
choline
FIGURE 10–9 Ether lipids. Plasmalogens have an ether-linked alkenyl
chain where most glycerophospholipids have an ester-linked fatty acid
(compare Fig. 10–8). Platelet-activating factor has a long ether-linked
alkyl chain at C-1 of glycerol, but C-2 is ester-linked to acetic acid,
which makes the compound much more water-soluble than most glyc-
erophospholipids and plasmalogens. The head-group alcohol is choline
in plasmalogens and in platelet-activating factor.
Monogalactosyldiacylglycerol
(MGDG)
CH
2
OH
CH
2
CH
HO
H
O
O
O
O C
O
O CCH
2
H
H
H
H
OH
OH
6-Sulfo-6-deoxy-H9251-D-glucopyranosyldiacylglycerol
(a sulfolipid)
CH
2
CH
2
CH
HO
O
O
O
O C
SO O
O
H11002
O
O CCH
2
H
H H
H
H
OH
OH
CH
2
OH
CH
2
HO
H
O
O
H
H
H
H
OH
OH
Digalactosyldiacylglycerol
(DGDG)
CH
2
CH
HO
H
O
O
O
O C
O
O CCH
2
H
H
H
H
OH
OH
FIGURE 10–10 Three glycolipids of chloroplast
membranes. In monogalactosyldiacylglycerols
(MGDGs) and digalactosyldiacylglycerols
(DGDGs), almost all the acyl groups are derived
from linoleic acid (18:2(H9004
9,12
)) and the head
groups are uncharged. In the sulfolipid 6-sulfo-
6-deoxy-H9251-D-glucopyranosyldiacylglycerol, the
sulfonate carries a fixed negative charge.
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Archaebacteria Contain Unique Membrane Lipids
The archaebacteria, most of which live in ecological
niches with extreme conditions—high temperatures
(boiling water), low pH, high ionic strength, for ex-
ample—have membrane lipids containing long-chain
(32 carbons) branched hydrocarbons linked at each end
to glycerol (Fig. 10–11). These linkages are through
ether bonds, which are much more stable to hydrolysis
at low pH and high temperature than are the ester bonds
found in the lipids of eubacteria and eukaryotes. In their
fully extended form, these archaebacterial lipids are
twice the length of phospholipids and sphingolipids and
span the width of the surface membrane. At each end
of the extended molecule is a polar head consisting of
glycerol linked to either phosphate or sugar residues.
The general name for these compounds, glycerol dialkyl
glycerol tetraethers (GDGTs), reflects their unique
structure. The glycerol moiety of the archaebacterial
lipids is not the same stereoisomer as that in the lipids
of eubacteria and eukaryotes; the central carbon is in
the R configuration in archaebacteria, in the S configu-
ration in the other kingdoms (Fig. 10–7).
Sphingolipids Are Derivatives of Sphingosine
Sphingolipids, the fourth large class of membrane
lipids, also have a polar head group and two nonpolar
tails, but unlike glycerophospholipids and galactolipids
they contain no glycerol. Sphingolipids are composed of
one molecule of the long-chain amino alcohol sphingo-
sine (also called 4-sphingenine) or one of its derivatives,
one molecule of a long-chain fatty acid, and a polar head
group that is joined by a glycosidic linkage in some cases
and by a phosphodiester in others (Fig. 10–12).
Carbons C-1, C-2, and C-3 of the sphingosine mol-
ecule are structurally analogous to the three carbons of
glycerol in glycerophospholipids. When a fatty acid is
attached in amide linkage to the ONH
2
on C-2, the re-
sulting compound is a ceramide, which is structurally
similar to a diacylglycerol. Ceramide is the structural
parent of all sphingolipids.
There are three subclasses of sphingolipids, all de-
rivatives of ceramide but differing in their head groups:
sphingomyelins, neutral (uncharged) glycolipids, and
gangliosides. Sphingomyelins contain phosphocholine
or phosphoethanolamine as their polar head group and
are therefore classified along with glycerophospholipids
as phospholipids (Fig. 10–6). Indeed, sphingomyelins
resemble phosphatidylcholines in their general proper-
ties and three-dimensional structure, and in having no
net charge on their head groups (Fig. 10–13). Sphingo-
myelins are present in the plasma membranes of animal
cells and are especially prominent in myelin, a mem-
branous sheath that surrounds and insulates the axons
of some neurons—thus the name “sphingomyelins.”
Glycosphingolipids, which occur largely in the
outer face of plasma membranes, have head groups with
one or more sugars connected directly to the OOH at
C-1 of the ceramide moiety; they do not contain phos-
phate. Cerebrosides have a single sugar linked to ce-
ramide; those with galactose are characteristically found
in the plasma membranes of cells in neural tissue, and
those with glucose in the plasma membranes of cells in
nonneural tissues. Globosides are neutral (uncharged)
glycosphingolipids with two or more sugars, usually D-
glucose, D-galactose, or N-acetyl-D-galactosamine. Cere-
brosides and globosides are sometimes called neutral
glycolipids, as they have no charge at pH 7.
Gangliosides, the most complex sphingolipids,
have oligosaccharides as their polar head groups and one
or more residues of N-acetylneuraminic acid (Neu5Ac),
a sialic acid (often simply called “sialic acid”), at the
Chapter 10 Lipids352
OH
2
C
CH
2
CH
2
CH
2
C
H
O
H
2
C
HC
O
CH
2
OH
O HCOH
P
H9251Glc(H92521—?2)Gal-1
Glycerol
Diphytanyl groups
Glycerol phosphate
Glycerol
O
H11002
OO
O
O
2 1
3
FIGURE 10–11 A typical membrane lipid of archaebacteria. In this
diphytanyl tetraether lipid, the diphytanyl moieties (yellow) are long
hydrocarbons composed of eight five-carbon isoprene groups con-
densed end-to-end (on the condensation of isoprene units, see Fig.
21–36; also, compare the diphytanyl groups with the 20-carbon phy-
tol side chain of chlorophylls in Fig. 19–40a). In this extended form,
the diphytanyl groups are about twice the length of a 16-carbon fatty
acid typically found in the membrane lipids of eubacteria and eu-
karyotes. The glycerol moieties in the archaebacterial lipids are in the
R configuration, in contrast to those of eubacteria and eukaryotes,
which have the S configuration. Archaebacterial lipids differ in the sub-
stituents on the glycerols. In the molecule shown here, one glycerol is
linked to the disaccharide H9251-glucopyranosyl-(1n2)-H9252-galactofuranose;
the other glycerol is linked to a glycerol phosphate head group.
8885d_c10_343-368 1/12/04 1:06 PM Page 352 mac76 mac76:385_reb:
termini. Sialic acid gives gangliosides the negative charge
at pH 7 that distinguishes them from globosides.
Gangliosides with one sialic acid residue are in the GM
(M for mono-) series, those with two are in the GD (D
for di-) series, and so on (GT, three sialic acid residues;
GQ, four).
H
OH
COO
H11002
O
C
OH
H C
OH
H CH
2
OH
HN C
O
CH
3
N-Acetylneuraminic acid (a sialic acid)
(Neu5Ac)
H
H
H
HOH
Sphingolipids at Cell Surfaces Are Sites of
Biological Recognition
When sphingolipids were discovered a century ago by
the physician-chemist Johann Thudichum, their biolog-
ical role seemed as enigmatic as the Sphinx, for which
he therefore named them. In humans, at least 60 dif-
ferent sphingolipids have been identified in cellular
membranes. Many of these are especially prominent in
the plasma membranes of neurons, and some are clearly
recognition sites on the cell surface, but a specific func-
tion for only a few sphingolipids has been discovered
thus far. The carbohydrate moieties of certain sphin-
golipids define the human blood groups and therefore
determine the type of blood that individuals can safely
receive in blood transfusions (Fig. 10–14).
10.2 Structural Lipids in Membranes 353
Sphingolipid
Sphingosine
Fatty acid
(general
structure)
2
C
HO
3
CH CH CH (CH
2
)
12
CH
3
1
CH
2
O X
H N
H
C
O
Name of X Formula of XName of sphingolipid
Ceramide
PhosphocholineSphingomyelin
Neutral glycolipids
Glucosylcerebroside Glucose
Di-, tri-, or
tetrasaccharide
Lactosylceramide
(a globoside)
Complex
oligosaccharide
Ganglioside GM2
P
O
O
H11002
O CH
2
CH
2
N
H11001
(CH
3
)
3
CH
2
OH
HO
Glc
GalGlc
Gal
GalNAc
Neu5Ac
H
H
OH
H
O
OH
H
H
H
FIGURE 10–12 Sphingolipids. The first three carbons at the polar end
of sphingosine are analogous to the three carbons of glycerol in glyc-
erophospholipids. The amino group at C-2 bears a fatty acid in amide
linkage. The fatty acid is usually saturated or monounsaturated, with
16, 18, 22, or 24 carbon atoms. Ceramide is the parent compound
for this group. Other sphingolipids differ in the polar head group (X)
attached at C-1. Gangliosides have very complex oligosaccharide head
groups. Standard abbreviations for sugars are used in this figure: Glc,
D-glucose; Gal, D-galactose; GalNAc, N-acetyl-D-galactosamine;
Neu5Ac, N-acetylneuraminic acid (sialic acid).
Johann Thudichum,
1829–1901
8885d_c10_343-368 1/12/04 1:06 PM Page 353 mac76 mac76:385_reb:
Gangliosides are concentrated in the outer surface
of cells, where they present points of recognition for ex-
tracellular molecules or surfaces of neighboring cells.
The kinds and amounts of gangliosides in the plasma
membrane change dramatically during embryonic de-
velopment. Tumor formation induces the synthesis of a
new complement of gangliosides, and very low concen-
trations of a specific ganglioside have been found to in-
duce differentiation of cultured neuronal tumor cells.
Investigation of the biological roles of diverse ganglio-
sides remains fertile ground for future research.
Phospholipids and Sphingolipids Are Degraded in
Lysosomes
Most cells continually degrade and replace their mem-
brane lipids. For each hydrolyzable bond in a glyc-
erophospholipid, there is a specific hydrolytic enzyme
in the lysosome (Fig. 10–15). Phospholipases of the A
type remove one of the two fatty acids, producing a
lysophospholipid. (These esterases do not attack the
ether link of plasmalogens.) Lysophospholipases remove
the remaining fatty acid.
Gangliosides are degraded by a set of lysosomal en-
zymes that catalyze the stepwise removal of sugar units,
finally yielding a ceramide. A genetic defect in any of
these hydrolytic enzymes leads to the accumulation of
gangliosides in the cell, with severe medical conse-
quences (Box 10–2).
Sterols Have Four Fused Carbon Rings
Sterols are structural lipids present in the membranes
of most eukaryotic cells. The characteristic structure of
this fifth group of membrane lipids is the steroid nu-
Chapter 10 Lipids354
CH
3
N
CH
3
CH
3
H11001
C
CH
2
O
O
O
H11002
H
2 O C
P
H
2
CH
NH
O
C
OH
H
C
Sphingomyelin
NCH
3
CH
3
H11001
C
CH
2
O
O
O
H11002
H
2
O
H
2
Phosphatidylcholine
CH
O C
O
C C
O
P
H
2
O
C
CH
3
FIGURE 10–13 The similarities in shape and in molecular structure
of phosphatidylcholine (a glycerophospholipid) and sphingomyelin (a
sphingolipid) are clear when their space-filling and structural formu-
las are drawn as here.
Ceramide
Gal
O Antigen
A Antigen
B Antigen
Sphingosine
Fatty acid
Glc Gal GalNAc Gal
Fuc
GalNAc
FIGURE 10–14 Glycosphingolipids as determinants of blood
groups. The human blood groups (O, A, B) are determined in
part by the oligosaccharide head groups (blue) of these glycosphin-
golipids. The same three oligosaccharides are also found attached to
certain blood proteins of individuals of blood types O, A, and B, re-
spectively. (Fuc represents the sugar fucose.)
8885d_c10_343-368 1/12/04 1:06 PM Page 354 mac76 mac76:385_reb:
cleus, consisting of four fused rings, three with six car-
bons and one with five (Fig. 10–16). The steroid nucleus
is almost planar and is relatively rigid; the fused rings
do not allow rotation about COC bonds. Cholesterol,
the major sterol in animal tissues, is amphipathic, with
a polar head group (the hydroxyl group at C-3) and a
nonpolar hydrocarbon body (the steroid nucleus and the
hydrocarbon side chain at C-17), about as long as a 16-
carbon fatty acid in its extended form. Similar sterols
are found in other eukaryotes: stigmasterol in plants and
ergosterol in fungi, for example. Bacteria cannot syn-
thesize sterols; a few bacterial species, however, can in-
corporate exogenous sterols into their membranes. The
sterols of all eukaryotes are synthesized from simple five-
carbon isoprene subunits, as are the fat-soluble vitamins,
quinones, and dolichols described in Section 10.3.
In addition to their roles as membrane constituents,
the sterols serve as precursors for a variety of products
with specific biological activities. Steroid hormones, for
example, are potent biological signals that regulate gene
expression. Bile acids are polar derivatives of choles-
terol that act as detergents in the intestine, emulsifying
dietary fats to make them more readily accessible to di-
gestive lipases. We return to cholesterol and other sterols
in later chapters, to consider the structural role of cho-
lesterol in biological membranes (Chapter 11), signal-
ing by steroid hormones (Chapter 12), the remarkable
biosynthetic pathway to cholesterol, and the transport
of cholesterol by lipoprotein carriers (Chapter 21).
SUMMARY 10.2 Structural Lipids in Membranes
■ The polar lipids, with polar heads and nonpolar
tails, are major components of membranes. The
most abundant are the glycerophospholipids,
which contain fatty acids esterified to two of
the hydroxyl groups of glycerol, and a second
alcohol, the head group, esterified to the third
hydroxyl of glycerol via a phosphodiester bond.
Other polar lipids are the sterols.
■ Glycerophospholipids differ in the structure of
their head group; common glycerophospholipids
are phosphatidylethanolamine and
phosphatidylcholine. The polar heads of the
glycerophospholipids carry electric charges at
pH near 7.
■ Chloroplast membranes are remarkably rich in
galactolipids, composed of a diacylglycerol with
10.2 Structural Lipids in Membranes 355
Phospholipase A
1
Phospholipase C
Phospholipase A
2
Phospholipase D
O P
O
3
C
2
C
1
CH
2
O C
O
H O C
O
H
2
O
H11002
O
H
OH
HOOH
H
H
H
OP
OP
HH
FIGURE 10–15 The specificities of phospholipases. Phospholipases
A
1
and A
2
hydrolyze the ester bonds of intact glycerophospholipids
at C-1 and C-2 of glycerol, respectively. Phospholipases C and D
each split one of the phosphodiester bonds in the head group. Some
phospholipases act on only one type of glycerophospholipid, such as
phosphatidylinositol 4,5-bisphosphate (shown here) or phosphatidyl-
choline; others are less specific. When one of the fatty acids has been
removed by a type A phospholipase, the second fatty acid is cleaved
from the molecule by a lysophospholipase (not shown).
Alkyl
Polar
Steroid
side
head
nucleus
chain
20
C
22
C
23
C
24
C
25
C
19
CH
3
H
27
CH
3
H
2
H
2
H
2
H
21
CH
3
AB
C
D
HO
26
CH
3
18
CH
3
5
11
4
2
1
8
3
9
7
6
10
13
12 17
16
1514
FIGURE 10–16 Cholesterol. The stick structure of cholesterol is visi-
ble through a transparent surface contour model of the molecule (from
coordinates supplied by Dave Woodcock). In the chemical structure,
the rings are labeled A through D to simplify reference to derivatives
of the steroid nucleus, and the carbon atoms are numbered in blue.
The C-3 hydroxyl group (pink in both representations) is the polar head
group. For storage and transport of the sterol, this hydroxyl group con-
denses with a fatty acid to form a sterol ester.
HO
CH
3
OH
17
C
O
NH CH
2
CH
2
SO
3
H11002
Taurocholic acid
(a bile acid)
OH
CH
3
CH
3
8885d_c10_343-368 1/12/04 1:06 PM Page 355 mac76 mac76:385_reb:
BOX 10–2 BIOCHEMISTRY IN MEDICINE
Inherited Human Diseases Resulting from
Abnormal Accumulations of Membrane Lipids
The polar lipids of membranes undergo constant meta-
bolic turnover, the rate of their synthesis normally
counterbalanced by the rate of breakdown. The
breakdown of lipids is promoted by hydrolytic en-
zymes in lysosomes, each enzyme capable of hy-
drolyzing a specific bond. When sphingolipid degra-
dation is impaired by a defect in one of these enzymes
(Fig. 1), partial breakdown products accumulate in
the tissues, causing serious disease.
For example, Niemann-Pick disease is caused by
a rare genetic defect in the enzyme sphingomyelinase,
which cleaves phosphocholine from sphingomyelin.
Sphingomyelin accumulates in the brain, spleen, and
liver. The disease becomes evident in infants, and
causes mental retardation and early death. More com-
mon is Tay-Sachs disease, in which ganglioside GM2
accumulates in the brain and spleen (Fig. 2) owing to
lack of the enzyme hexosaminidase A. The symptoms
of Tay-Sachs disease are progressive retardation in de-
velopment, paralysis, blindness, and death by the age
of 3 or 4 years.
Genetic counseling can predict and avert many in-
heritable diseases. Tests on prospective parents can
detect abnormal enzymes, then DNA testing can de-
termine the exact nature of the defect and the risk it
poses for offspring. Once a pregnancy occurs, fetal
cells obtained by sampling a part of the placenta
(chorionic villus sampling) or the fluid surrounding
the fetus (amniocentesis) can be tested in the same
way.
H9252-galactosidase
H9251-galacto-
sidase A
H9252-galactosidase
Ceramide
GM1
Generalized gangliosidosis
glucocerebrosidase
Ceramide
Ceramide
Gaucher’s disease
hexosaminidase A
Ceramide
Tay-Sachs disease
GM2
hexosaminidase
A and B
Ceramide
Sandhoff’s disease
Globoside
Ceramide
Fabry’s disease
ganglioside
neuraminidase
Ceramide
GM3
sphingo-
myelinase
Ceramide Phosphocholine
Sphingomyelin
Phosphocholine
Niemann-Pick disease
Ceramide
Glc
Gal
GalNAc
Neu5Ac
1mH9262
FIGURE 1 Pathways for the breakdown of GM1, globoside, and
sphingomyelin to ceramide. A defect in the enzyme hydrolyzing a
particular step is indicated by H11538, and the disease that results from
accumulation of the partial breakdown product is noted.
FIGURE 2 Electron micrograph
of a portion of a brain cell from
an infant with Tay-Sachs disease,
showing abnormal ganglioside
deposits in the lysosomes.
8885d_c10_343-368 1/12/04 1:06 PM Page 356 mac76 mac76:385_reb:
one or two linked galactose residues, and
sulfolipids, diacylglycerols with a linked
sulfonated sugar residue and thus a negatively
charged head group.
■ Archaebacteria have unique membrane lipids,
with long-chain alkyl groups ether-linked to
glycerol at both ends and with sugar residues
and/or phosphate joined to the glycerol to
provide a polar or charged head group. These
lipids are stable under the harsh conditions in
which archaebacteria live.
■ The sphingolipids contain sphingosine, a long-
chain aliphatic amino alcohol, but no glycerol.
Sphingomyelin has, in addition to phosphoric
acid and choline, two long hydrocarbon chains,
one contributed by a fatty acid and the other
by sphingosine. Three other classes of
sphingolipids are cerebrosides, globosides, and
gangliosides, which contain sugar components.
■ Sterols have four fused rings and a hydroxyl
group. Cholesterol, the major sterol in animals,
is both a structural component of membranes
and precursor to a wide variety of steroids.
10.3 Lipids as Signals, Cofactors,
and Pigments
The two functional classes of lipids considered thus far
(storage lipids and structural lipids) are major cellular
components; membrane lipids make up 5% to 10% of
the dry mass of most cells, and storage lipids more than
80% of the mass of an adipocyte. With some important
exceptions, these lipids play a passive role in the cell;
lipid fuels are stored until oxidized by enzymes, and
membrane lipids form impermeable barriers around
cells and cellular compartments. Another group of
lipids, present in much smaller amounts, have active
roles in the metabolic traffic as metabolites and mes-
sengers. Some serve as potent signals—as hormones,
carried in the blood from one tissue to another, or as in-
tracellular messengers generated in response to an ex-
tracellular signal (hormone or growth factor). Others
function as enzyme cofactors in electron-transfer
reactions in chloroplasts and mitochondria, or in the
transfer of sugar moieties in a variety of glycosylation
(addition of sugar) reactions. A third group consists of
lipids with a system of conjugated double bonds: pig-
ment molecules that absorb visible light. Some of these
act as light-capturing pigments in vision and photosyn-
thesis; others produce natural colorations, such as the
orange of pumpkins and carrots and the yellow of ca-
nary feathers. Specialized lipids such as these are de-
rived from lipids of the plasma membrane or from the
fat-soluble vitamins A, D, E, and K. We describe in this
section a few of these biologically active lipids. In later
chapters, their synthesis and biological roles are con-
sidered in more detail.
Phosphatidylinositols and Sphingosine Derivatives Act
as Intracellular Signals
Phosphatidylinositol and its phosphorylated derivatives
act at several levels to regulate cell structure and me-
tabolism (Fig. 10–17). Phosphatidylinositol 4,5-bisphos-
phate (Fig. 10–8) in the cytoplasmic (inner) face of
plasma membranes serves as a specific binding site for
certain cytoskeletal proteins and for some soluble pro-
teins involved in membrane fusion during exocytosis. It
also serves as a reservoir of messenger molecules that
are released inside the cell in response to extracellular
signals interacting with specific receptors on the outer
surface of the plasma membrane. The signals act through
a series of steps (Fig. 10–17) that begins with enzymatic
removal of a phospholipid head group and ends with ac-
tivation of an enzyme (protein kinase C). For example,
when the hormone vasopressin binds to plasma mem-
brane receptors on the epithelial cells of the renal col-
lecting duct, a specific phospholipase C is activated.
Phospholipase C hydrolyzes the bond between glyc-
erol and phosphate in phosphatidylinositol 4,5-bisphos-
phate, releasing two products: inositol 1,4,5-trisphos-
phate (IP
3
), which is water-soluble, and diacylglycerol,
which remains associated with the plasma membrane.
IP
3
triggers release of Ca
2H11001
from the endoplasmic retic-
ulum, and the combination of diacylglycerol and elevated
cytosolic Ca
2H11001
activates the enzyme protein kinase C.
10.3 Lipids as Signals, Cofactors, and Pigments 357
Phosphatidylinositol
phosphorylation 2ATP
in plasma
in plasma
membrane 2ADP
Phosphatidylinositol 4,5-bisphosphate
hormone-sensitive
H
2
O
phospholipase C
membrane
DiacylglycerolInositol 1,4,5-trisphosphate
Activation of
protein kinase C
Release of intracellular Ca
2H11001
Regulation of other enzymes
(by protein phosphorylation)
Regulation of other enzymes
(by Ca
2H11001
)
FIGURE 10–17 Phosphatidylinositols in cellular regulation. Phos-
phatidylinositol 4,5-bisphosphate in the plasma membrane is hy-
drolyzed by a specific phospholipase C in response to hormonal
signals. Both products of hydrolysis act as intracellular messengers.
8885d_c10_343-368 1/12/04 1:06 PM Page 357 mac76 mac76:385_reb:
This enzyme catalyzes the transfer of a phosphoryl
group from ATP to a specific residue in one or more tar-
get proteins, thereby altering their activity and conse-
quently the cell’s metabolism. This signaling mechanism
is described more fully in Chapter 12 (see Fig. 12–19).
Inositol phospholipids also serve as points of nu-
cleation for certain supramolecular complexes involved
in signaling or in exocytosis. Proteins that contain cer-
tain structural motifs, called PH and PX domains (for
pleckstrin homology and Phox homology, respectively),
bind phosphatidylinositols in the membrane with high
specificity and affinity, initiating the formation of mul-
tienzyme complexes at the membrane’s cytosolic sur-
face. A number of proteins bind specifically to phos-
phatidylinositol 3,4,5-trisphosphate, and the formation
of this phospholipid in response to extracellular signals
brings the proteins together at the surface of the plasma
membrane (see Fig. 12–8).
Membrane sphingolipids also can serve as sources
of intracellular messengers. Both ceramide and sphin-
gomyelin (Fig. 10–12) are potent regulators of protein
kinases, and ceramide or its derivatives are known to be
involved in the regulation of cell division, differentiation,
migration, and programmed cell death (also called apop-
tosis; see Chapter 12).
Eicosanoids Carry Messages to Nearby Cells
Eicosanoids are paracrine hormones, substances
that act only on cells near the point of hormone
synthesis instead of being transported in the blood to
act on cells in other tissues or organs. These fatty acid
derivatives have a variety of dramatic effects on verte-
brate tissues. They are known to be involved in repro-
ductive function; in the inflammation, fever, and pain
associated with injury or disease; in the formation of
blood clots and the regulation of blood pressure; in gas-
tric acid secretion; and in a variety of other processes
important in human health or disease.
All eicosanoids are derived from arachidonic acid
(20:4(H9004
5,8,11,14
)) (Fig. 10–18), the 20-carbon polyun-
saturated fatty acid from which they take their gen-
Chapter 10 Lipids358
Arachidonic acid
1
C
O
OH
CH
3
O
C
O
O
H11002 O
H
CH
3
Prostaglandin E
1
(PGE
1
)
C
O
O
H11002
CH
3
Leukotriene A
4
O
CH
3
OH
O
NSAIDs
C
O
O
H11002
8
12
O
HO
8
12
8
11
5
14
Thromboxane A
2
(b) Eicosanoids
C
C
C
O
X
H
2
H O C
O
H
2
O C
O
Membrane
Polar
phospholipid
head
group
(a)
Phospholipase A
2
851114
FIGURE 10–18 Arachidonic acid and some eicosanoid de-
rivatives. (a) In response to hormonal signals, phospholipase
A
2
cleaves arachidonic acid–containing membrane phospholipids to
release arachidonic acid (arachidonate at pH 7), the precursor to var-
ious eicosanoids. (b) These compounds include prostaglandins such
as PGE
1
, in which C-8 and C-12 of arachidonate are joined to form
the characteristic five-membered ring. In thromboxane A
2
, the C-8 and
C-12 are joined and an oxygen atom is added to form the six-
membered ring. Leukotriene A
4
has a series of three conjugated dou-
ble bonds. Nonsteroidal antiinflammatory drugs (NSAIDs) such as
aspirin and ibuprofen block the formation of prostaglandins and throm-
boxanes from arachidonate by inhibiting the enzyme cyclooxygenase
(prostaglandin H
2
synthase).
8885d_c10_343-368 1/12/04 1:06 PM Page 358 mac76 mac76:385_reb:
eral name (Greek eikosi, “twenty”). There are three
classes of eicosanoids: prostaglandins, thromboxanes,
and leukotrienes.
Prostaglandins (PG) contain a five-carbon ring
originating from the chain of arachidonic acid. Their name
derives from the prostate gland, the tissue from which
they were first isolated by Bengt Samuelsson and Sune
Bergstr?m. Two groups of prostaglandins were originally
defined: PGE, for ether-soluble, and PGF, for phosphate
( fosfat in Swedish) buffer–soluble. Each group contains
numerous subtypes, named PGE
1
, PGE
2
, and so forth.
Prostaglandins act in many tissues by regulating the syn-
thesis of the intracellular messenger 3H11032,5H11032-cyclic AMP
(cAMP). Because cAMP mediates the action of diverse
hormones, the prostaglandins affect a wide range of
cellular and tissue functions. Some prostaglandins stim-
ulate contraction of the smooth muscle of the uterus
during menstruation and labor. Others affect blood flow
to specific organs, the wake-sleep cycle, and the re-
sponsiveness of certain tissues to hormones such as
epinephrine and glucagon. Prostaglandins in a third
group elevate body temperature (producing fever) and
cause inflammation and pain.
The thromboxanes have a six-membered ring con-
taining an ether. They are produced by platelets (also
called thrombocytes) and act in the formation of blood
clots and the reduction of blood flow to the site of a clot.
The nonsteroidal antiinflammatory drugs (NSAIDs)—
aspirin, ibuprofen, and meclofenamate, for example—
were shown by John Vane to inhibit the enzyme
prostaglandin H
2
synthase (also called cyclooxygenase
or COX), which catalyzes an early step in the pathway
from arachidonate to prostaglandins and thromboxanes
(Fig. 10–18; see also Box 21–2).
Leukotrienes, first found in leukocytes, contain
three conjugated double bonds. They are powerful bio-
logical signals. For example, leukotriene D
4
, derived
from leukotriene A
4
, induces contraction of the muscle
lining the airways to the lung. Overproduction of
leukotrienes causes asthmatic attacks, and leukotriene
synthesis is one target of antiasthmatic drugs such as
prednisone. The strong contraction of the smooth mus-
cles of the lung that occurs during anaphylactic shock
is part of the potentially fatal allergic reaction in indi-
viduals hypersensitive to bee stings, penicillin, or other
agents. ■
Steroid Hormones Carry Messages between Tissues
Steroids are oxidized derivatives of sterols; they
have the sterol nucleus but lack the alkyl chain
attached to ring D of cholesterol, and they are more po-
lar than cholesterol. Steroid hormones move through
the bloodstream (on protein carriers) from their site of
production to target tissues, where they enter cells, bind
to highly specific receptor proteins in the nucleus, and
trigger changes in gene expression and metabolism. Be-
cause hormones have very high affinity for their recep-
tors, very low concentrations of hormones (nanomolar
or less) are sufficient to produce responses in target tis-
sues. The major groups of steroid hormones are the male
and female sex hormones and the hormones produced
by the adrenal cortex, cortisol and aldosterone (Fig.
10–19). Prednisone and prednisolone are steroid drugs
with potent antiinflammatory activities, mediated in part
by the inhibition of arachidonate release by phospholi-
pase A
2
(Fig. 10–18) and consequent inhibition of the
10.3 Lipids as Signals, Cofactors, and Pigments 359
John Vane, Sune Bergstr?m, and Bengt Samuelsson
C
CH
2
OH
O
Cortisol
C
O
H
2
O
H
O
Aldosterone
H
O
O
OH
Testosterone Estradiol
H
3
H
OH
HC
3
HC
3
HC
3
HC
3
HC
3
O
O
O
OH
C
CH
HO
C
CH
2
OH
O
Prednisolone Prednisone
O
HC
3
HC
3
OH
HO
C
CH
2
OH
O
O
HC
3
HC
3
OH
O
C
FIGURE 10–19 Steroids derived from cholesterol. Testos-
terone, the male sex hormone, is produced in the testes. Estra-
diol, one of the female sex hormones, is produced in the ovaries and
placenta. Cortisol and aldosterone are hormones synthesized in the
cortex of the adrenal gland; they regulate glucose metabolism and salt
excretion, respectively. Prednisolone and prednisone are synthetic
steroids used as antiinflammatory agents.
8885d_c10_343-368 1/12/04 1:06 PM Page 359 mac76 mac76:385_reb:
synthesis of leukotrienes, prostaglandins, and throm-
boxanes. They have a variety of medical applications,
including the treatment of asthma and rheumatoid
arthritis. ■
Plants Use Phosphatidylinositols, Steroids, and
Eicosanoidlike Compounds in Signaling
Vascular plants contain phosphatidylinositol 4,5-bisphos-
phate, as well as the phospholipase that releases IP
3
,
and they use IP
3
to regulate the intracellular concen-
tration of Ca
2H11001
. Brassinolide and the related group of
brassinosteroids are potent growth regulators in plants,
increasing the rate of stem elongation and influencing
the orientation of cellulose microfibrils in the cell wall
during growth. Jasmonate, derived from the fatty acid
18:3(H9004
9,12,15
) in membrane lipids, is chemically similar
to the eicosanoids of animal tissues and also serves as
a powerful signal, triggering the plant’s defenses in re-
sponse to insect-inflicted damage. The methyl ester of
jasmonate gives the characteristic fragrance of jasmine
oil, which is widely used in the perfume industry.
Vitamins A and D Are Hormone Precursors
During the first third of the twentieth century, a
major focus of research in physiological chem-
istry was the identification of vitamins, compounds that
are essential to the health of humans and other verte-
brates but cannot be synthesized by these animals and
must therefore be obtained in the diet. Early nutritional
O
COO
H11002
Jasmonate
Brassinolide
(a brassinosteroid)
HO
H
O
O
HO
OH
OH
Chapter 10 Lipids360
7-Dehydrocholesterol
1,25-Dihydroxycholecalciferol
(1,25-dihydroxyvitamin D
3
)
CH
3
CH
3
CH
3
CH
3
CH
3
HO
7
64
1
53
210
9
8
HO
CH
2
7
6
4
5
3
2
1
CH
3
CH
3 CH
3
CH
3
25
OH
25
HO
CH
2
7
6
4
5
3
2
1
CH
3
CH
3
CH
3
CH
3
OH
UV light
2 steps (in skin)
(a)
Cholecalciferol (vitamin D
3
)
1 step in the liver
1 step in the kidney
FIGURE 10–20 Vitamin D
3
production
and metabolism. (a) Cholecalciferol
(vitamin D
3
) is produced in the skin by UV
irradiation of 7-dehydrocholesterol, which breaks
the bond shaded pink. In the liver, a hydroxyl
group is added at C-25 (pink); in the kidney, a
second hydroxylation at C-1 (pink) produces the
active hormone, 1,25-dihydroxycholecalciferol.
This hormone regulates the metabolism of
Ca
2H11001
in kidney, intestine, and bone. (b) Dietary
vitamin D prevents rickets, a disease once
common in cold climates where heavy clothing
blocks the UV component of sunlight necessary
for the production of vitamin D
3
in skin. On the
left is a 2
1
?2-year-old boy with severe rickets; on
the right, the same boy at age 5, after 14 months
of vitamin D therapy.
Before vitamin D treatment After 14 months of vitamin D treatment
(b)
8885d_c10_343-368 1/12/04 1:06 PM Page 360 mac76 mac76:385_reb:
10.3 Lipids as Signals, Cofactors, and Pigments 361
H9252-Carotene
Vitamin A
1
point of
cleavage
(retinol)
C
oxidation of
alcohol to
aldehyde
11-cis-Retinal
Neuronal
all-trans-Retinal
signal
to brain
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
12
CH
3
CH
3
CH
3
CH
3
CH
2
OH
CH
3
15
11
CH
3
CH
3
CH
3
CH
3
2
7
6
12
C
11
11
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
visible
light
OH
OH
(visual pigment)
(a)
(b) (e)
(d)
oxidation
of aldehyde
to acid
Retinoic acid Hormonal
signal to
epithelial
cells
(c)
FIGURE 10–21 Vitamin A
1
and its precursor and derivatives.
(a) H9252-Carotene is the precursor of vitamin A
1
. Isoprene struc-
tural units are set off by dashed red lines. Cleavage of H9252-carotene yields
two molecules of vitamin A
1
(retinol) (b). Oxidation at C-15 converts
retinol to the aldehyde, retinal (c), and further oxidation produces
retinoic acid (d), a hormone that regulates gene expression. Retinal
combines with the protein opsin to form rhodopsin (not shown), a vi-
sual pigment widespread in nature. In the dark, retinal of rhodopsin
is in the 11-cis form (c). When a rhodopsin molecule is excited by
visible light, the 11-cis-retinal undergoes a series of photochemical re-
actions that convert it to all-trans-retinal (e), forcing a change in the
shape of the entire rhodopsin molecule. This transformation in the rod
cell of the vertebrate retina sends an electrical signal to the brain that
is the basis of visual transduction, a topic we address in more detail
in Chapter 12.
studies identified two general classes of such com-
pounds: those soluble in nonpolar organic solvents (fat-
soluble vitamins) and those that could be extracted from
foods with aqueous solvents (water-soluble vitamins).
Eventually the fat-soluble group was resolved into the
four vitamin groups A, D, E, and K, all of which are iso-
prenoid compounds synthesized by the condensation of
multiple isoprene units. Two of these (D and A) serve
as hormone precursors.
Vitamin D
3
, also called cholecalciferol, is nor-
mally formed in the skin from 7-dehydrocholesterol in
a photochemical reaction driven by the UV component
of sunlight (Fig. 10–20). Vitamin D
3
is not itself biolog-
ically active, but it is converted by enzymes in the liver
and kidney to 1,25-dihydroxycholecalciferol, a hormone
that regulates calcium uptake in the intestine and cal-
cium levels in kidney and bone. Deficiency of vitamin D
CH
3
C
Isoprene
CH CH
2
CH
2
leads to defective bone formation and the disease rick-
ets, for which administration of vitamin D produces a
dramatic cure. Vitamin D
2
(ergocalciferol) is a com-
mercial product formed by UV irradiation of the ergos-
terol of yeast. Vitamin D
2
is structurally similar to D
3
,
with slight modification to the side chain attached to the
sterol D ring. Both have the same biological effects, and
D
2
is commonly added to milk and butter as a dietary
supplement. Like steroid hormones, the product of vi-
tamin D metabolism, 1,25-dihydroxycholecalciferol, reg-
ulates gene expression—for example, turning on the
synthesis of an intestinal Ca
2H11001
-binding protein.
Vitamin A (retinol) in its various forms functions
as a hormone and as the visual pigment of the verte-
brate eye (Fig. 10–21). Acting through receptor proteins
in the cell nucleus, the vitamin A derivative retinoic acid
regulates gene expression in the development of ep-
ithelial tissue, including skin. Retinoic acid is the active
ingredient in the drug tretinoin (Retin-A), used in the
treatment of severe acne and wrinkled skin. The vita-
min A derivative retinal is the pigment that initiates the
8885d_c10_343-368 1/12/04 1:06 PM Page 361 mac76 mac76:385_reb:
response of rod and cone cells of the retina to light,
producing a neuronal signal to the brain. This role of
retinal is described in detail in Chapter 12.
Vitamin A was first isolated from fish liver oils; liver,
eggs, whole milk, and butter are good dietary sources.
In vertebrates, H9252-carotene, the pigment that gives car-
rots, sweet potatoes, and other yellow vegetables their
characteristic color, can be enzymatically converted to
vitamin A. Deficiency of vitamin A leads to a variety of
symptoms in humans, including dryness of the skin,
eyes, and mucous membranes; retarded development
and growth; and night blindness, an early symptom com-
monly used in diagnosing vitamin A deficiency. ■
Vitamins E and K and the Lipid Quinones Are
Oxidation-Reduction Cofactors
Vitamin E is the collective name for a group of
closely related lipids called tocopherols, all of
which contain a substituted aromatic ring and a long iso-
prenoid side chain (Fig. 10–22a). Because they are hy-
drophobic, tocopherols associate with cell membranes,
lipid deposits, and lipoproteins in the blood. Tocopherols
are biological antioxidants. The aromatic ring reacts
with and destroys the most reactive forms of oxygen
radicals and other free radicals, protecting unsaturated
fatty acids from oxidation and preventing oxidative
Chapter 10 Lipids362
FIGURE 10–22 Some other biologically active isoprenoid com-
pounds or derivatives. Isoprene structural units are set off by dashed
red lines. In most mammalian tissues, ubiquinone (also called coen-
zyme Q) has 10 isoprene units. Dolichols of animals have 17 to 21
isoprene units (85 to 105 carbon atoms), bacterial dolichols have 11,
and those of plants and fungi have 14 to 24.
HO
CH
3
O
CH
2
CH
2
CH
2
C
CH
3
H CH
2
CH
2
CH
2
C
CH
3
H CH
2
CH
2
CH
2
C
CH
3
H CH
3
OCH
3
CH
2
CH C
CH
3
CH
2
CH
2
CH
2
C
CH
3
H CH
22
CH
2
CH
2
C
CH
3
H CH
3
O
OH
C
CH
H
2
C
O
CH
3
CH
3
O
O
CH
3
CH
2
CH C
CH
3
CH
2
CH
2
CH C
CH
3
CH
2 n
CH
2
CH C
CH
3
CH
3
CH
3
O
CH
2
CH C
CH
3
CH
2
HO CH
2
CH
2
C
CH
3
H CH
2
CH
2
CH C
CH
3
CH
2 n
CH
2
CH C
CH
3
CH
3
CH
3
CH
3
O
CH
3
O
O
O
CH
3
O
CH
3
CH
2
CH C
CH
3
CH
2 n
CH
2
CH C
CH
3
CH
3
H20899H20898
H20898
H20899
H20899
H20898
H20899H20898
(b)
Vitamin K
1
: a blood-clotting
cofactor (phylloquinone)
(d)
Ubiquinone: a mitochondrial
electron carrier (coenzyme Q)
(n H11005 4 to 8)
(e)
Plastoquinone: a chloroplast
electron carrier (n H11005 4 to 8)
(f)
Dolichol: a sugar carrier
(n H11005 9 to 22)
(a)
Vitamin E: an antioxidant
(c)
Warfarin: a blood
anticoagulant
8885d_c10_343-368 1/12/04 1:06 PM Page 362 mac76 mac76:385_reb:
damage to membrane lipids, which can cause cell
fragility. Tocopherols are found in eggs and vegetable
oils and are especially abundant in wheat germ. Labo-
ratory animals fed diets depleted of vitamin E develop
scaly skin, muscular weakness and wasting, and steril-
ity. Vitamin E deficiency in humans is very rare; the prin-
cipal symptom is fragile erythrocytes.
The aromatic ring of vitamin K (Fig. 10–22b) un-
dergoes a cycle of oxidation and reduction during the
formation of active prothrombin, a blood plasma protein
essential in blood clot formation. Prothrombin is a pro-
teolytic enzyme that splits peptide bonds in the blood
protein fibrinogen to convert it to fibrin, the insoluble
fibrous protein that holds blood clots together. Henrik
Dam and Edward A. Doisy independently discovered
that vitamin K deficiency slows blood clotting, which
can be fatal. Vitamin K deficiency is very uncommon in
humans, aside from a small percentage of infants who
suffer from hemorrhagic disease of the newborn, a po-
tentially fatal disorder. In the United States, newborns
are routinely given a 1 mg injection of vitamin K. Vita-
min K
1
(phylloquinone) is found in green plant leaves;
a related form, vitamin K
2
(menaquinone), is formed by
bacteria residing in the vertebrate intestine.
Warfarin (Fig. 10–22c) is a synthetic compound that
inhibits the formation of active prothrombin. It is par-
ticularly poisonous to rats, causing death by internal
bleeding. Ironically, this potent rodenticide is also an in-
valuable anticoagulant drug for treating humans at risk
for excessive blood clotting, such as surgical patients
and those with coronary thrombosis. ■
Ubiquinone (also called coenzyme Q) and plasto-
quinone (Fig. 10–22d, e) are isoprenoids that function
as lipophilic electron carriers in the oxidation-reduction
reactions that drive ATP synthesis in mitochondria and
chloroplasts, respectively. Both ubiquinone and plasto-
quinone can accept either one or two electrons and ei-
ther one or two protons (see Fig. 19–54).
Dolichols Activate Sugar Precursors for Biosynthesis
During assembly of the complex carbohydrates of bac-
terial cell walls, and during the addition of polysaccha-
ride units to certain proteins (glycoproteins) and lipids
(glycolipids) in eukaryotes, the sugar units to be added
are chemically activated by attachment to isoprenoid al-
cohols called dolichols (Fig. 10–22f). These compounds
have strong hydrophobic interactions with membrane
lipids, anchoring the attached sugars to the membrane,
where they participate in sugar-transfer reactions.
SUMMARY 10.3 Lipids as Signals, Cofactors,
and Pigments
■ Some types of lipids, although present in
relatively small quantities, play critical roles as
cofactors or signals.
■ Phosphatidylinositol bisphosphate is hydrolyzed
to yield two intracellular messengers,
diacylglycerol and inositol 1,4,5-trisphosphate.
Phosphatidylinositol 3,4,5-trisphosphate is a
nucleation point for supramolecular protein
complexes involved in biological signaling.
■ Prostaglandins, thromboxanes, and leukotrienes
(the eicosanoids), derived from arachidonate,
are extremely potent hormones.
■ Steroid hormones, derived from sterols, serve
as powerful biological signals, such as the sex
hormones.
■ Vitamins D, A, E, and K are fat-soluble
compounds made up of isoprene units. All play
essential roles in the metabolism or physiology
of animals. Vitamin D is precursor to a hormone
that regulates calcium metabolism. Vitamin A
furnishes the visual pigment of the vertebrate
eye and is a regulator of gene expression during
epithelial cell growth. Vitamin E functions
in the protection of membrane lipids from
oxidative damage, and vitamin K is essential in
the blood-clotting process.
■ Ubiquinones and plastoquinones, also
isoprenoid derivatives, function as electron
carriers in mitochondria and chloroplasts,
respectively.
■ Dolichols activate and anchor sugars on cellular
membranes for use in the synthesis of certain
complex carbohydrates, glycolipids, and
glycoproteins.
10.4 Working with Lipids
In exploring the biological role of lipids in cells and tis-
sues, it is essential to know which lipids are present and
in what proportions. Because lipids are insoluble in wa-
ter, their extraction and subsequent fractionation re-
quire the use of organic solvents and some techniques
10.4 Working with Lipids 363
Henrik Dam,
1895–1976
Edward A. Doisy,
1893–1986
8885d_c10_343-368 1/12/04 1:06 PM Page 363 mac76 mac76:385_reb:
not commonly used in the purification of water-soluble
molecules such as proteins and carbohydrates. In gen-
eral, complex mixtures of lipids are separated by dif-
ferences in the polarity or solubility of the components
in nonpolar solvents. Lipids that contain ester- or amide-
linked fatty acids can be hydrolyzed by treatment with
acid or alkali or with highly specific hydrolytic enzymes
(phospholipases, glycosidases) to yield their component
parts for analysis. Some methods commonly used in lipid
analysis are shown in Figure 10–23 and discussed below.
Lipid Extraction Requires Organic Solvents
Neutral lipids (triacylglycerols, waxes, pigments, and so
forth) are readily extracted from tissues with ethyl
ether, chloroform, or benzene, solvents that do not per-
mit lipid clustering driven by hydrophobic interactions.
Membrane lipids are more effectively extracted by more
polar organic solvents, such as ethanol or methanol,
which reduce the hydrophobic interactions among lipid
molecules while also weakening the hydrogen bonds and
electrostatic interactions that bind membrane lipids to
membrane proteins. A commonly used extractant is a
mixture of chloroform, methanol, and water, initially in
volume proportions (1:2:0.8) that are miscible, produc-
ing a single phase. After tissue is homogenized in this
solvent to extract all lipids, more water is added to the
resulting extract and the mixture separates into two
phases, methanol/water (top phase) and chloroform
(bottom phase). The lipids remain in the chloroform
layer, and more polar molecules such as proteins and
sugars partition into the methanol/water layer.
Chapter 10 Lipids364
Concentration
Elution time
homogenized in
chloroform/methanol/water
Water
Methanol/water
Chloroform
Adsorption
chromatography
(d)
(e) (f)
(c)
NaOH/methanol
Fatty acyl methyl esters
Polar
lipids
Charged
lipids
Neutral
lipids
Gas-liquid
chromatography
High-
performance
liquid
chromatography
Tissue
18:0 16:1
14:0
16:0
(b)
Thin-layer
chromatography
123456789
(a)
FIGURE 10–23 Common procedures in the extraction, separation,
and identification of cellular lipids. (a) Tissue is homogenized in a
chloroform/methanol/water mixture, which on addition of water and
removal of unextractable sediment by centrifugation yields two phases.
Different types of extracted lipids in the chloroform phase may be sep-
arated by (b) adsorption chromatography on a column of silica gel,
through which solvents of increasing polarity are passed, or (c) thin-
layer chromatography (TLC), in which lipids are carried up a silica gel-
coated plate by a rising solvent front, less polar lipids traveling farther
than more polar or charged lipids. TLC with appropriate solvents can
also be used to separate closely related lipid species; for example, the
charged lipids phosphatidylserine, phosphatidylglycerol, and phos-
phatidylinositol are easily separated by TLC.
For the determination of fatty acid composition, a lipid fraction
containing ester-linked fatty acids is transesterified in a warm aque-
ous solution of NaOH and methanol (d), producing a mixture of fatty
acyl methyl esters. These methyl esters are then separated on the ba-
sis of chain length and degree of saturation by (e) gas-liquid chro-
matography (GLC) or (f ) high-performance liquid chromatography
(HPLC). Precise determination of molecular mass by mass spectrom-
etry allows unambiguous identification of individual lipids.
8885d_c10_343-368 1/12/04 1:06 PM Page 364 mac76 mac76:385_reb:
Adsorption Chromatography Separates Lipids of
Different Polarity
Complex mixtures of tissue lipids can be fractionated
by chromatographic procedures based on the different
polarities of each class of lipid. In adsorption chro-
matography (Fig. 10–23b), an insoluble, polar material
such as silica gel (a form of silicic acid, Si(OH)
4
) is
packed into a glass column, and the lipid mixture (in
chloroform solution) is applied to the top of the col-
umn. (In high-performance liquid chromatography, the
column is of smaller diameter and solvents are forced
through the column under high pressure.) The polar
lipids bind tightly to the polar silicic acid, but the neu-
tral lipids pass directly through the column and emerge
in the first chloroform wash. The polar lipids are then
eluted, in order of increasing polarity, by washing the
column with solvents of progressively higher polarity.
Uncharged but polar lipids (cerebrosides, for example)
are eluted with acetone, and very polar or charged
lipids (such as glycerophospholipids) are eluted with
methanol.
Thin-layer chromatography on silicic acid employs
the same principle (Fig. 10–23c). A thin layer of silica
gel is spread onto a glass plate, to which it adheres. A
small sample of lipids dissolved in chloroform is applied
near one edge of the plate, which is dipped in a shallow
container of an organic solvent or solvent mixture—all
of which is enclosed within a chamber saturated with
the solvent vapor. As the solvent rises on the plate by
capillary action, it carries lipids with it. The less polar
lipids move farthest, as they have less tendency to bind
to the silicic acid. The separated lipids can be detected
by spraying the plate with a dye (rhodamine) that flu-
oresces when associated with lipids or by exposing the
plate to iodine fumes. Iodine reacts reversibly with the
double bonds in fatty acids, such that lipids containing
unsaturated fatty acids develop a yellow or brown color.
A number of other spray reagents are also useful in de-
tecting specific lipids. For subsequent analysis, regions
containing separated lipids can be scraped from the
plate and the lipids recovered by extraction with an or-
ganic solvent.
Gas-Liquid Chromatography Resolves Mixtures of
Volatile Lipid Derivatives
Gas-liquid chromatography separates volatile compo-
nents of a mixture according to their relative tenden-
cies to dissolve in the inert material packed in the chro-
matography column and to volatilize and move through
the column, carried by a current of an inert gas such as
helium. Some lipids are naturally volatile, but most must
first be derivatized to increase their volatility (that is,
lower their boiling point). For an analysis of the fatty
acids in a sample of phospholipids, the lipids are first
heated in a methanol/HCl or methanol/NaOH mixture,
which converts fatty acids esterified to glycerol into
their methyl esters (in a process of transesterification;
Fig. 10–23d). These fatty acyl methyl esters are then
loaded onto the gas-liquid chromatography column, and
the column is heated to volatilize the compounds. Those
fatty acyl esters most soluble in the column material par-
tition into (dissolve in) that material; the less soluble
lipids are carried by the stream of inert gas and emerge
first from the column. The order of elution depends on
the nature of the solid adsorbant in the column and on
the boiling point of the components of the lipid mixture.
Using these techniques, mixtures of fatty acids of vari-
ous chain lengths and various degrees of unsaturation
can be completely resolved (Fig. 10–23e).
Specific Hydrolysis Aids in Determination of
Lipid Structure
Certain classes of lipids are susceptible to degradation
under specific conditions. For example, all ester-linked
fatty acids in triacylglycerols, phospholipids, and sterol
esters are released by mild acid or alkaline treatment,
and somewhat harsher hydrolysis conditions release
amide-bound fatty acids from sphingolipids. Enzymes
that specifically hydrolyze certain lipids are also useful
in the determination of lipid structure. Phospholipases
A, C, and D (Fig. 10–15) each split particular bonds in
phospholipids and yield products with characteristic sol-
ubilities and chromatographic behaviors. Phospholipase
C, for example, releases a water-soluble phosphoryl al-
cohol (such as phosphocholine from phosphatidyl-
choline) and a chloroform-soluble diacylglycerol, each
of which can be characterized separately to determine
the structure of the intact phospholipid. The combina-
tion of specific hydrolysis with characterization of the
products by thin-layer, gas-liquid, or high-performance
liquid chromatography often allows determination of a
lipid structure.
Mass Spectrometry Reveals Complete Lipid Structure
To establish unambiguously the length of a hydrocarbon
chain or the position of double bonds, mass spectral
analysis of lipids or their volatile derivatives is invalu-
able. The chemical properties of similar lipids (for ex-
ample, two fatty acids of similar length unsaturated at
different positions, or two isoprenoids with different
numbers of isoprene units) are very much alike, and
their positions of elution from the various chromato-
graphic procedures often do not distinguish between
them. When the effluent from a chromatography column
is sampled by mass spectrometry, however, the compo-
nents of a lipid mixture can be simultaneously separated
and identified by their unique pattern of fragmentation
(Fig. 10–24).
10.4 Working with Lipids 365
8885d_c10_343-368 1/12/04 1:06 PM Page 365 mac76 mac76:385_reb:
Chapter 10 Lipids366
Key Terms
fatty acid 343
triacylglycerol 345
lipases 346
phospholipid 348
glycolipid 348
glycerophospholipid
349
ether lipid 349
plasmalogen 349
galactolipid 351
sphingolipid 352
ceramide 352
sphingomyelin 352
glycosphingolipid 352
cerebroside 352
globoside 352
neutral glycolipids 352
gangliosides 352
sterols 354
cholesterol 355
prostaglandins 359
thromboxanes 359
leukotrienes 359
vitamin 360
vitamin D
3
361
cholecalciferol 361
vitamin A (retinol) 361
vitamin E 362
tocopherols 362
vitamin K 363
dolichol 363
Terms in bold are defined in the glossary.
Abundance (%)
90
80
70
60
60 80 100 120 140 160
55
67
92
108
123
151
164
178
206
220
260
234
274
300
314
356
371
92
108
164 206 234 274 314 342
178 220 260 300 328 356
328
M
+
N
180 200 220 240 260 280 300 320 340 360 380
m/z
50
40
30
20
10
H
C
H
O
O
C
FIGURE 10–24 Determination of the structure of a fatty acid by mass
spectrometry. The fatty acid is first converted to a derivative that min-
imizes migration of the double bonds when the molecule is fragmented
by electron bombardment. The derivative shown here is a picolinyl
ester of linoleic acid—18:2(H9004
9,12
) (M
r
371)—in which the alcohol is
picolinol (red). When bombarded with a stream of electrons, this mol-
ecule is volatilized and converted to a parent ion (M
H11001
; M
r
371), in
which the N atom bears the positive charge, and a series of smaller
fragments produced by breakage of COC bonds in the fatty acid. The
mass spectrometer separates these charged fragments according to
their mass/charge ratio (m/z). (To review the principles of mass spec-
trometry, see Box 3–2.)
The prominent ions at m/z H11005 92, 108, 151, and 164 contain the
pyridine ring of the picolinol and various fragments of the carboxyl group,
showing that the compound is indeed a picolinyl ester. The molecular
ion (m/z H11005 371) confirms the presence of a C-18 fatty acid with two
double bonds. The uniform series of ions 14 atomic mass units (amu)
apart represents loss of each successive methyl and methylene group
from the right end of the molecule (C-18 of the fatty acid), until the ion
at m/z H11005 300 is reached. This is followed by a gap of 26 amu for the
carbons of the terminal double bond, at m/z H11005 274; a further gap of 14
amu for the C-11 methylene group, at m/z H11005 260, and so forth. By this
means the entire structure is determined, although these data alone do
not reveal the configuration (cis or trans) of the double bonds.
SUMMARY 10.4 Working with Lipids
■ In the determination of lipid composition, the
lipids are first extracted from tissues with
organic solvents and separated by thin-layer,
gas-liquid, or high-performance liquid
chromatography.
■ Phospholipases specific for one of the bonds in
a phospholipid can be used to generate simpler
compounds for subsequent analysis.
■ Individual lipids are identified by their
chromatographic behavior, their susceptibility
to hydrolysis by specific enzymes, or mass
spectrometry.
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Chapter 10 Problems 367
Further Reading
General
Gurr, M.I. & Harwood, J.L. (1991) Lipid Biochemistry: An
Introduction, 4th edn, Chapman & Hall, London.
A good general resource on lipid structure and metabolism, at
the intermediate level.
Vance, D.E. & Vance, J.E. (eds) (2002) Biochemistry of Lipids,
Lipoproteins, and Membranes, New Comprehensive Biochemistry,
Vol. 36, Elsevier Science Publishing Co., Inc., New York.
An excellent collection of reviews on various aspects of lipid
structure, biosynthesis, and function.
Structural Lipids in Membranes
Bogdanov, M. & Dowhan, W. (1999) Lipid-assisted protein
folding. J. Biol. Chem. 274, 36,827–36,830.
A minireview of the role of membrane lipids in the folding of
membrane proteins.
De Rosa, M. & Gambacorta, A. (1988) The lipids of archaebac-
teria. Prog. Lipid Res. 27, 153–175.
Dowhan, W. (1997) Molecular basis for membrane phospholipid
diversity: why are there so many lipids? Annu. Rev. Biochem. 66,
199–232.
Gravel, R.A., Kaback, M.M., Proia, R., Sandhoff, K., Suzuki,
K., & Suzuki, K. (2001) The GM
2
gangliosidoses. In The Metabolic
and Molecular Bases of Inherited Disease, 8th edn (Scriver, C.R.,
Sly, W.S., Childs, B., Beaudet, A.L., Valle, D., Kinzler, K.W., &
Vogelstein, B., eds), pp. 3827–3876, McGraw-Hill, Inc., New York.
This article is one of many in a four-volume set that contains
definitive descriptions of the clinical, biochemical, and genetic
aspects of hundreds of human metabolic diseases—an authori-
tative source and fascinating reading.
Hoekstra, D. (ed.) (1994) Cell Lipids, Current Topics in
Membranes, Vol. 4, Academic Press, Inc., San Diego.
Lipids as Signals, Cofactors, and Pigments
Bell, R.M., Exton, J.H., & Prescott, S.M. (eds) (1996) Lipid
Second Messengers, Handbook of Lipid Research, Vol. 8, Plenum
Press, New York.
Binkley, N.C. & Suttie, J.W. (1995) Vitamin K nutrition and
osteoporosis. J. Nutr. 125, 1812–1821.
Brigelius-Flohé, R. & Traber, M.G. (1999) Vitamin E: function
and metabolism. FASEB J. 13, 1145–1155.
Chojnacki, T. & Dallner, G. (1988) The biological role of
dolichol. Biochem. J. 251, 1–9.
Clouse, S.D. (2002) Brassinosteroid signal transduction: clarifying
the pathway from ligand perception to gene expression. Mol. Cell
10, 973–982.
Lemmon, M.A. & Ferguson, K.M. (2000) Signal-dependent
membrane targeting by pleckstrin homology (PH) domains.
Biochem. J. 350, 1–18.
Prescott, S.M., Zimmerman, G.A., Stafforini, D.M., &
McIntyre, T.M. (2000) Platelet-activating factor and related lipid
mediators. Annu. Rev. Biochem. 69, 419–445.
Schneiter, R. (1999) Brave little yeast, please guide us to Thebes:
sphingolipid function in S. cerevisiae. BioEssays 21, 1004–1010.
Suttie, J.W. (1993) Synthesis of vitamin K-dependent proteins.
FASEB J. 7, 445–452.
Vermeer, C. (1990) H9253-Carboxyglutamate-containing proteins and
the vitamin K-dependent carboxylase. Biochem. J. 266, 625–636.
Describes the biochemical basis for the requirement of vitamin
K in blood clotting and the importance of carboxylation in the
synthesis of the blood-clotting protein thrombin.
Viitala, J. & J?rnefelt, J. (1985) The red cell surface revisited.
Trends Biochem. Sci. 10, 392–395.
Includes discussion of the human A, B, and O blood type deter-
minants.
Weber, H. (2002) Fatty acid-derived signals in plants. Trends
Plant Sci. 7, 217–224.
Zittermann, A. (2001) Effects of vitamin K on calcium and bone
metabolism. Curr. Opin. Clin. Nutr. Metab. Care 4, 483–487.
Working with Lipids
Christie, W.W. (1998) Gas chromatography-mass spectrometry
methods for structural analysis of fatty acids. Lipids 33, 343–353.
A detailed description of the methods used to obtain data such
as those presented in Figure 10–24.
Christie, W.W. (2003) Lipid Analysis, 3rd edn, The Oily Press,
Bridgwater, England.
Hamilton, R.J. & Hamilton, S. (eds) (1992) Lipid Analysis: A
Practical Approach, IRL Press at Oxford University Press, New
York.
This text, though out of print, is available as part of the IRL
Press Practical Approach Series on CD-ROM, from Oxford
University Press (www.oup-usa/acadsci/pasbooks.html).
Matsubara, T. & Hagashi, A. (1991) FAB/mass spectrometry of
lipids. Prog. Lipid Res. 30, 301–322.
An advanced discussion of the identification of lipids by fast
atom bombardment (FAB) mass spectrometry, a powerful tech-
nique for structure determination.
1. Operational Definition of Lipids How is the defini-
tion of “lipid” different from the types of definitions used for
other biomolecules that we have considered, such as amino
acids, nucleic acids, and proteins?
2. Melting Points of Lipids The melting points of a se-
ries of 18-carbon fatty acids are: stearic acid, 69.6 H11034C; oleic
acid, 13.4 H11034C; linoleic acid, H110025 H11034C; and linolenic acid, H1100211 H11034C.
(a) What structural aspect of these 18-carbon fatty acids
Problems
8885d_c10_367 1/16/04 8:18 AM Page 367 mac76 mac76:385_reb:
Chapter 10 Lipids368
can be correlated with the melting point? Provide a molecu-
lar explanation for the trend in melting points.
(b) Draw all the possible triacylglycerols that can be
constructed from glycerol, palmitic acid, and oleic acid. Rank
them in order of increasing melting point.
(c) Branched-chain fatty acids are found in some bac-
terial membrane lipids. Would their presence increase or de-
crease the fluidity of the membranes (that is, give them a
lower or higher melting point)? Why?
3. Preparation of Béarnaise Sauce During the prepa-
ration of béarnaise sauce, egg yolks are incorporated into
melted butter to stabilize the sauce and avoid separation. The
stabilizing agent in the egg yolks is lecithin (phosphatidyl-
choline). Suggest why this works.
4. Hydrophobic and Hydrophilic Components of Mem-
brane Lipids A common structural feature of membrane
lipids is their amphipathic nature. For example, in phos-
phatidylcholine, the two fatty acid chains are hydrophobic
and the phosphocholine head group is hydrophilic. For each
of the following membrane lipids, name the components that
serve as the hydrophobic and hydrophilic units: (a) phos-
phatidylethanolamine; (b) sphingomyelin; (c) galactosyl-
cerebroside; (d) ganglioside; (e) cholesterol.
5. Alkali Lability of Triacylglycerols A common pro-
cedure for cleaning the grease trap in a sink is to add a prod-
uct that contains sodium hydroxide. Explain why this works.
6. The Action of Phospholipases The venom of
the Eastern diamondback rattler and the Indian co-
bra contains phospholipase A
2
, which catalyzes the hydroly-
sis of fatty acids at the C-2 position of glycerophospholipids.
The phospholipid breakdown product of this reaction is
lysolecithin (lecithin is phosphatidylcholine). At high con-
centrations, this and other lysophospholipids act as deter-
gents, dissolving the membranes of erythrocytes and lysing
the cells. Extensive hemolysis may be life-threatening.
(a) Detergents are amphipathic. What are the hy-
drophilic and hydrophobic portions of lysolecithin?
(b) The pain and inflammation caused by a snake bite
can be treated with certain steroids. What is the basis of this
treatment?
(c) Though high levels of phospholipase A
2
can be
deadly, this enzyme is necessary for a variety of normal meta-
bolic processes. What are these processes?
7. Intracellular Messengers from Phosphatidylinosi-
tols When the hormone vasopressin stimulates cleavage of
phosphatidylinositol 4,5-bisphosphate by hormone-sensitive
phospholipase C, two products are formed. What are they?
Compare their properties and their solubilities in water, and
predict whether either would diffuse readily through the
cytosol.
8. Storage of Fat-Soluble Vitamins In contrast to
water-soluble vitamins, which must be a part of our daily diet,
fat-soluble vitamins can be stored in the body in amounts suf-
ficient for many months. Suggest an explanation for this dif-
ference, based on solubilities.
9. Hydrolysis of Lipids Name the products of mild hy-
drolysis with dilute NaOH of (a) 1-stearoyl-2,3-dipalmitoyl-
glycerol; (b) 1-palmitoyl-2-oleoylphosphatidylcholine.
10. Effect of Polarity on Solubility Rank the following
in order of increasing solubility in water: a triacylglycerol, a
diacylglycerol, and a monoacylglycerol, all containing only
palmitic acid.
11. Chromatographic Separation of Lipids A mixture
of lipids is applied to a silica gel column, and the column is
then washed with increasingly polar solvents. The mixture
consists of phosphatidylserine, phosphatidylethanolamine,
phosphatidylcholine, cholesteryl palmitate (a sterol ester),
sphingomyelin, palmitate, n-tetradecanol, triacylglycerol, and
cholesterol. In what order do you expect the lipids to elute
from the column? Explain your reasoning.
12. Identification of Unknown Lipids Johann Thu-
dichum, who practiced medicine in London about 100 years
ago, also dabbled in lipid chemistry in his spare time. He iso-
lated a variety of lipids from neural tissue, and characterized
and named many of them. His carefully sealed and labeled
vials of isolated lipids were rediscovered many years later.
(a) How would you confirm, using techniques not avail-
able to Thudichum, that the vials labeled “sphingomyelin” and
“cerebroside” actually contain these compounds?
(b) How would you distinguish sphingomyelin from
phosphatidylcholine by chemical, physical, or enzymatic
tests?
13. Ninhydrin to Detect Lipids on TLC Plates Ninhy-
drin reacts specifically with primary amines to form a
purplish-blue product. A thin-layer chromatogram of rat liver
phospholipids is sprayed with ninhydrin, and the color is al-
lowed to develop. Which phospholipids can be detected in
this way?
8885d_c10_343-368 1/12/04 1:06 PM Page 368 mac76 mac76:385_reb:
chapter
T
he first cell probably came into being when a mem-
brane formed, enclosing a small volume of aqueous
solution and separating it from the rest of the universe.
Membranes define the external boundaries of cells and
regulate the molecular traffic across that boundary
(Fig. 11–1); in eukaryotic cells, they divide the internal
space into discrete compartments to segregate processes
and components. They organize complex reaction se-
quences and are central to both biological energy con-
servation and cell-to-cell communication. The biological
activities of membranes flow from their remarkable
physical properties. Membranes are flexible, self-sealing,
and selectively permeable to polar solutes. Their flexi-
bility permits the shape changes that accompany cell
growth and movement (such as amoeboid movement).
With their ability to break and reseal, two membranes
can fuse, as in exocytosis, or a single membrane-enclosed
compartment can undergo fission to yield two sealed
compartments, as in endocytosis or cell division, without
creating gross leaks through cellular surfaces. Because
membranes are selectively permeable, they retain certain
compounds and ions within cells and within specific cel-
lular compartments, while excluding others.
Membranes are not merely passive barriers. They in-
clude an array of proteins specialized for promoting or
catalyzing various cellular processes. At the cell surface,
transporters move specific organic solutes and inorganic
ions across the membrane; receptors sense extracellular
signals and trigger molecular changes in the cell; adhe-
sion molecules hold neighboring cells together. Within
the cell, membranes organize cellular processes such as
the synthesis of lipids and certain proteins, and the en-
ergy transductions in mitochondria and chloroplasts.
Because membranes consist of just two layers of mole-
cules, they are very thin—essentially two-dimensional.
Intermolecular collisions are far more probable in this
two-dimensional space than in three-dimensional space,
so the efficiency of enzyme-catalyzed processes organ-
ized within membranes is vastly increased.
11
369
BIOLOGICAL MEMBRANES
AND TRANSPORT
Good fences make good neighbors.
—Robert Frost, “Mending Wall,” in North of Boston, 1914
11.1 The Composition and Architecture of
Membranes 370
11.2 Membrane Dynamics 380
11.3 Solute Transport across Membranes 389
Membrane
bilayer
FIGURE 11–1 Biological membranes. Viewed in cross section, all cell
membranes share a characteristic trilaminar appearance. When an
erythrocyte is stained with osmium tetroxide and viewed with an elec-
tron microscope, the plasma membrane appears as a three-layer struc-
ture, 5 to 8 nm (50 to 80 ?) thick. The trilaminar image consists of
two electron-dense layers (the osmium, bound to the inner and outer
surfaces of the membrane) separated by a less dense central region.
8885d_c11_369-420 2/7/04 6:58 AM Page 369 mac76 mac76:385_reb:
In this chapter we first describe the composition of
cellular membranes and their chemical architecture—
the molecular structures that underlie their biological
functions. Next, we consider the remarkable dynamic
features of membranes, in which lipids and proteins
move relative to each other. Cell adhesion, endocytosis,
and the membrane fusion accompanying neurotrans-
mitter secretion illustrate the dynamic role of membrane
proteins. We then turn to the protein-mediated passage
of solutes across membranes via transporters and ion
channels. In later chapters we discuss the role of mem-
branes in signal transduction (Chapters 12 and 23),
energy transduction (Chapter 19), lipid synthesis
(Chapter 21), and protein synthesis (Chapter 27).
11.1 The Composition and Architecture
of Membranes
One approach to understanding membrane function is
to study membrane composition—to determine, for ex-
ample, which components are common to all mem-
branes and which are unique to membranes with
specific functions. So before describing membrane
structure and function we consider the molecular com-
ponents of membranes: proteins and polar lipids, which
account for almost all the mass of biological membranes,
and carbohydrates, present as part of glycoproteins and
glycolipids.
Each Type of Membrane Has Characteristic
Lipids and Proteins
The relative proportions of protein and lipid vary with
the type of membrane (Table 11–1), reflecting the di-
versity of biological roles. For example, certain neurons
have a myelin sheath, an extended plasma membrane
that wraps around the cell many times and acts as a pas-
sive electrical insulator. The myelin sheath consists
primarily of lipids, whereas the plasma membranes of
bacteria and the membranes of mitochondria and
chloroplasts, the sites of many enzyme-catalyzed
processes, contain more protein than lipid (in mass per
total mass).
For studies of membrane composition, the first task
is to isolate a selected membrane. When eukaryotic cells
are subjected to mechanical shear, their plasma mem-
branes are torn and fragmented, releasing cytoplasmic
components and membrane-bounded organelles such as
mitochondria, chloroplasts, lysosomes, and nuclei.
Plasma membrane fragments and intact organelles can
be isolated by centrifugal techniques described in
Chapter 1 (see Fig. 1–8).
Chemical analyses of membranes isolated from var-
ious sources reveal certain common properties. Each
kingdom, each species, each tissue or cell type, and the
organelles of each cell type have a characteristic set of
membrane lipids. Plasma membranes, for example, are
enriched in cholesterol and contain no detectable
cardiolipin (Fig. 11–2); in the inner mitochondrial mem-
brane of the hepatocyte, this distribution is reversed:
very low cholesterol and high cardiolipin. Cardiolipin is
essential to the function of certain proteins of the inner
mitochondrial membrane. Cells clearly have mecha-
nisms to control the kinds and amounts of membrane
lipids they synthesize and to target specific lipids to
particular organelles. In many cases, we can surmise the
adaptive advantages of distinct combinations of mem-
brane lipids; in other cases, the functional significance
of these combinations is as yet unknown.
The protein composition of membranes from dif-
ferent sources varies even more widely than their lipid
composition, reflecting functional specialization. In a
rod cell of the vertebrate retina, one portion of the cell
is highly specialized for the reception of light; more than
90% of the plasma membrane protein in this region is
the light-absorbing glycoprotein rhodopsin. The less-
specialized plasma membrane of the erythrocyte has
about 20 prominent types of proteins as well as scores
of minor ones; many of these are transporters, each
moving a specific solute across the membrane. The
plasma membrane of Escherichia coli contains hun-
Chapter 11 Biological Membranes and Transport370
TABLE 11–1 Major Components of Plasma Membranes in Various Organisms
Components (% by weight)
Protein Phospholipid Sterol Sterol type Other lipids
Human myelin sheath 30 30 19 Cholesterol Galactolipids, plasmalogens
Mouse liver 45 27 25 Cholesterol —
Maize leaf 47 26 7 Sitosterol Galactolipids
Yeast 52 7 4 Ergosterol Triacylglycerols, steryl esters
Paramecium (ciliated protist) 56 40 4 Stigmasterol —
E. coli 75 25 0 — —
Note: Values do not add up to 100% in every case, because there are components other than protein, phospholipids, and sterol; plants, for
example, have high levels of glycolipids.
8885d_c11_369-420 2/7/04 6:58 AM Page 370 mac76 mac76:385_reb:
dreds of different proteins, including transporters and
many enzymes involved in energy-conserving metabo-
lism, lipid synthesis, protein export, and cell division.
The outer membrane of E. coli, which encloses the
plasma membrane, has a different function (protection)
and a different set of proteins.
Some membrane proteins are covalently linked to
complex arrays of carbohydrate. For example, in gly-
cophorin, a glycoprotein of the erythrocyte plasma
membrane, 60% of the mass consists of complex
oligosaccharide units covalently attached to specific
amino acid residues. Ser, Thr, and Asn residues are the
most common points of attachment (see Fig. 7–31). At
the other end of the scale is rhodopsin of the rod cell
plasma membrane, which contains just one hexasac-
charide. The sugar moieties of surface glycoproteins
influence the folding of the proteins, as well as their sta-
bilities and intracellular destinations, and they play a
significant role in the specific binding of ligands to gly-
coprotein surface receptors (see Fig. 7–37).
Some membrane proteins are covalently attached
to one or more lipids, which serve as hydrophobic an-
chors that hold the proteins to the membrane, as we
shall see.
All Biological Membranes Share Some
Fundamental Properties
Membranes are impermeable to most polar or charged
solutes, but permeable to nonpolar compounds; they are
5 to 8 nm (50 to 80 ?) thick and appear trilaminar when
viewed in cross section with the electron microscope
(Fig. 11–1). The combined evidence from electron mi-
croscopy and studies of chemical composition, as well
as physical studies of permeability and the motion of in-
dividual protein and lipid molecules within membranes,
led to the development of the fluid mosaic model for
the structure of biological membranes (Fig. 11–3).
Phospholipids form a bilayer in which the nonpolar re-
gions of the lipid molecules in each layer face the core
of the bilayer and their polar head groups face outward,
interacting with the aqueous phase on either side. Pro-
teins are embedded in this bilayer sheet, held by hy-
drophobic interactions between the membrane lipids
and hydrophobic domains in the proteins. Some proteins
protrude from only one side of the membrane; others
have domains exposed on both sides. The orientation of
proteins in the bilayer is asymmetric, giving the mem-
brane “sidedness”: the protein domains exposed on one
side of the bilayer are different from those exposed on
the other side, reflecting functional asymmetry. The in-
dividual lipid and protein units in a membrane form a
fluid mosaic with a pattern that, unlike a mosaic of ce-
ramic tile and mortar, is free to change constantly. The
membrane mosaic is fluid because most of the interac-
tions among its components are noncovalent, leaving
individual lipid and protein molecules free to move lat-
erally in the plane of the membrane.
We now look at some of these features of the fluid
mosaic model in more detail and consider the experi-
mental evidence that supports the basic model but has
necessitated its refinement in several ways.
A Lipid Bilayer Is the Basic Structural
Element of Membranes
Glycerophospholipids, sphingolipids, and sterols are vir-
tually insoluble in water. When mixed with water, they
spontaneously form microscopic lipid aggregates in a
phase separate from their aqueous surroundings, clus-
tering together, with their hydrophobic moieties in con-
tact with each other and their hydrophilic groups in-
teracting with the surrounding water. Recall that lipid
clustering reduces the amount of hydrophobic surface
11.1 The Composition and Architecture of Membranes 371
FIGURE 11–2 Lipid composition of the plasma membrane and or-
ganelle membranes of a rat hepatocyte. The functional specialization
of each membrane type is reflected in its unique lipid composition.
Cholesterol is prominent in plasma membranes but barely detectable
in mitochondrial membranes. Cardiolipin is a major component of the
inner mitochondrial membrane but not of the plasma membrane.
Phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol
are relatively minor components (yellow) of most membranes but serve
critical functions; phosphatidylinositol and its derivatives, for exam-
ple, are important in signal transductions triggered by hormones.
Sphingolipids, phosphatidylcholine, and phosphatidylethanolamine
are present in most membranes, but in varying proportions.
Glycolipids, which are major components of the chloroplast mem-
branes of plants, are virtually absent from animal cells.
0
Plasma
Lysosomal
Nuclear
Rat hepatocyte membrane type
Rough ER
Smooth ER
Golgi
Inner
mitochondrial
Outer
mitochondrial
20 40
Percent membrane lipid
Cholesterol
60 80 100
Cardiolipin
Minor lipids
Sphingolipids
Phosphatidylcholine
Phosphatidylethanolamine
8885d_c11_369-420 2/7/04 6:58 AM Page 371 mac76 mac76:385_reb:
Lipid
bilayer
Peripheral
protein
Integral protein
(single trans-
membrane helix)
Peripheral protein
covalently linked
to lipid
Oligosaccharide
chains of
glycoprotein
Integral protein
(multiple trans-
membrane helices)
Phospholipid
polar heads
Sterol
Glycolipid
Outside
Inside
FIGURE 11–3 Fluid mosaic model for membrane structure. The fatty
acyl chains in the interior of the membrane form a fluid, hydropho-
bic region. Integral proteins float in this sea of lipid, held by
hydrophobic interactions with their nonpolar amino acid side chains.
Both proteins and lipids are free to move laterally in the plane of the
bilayer, but movement of either from one face of the bilayer to the
other is restricted. The carbohydrate moieties attached to some
proteins and lipids of the plasma membrane are exposed on the ex-
tracellular surface of the membrane.
exposed to water and thus minimizes the number of mol-
ecules in the shell of ordered water at the lipid-water
interface (see Fig. 2–7), resulting in an increase in en-
tropy. Hydrophobic interactions among lipid molecules
provide the thermodynamic driving force for the for-
mation and maintenance of these clusters.
Depending on the precise conditions and the nature
of the lipids, three types of lipid aggregates can form
when amphipathic lipids are mixed with water (Fig.
11–4). Micelles are spherical structures that contain
anywhere from a few dozen to a few thousand amphi-
pathic molecules. These molecules are arranged with
Chapter 11 Biological Membranes and Transport372
Individual units are
wedge-shaped
(cross section of head
greater than that
of side chain)
Individual units are
cylindrical (cross section
of head equals that of side chain)
(c) Liposome(b) Bilayer(a) Micelle
Aqueous
cavity
FIGURE 11–4 Amphipathic lipid aggregates that form in water. (a) In
micelles, the hydrophobic chains of the fatty acids are sequestered at
the core of the sphere. There is virtually no water in the hydrophobic
interior. (b) In an open bilayer, all acyl side chains except those at the
edges of the sheet are protected from interaction with water. (c) When
a two-dimensional bilayer folds on itself, it forms a closed bilayer, a
three-dimensional hollow vesicle (liposome) enclosing an aqueous
cavity.
8885d_c11_369-420 2/7/04 6:58 AM Page 372 mac76 mac76:385_reb:
their hydrophobic regions aggregated in the interior,
where water is excluded, and their hydrophilic head
groups at the surface, in contact with water. Micelle for-
mation is favored when the cross-sectional area of the
head group is greater than that of the acyl side chain(s),
as in free fatty acids, lysophospholipids (phospholipids
lacking one fatty acid), and detergents such as sodium
dodecyl sulfate (SDS; p. 92).
A second type of lipid aggregate in water is the
bilayer, in which two lipid monolayers (leaflets) form
a two-dimensional sheet. Bilayer formation occurs most
readily when the cross-sectional areas of the head group
and acyl side chain(s) are similar, as in glycerophos-
pholipids and sphingolipids. The hydrophobic portions
in each monolayer, excluded from water, interact with
each other. The hydrophilic head groups interact with
water at each surface of the bilayer. Because the hy-
drophobic regions at its edges (Fig. 11–4b) are tran-
siently in contact with water, the bilayer sheet is rela-
tively unstable and spontaneously forms a third type of
aggregate: it folds back on itself to form a hollow sphere,
a vesicle or liposome (Fig. 11–4c). By forming vesicles,
bilayers lose their hydrophobic edge regions, achieving
maximal stability in their aqueous environment. These
bilayer vesicles enclose water, creating a separate aque-
ous compartment. It is likely that the precursors to the
first living cells resembled liposomes, their aqueous con-
tents segregated from the rest of the world by a hy-
drophobic shell.
Biological membranes are constructed of lipid bi-
layers 3 nm (30 ?) thick, with proteins protruding on
each side. The hydrocarbon core of the membrane, made
up of the OCH
2
O and OCH
3
of the fatty acyl groups,
is about as nonpolar as decane, and liposomes formed
in the laboratory from pure lipids are essentially imper-
meable to polar solutes, as are biological membranes
(although the latter, as we shall see, are permeable to
solutes for which they have specific transporters).
Plasma membrane lipids are asymmetrically dis-
tributed between the two monolayers of the bilayer, al-
though the asymmetry, unlike that of membrane pro-
teins, is not absolute. In the plasma membrane of the
erythrocyte, for example, choline-containing lipids
(phosphatidylcholine and sphingomyelin) are typically
found in the outer (extracellular or exoplasmic) leaflet
(Fig. 11–5), whereas phosphatidylserine, phosphatidyl-
ethanolamine, and the phosphatidylinositols are much
more common in the inner (cytoplasmic) leaflet.
Changes in the distribution of lipids between plasma
membrane leaflets have biological consequences. For
example, only when the phosphatidylserine in the
plasma membrane moves into the outer leaflet is a
platelet able to play its role in formation of a blood clot.
For many other cells types, phosphatidylserine expo-
sure on the outer surface marks a cell for destruction
by programmed cell death.
Peripheral Membrane Proteins Are Easily Solubilized
Membrane proteins may be divided operationally into
two groups (Fig. 11–6). Integral proteins are very
firmly associated with the membrane, removable only
by agents that interfere with hydrophobic interactions,
such as detergents, organic solvents, or denaturants.
Peripheral proteins associate with the membrane
through electrostatic interactions and hydrogen bond-
ing with the hydrophilic domains of integral proteins
and with the polar head groups of membrane lipids.
They can be released by relatively mild treatments that
interfere with electrostatic interactions or break hy-
drogen bonds; a commonly used agent is carbonate at
high pH. Peripheral proteins may serve as regulators of
membrane-bound enzymes or may limit the mobility of
integral proteins by tethering them to intracellular
structures.
Many Membrane Proteins Span the Lipid Bilayer
Membrane protein topology (localization relative to the
lipid bilayer) can be determined with reagents that
react with protein side chains but cannot cross
membranes—polar chemical reagents that react with
primary amines of Lys residues, for example, or enzymes
11.1 The Composition and Architecture of Membranes 373
Phosphatidylinositol
4-phosphate
Phosphatidylinositol
Phosphatidylinositol
4,5-bisphosphate
Phosphatidic acid
5
Phosphatidylserine
15
Sphingomyelin
23
Phosphatidylcholine
27
Phosphatidyl-
ethanolamine
30
100
Inner
monolayer
Outer
monolayer
0
Distribution in
membrane
Membrane
phospholipid
Percent of
total
membrane
phospholipid
100
FIGURE 11–5 Asymmetric distribution of phospholipids between the
inner and outer monolayers of the erythrocyte plasma membrane.
The distribution of a specific phospholipid is determined by treating
the intact cell with phospholipase C, which cannot reach lipids in the
inner monolayer (leaflet) but removes the head groups of lipids in the
outer monolayer. The proportion of each head group released provides
an estimate of the fraction of each lipid in the outer monolayer.
8885d_c11_369-420 2/7/04 6:58 AM Page 373 mac76 mac76:385_reb:
like trypsin that cleave proteins but cannot cross the
membrane. The human erythrocyte is convenient for
such studies because it has no membrane-bounded or-
ganelles; the plasma membrane is the only membrane
present. If a membrane protein in an intact erythrocyte
reacts with a membrane-impermeant reagent, that pro-
tein must have at least one domain exposed on the outer
(extracellular) face of the membrane. Trypsin is found
to cleave extracellular domains but does not affect do-
mains buried within the bilayer or exposed on the inner
surface only, unless the plasma membrane is broken to
make these domains accessible to the enzyme.
Experiments with such topology-specific reagents
show that the erythrocyte glycoprotein glycophorin
spans the plasma membrane. Its amino-terminal domain
(bearing the carbohydrate chains) is on the outer sur-
face and is cleaved by trypsin. The carboxyl terminus
protrudes on the inside of the cell, where it cannot re-
act with impermeant reagents. Both the amino-terminal
and carboxyl-terminal domains contain many polar or
charged amino acid residues and are therefore quite
hydrophilic. However, a segment in the center of the
protein (residues 75 to 93) contains mainly hydropho-
bic amino acid residues, suggesting that glycophorin
has a transmembrane segment arranged as shown in
Figure 11–7.
One further fact may be deduced from the results
of experiments with glycophorin: its disposition in the
membrane is asymmetric. Similar studies of other mem-
brane proteins show that each has a specific orientation
Chapter 11 Biological Membranes and Transport374
Peripheral
protein
Ca
2+
H11545
H11545
H11546H11546
H11545
detergent
phospholipase C
GPI-linked
protein
Protein-glycan
Integral protein
(hydrophobic domain
coated with detergent)
change in pH;
chelating agent;
urea; CO
3
2–
FIGURE 11–6 Peripheral and integral proteins. Membrane proteins
can be operationally distinguished by the conditions required to re-
lease them from the membrane. Most peripheral proteins are released
by changes in pH or ionic strength, removal of Ca
2H11001
by a chelating
agent, or addition of urea or carbonate. Integral proteins are ex-
tractable with detergents, which disrupt the hydrophobic interactions
with the lipid bilayer and form micelle-like clusters around individual
protein molecules. Integral proteins covalently attached to a mem-
brane lipid, such as a glycosyl phosphatidylinositol (GPI; see Fig.
11–14), can be released by treatment with phospholipase C.
Inside
Outside
Amino
terminus
Carboxyl
terminus
Leu
Ser Thr
Thr
Glu
Val
Ala Met
His
Thr Thr Thr
Ser
Ser
Ser
Val
Ser
Lys
Ser
TyrIle
Ser
SerGln
Thr
Asn
AspThr
HisLys
ArgAsp
Thr
Tyr
Ala
Ala
Thr
Pro Arg Ala
His
Glu
Val
Ser
Glu
Ile
Ser Val
Arg Thr Val
Tyr
Pro
Pro
Glu
GluGluThr
Glu
Glu
Arg
Val
Gln
Leu
Ala
His
Phe
Ser
Pro
Glu
Glu
Ile
Thr
Leu
Ile
Ile
Phe
Gly
Val
Met
Ala
Gly
Val
Ile
Gly
Thr
Ile
Leu
Leu
Ile
Ser
Tyr
Gly
Ile
ArgArg
LeuIle
Lys
Lys
Ser
Pro
Ser
Asp
Val
Lys
Pro
Leu Pro
Ser
Pro
Asp
Val
Thr
Asp
Pro Leu Ser Ser
Val
Glu
Ile
Glu
AsnPro
Glu
Thr
SerAsp
Gln
His
1
60
74
95
131
FIGURE 11–7 Transbilayer disposition of glycophorin in an
erythrocyte. One hydrophilic domain, containing all the sugar
residues, is on the outer surface, and another hydrophilic domain pro-
trudes from the inner face of the membrane. Each red hexagon rep-
resents a tetrasaccharide (containing two Neu5Ac (sialic acid), Gal,
and GalNAc) O-linked to a Ser or Thr residue; the blue hexagon rep-
resents an oligosaccharide chain N-linked to an Asn residue. The rel-
ative size of the oligosaccharide units is larger than shown here. A
segment of 19 hydrophobic residues (residues 75 to 93) forms an H9251
helix that traverses the membrane bilayer (see Fig. 11–11a). The seg-
ment from residues 64 to 74 has some hydrophobic residues and prob-
ably penetrates into the outer face of the lipid bilayer, as shown.
8885d_c11_369-420 2/7/04 6:58 AM Page 374 mac76 mac76:385_reb:
in the bilayer; one domain of a transmembrane protein
always faces out, the other always faces in. Further-
more, glycoproteins of the plasma membrane are in-
variably situated with their sugar residues on the outer
surface of the cell. As we shall see, the asymmetric
arrangement of membrane proteins results in functional
asymmetry. All the molecules of a given ion pump, for
example, have the same orientation in the membrane
and therefore pump in the same direction.
Integral Proteins Are Held in the Membrane
by Hydrophobic Interactions with Lipids
The firm attachment of integral proteins to membranes
is the result of hydrophobic interactions between mem-
brane lipids and hydrophobic domains of the protein.
Some proteins have a single hydrophobic sequence in
the middle (as in glycophorin) or at the amino or car-
boxyl terminus. Others have multiple hydrophobic se-
quences, each of which, when in the H9251-helical confor-
mation, is long enough to span the lipid bilayer (Fig.
11–8). The same techniques used to determine the
three-dimensional structures of soluble proteins can, in
principle, be applied to membrane proteins. In practice,
however, membrane proteins have until recently proved
difficult to crystallize. New techniques are overcoming
this obstacle, and crystallographic structures of mem-
brane proteins are regularly becoming available, yield-
ing deep insights into membrane events at the molecu-
lar level.
One of the best-studied membrane-spanning pro-
teins, bacteriorhodopsin, has seven very hydrophobic in-
ternal sequences and crosses the lipid bilayer seven
times. Bacteriorhodopsin is a light-driven proton pump
densely packed in regular arrays in the purple mem-
brane of the bacterium Halobacterium salinarum.
X-ray crystallography reveals a structure with seven H9251-
helical segments, each traversing the lipid bilayer, con-
nected by nonhelical loops at the inner and outer face
of the membrane (Fig. 11–9). In the amino acid se-
quence of bacteriorhodopsin, seven segments of about
20 hydrophobic residues can be identified, each seg-
ment just long enough to form an H9251 helix that spans the
bilayer. Hydrophobic interactions between the nonpolar
amino acids and the fatty acyl groups of the membrane
lipids firmly anchor the protein in the membrane. The
seven helices are clustered together and oriented not
quite perpendicular to the bilayer plane, providing a
transmembrane pathway for proton movement. As we
shall see in Chapter 12, this pattern of seven hy-
drophobic membrane-spanning helices is a common mo-
tif in membrane proteins involved in signal reception.
The photosynthetic reaction center of a purple bac-
terium was the first membrane protein structure solved
by crystallography. Although a more complex membrane
protein than bacteriorhodopsin, it is constructed on the
same principles. The reaction center has four protein
subunits, three of which contain H9251-helical segments that
span the membrane (Fig. 11–10). These segments are
rich in nonpolar amino acids, their hydrophobic side
chains oriented toward the outside of the molecule
where they interact with membrane lipids. The archi-
tecture of the reaction center protein is therefore the
inverse of that seen in most water-soluble proteins, in
11.1 The Composition and Architecture of Membranes 375
Inside Outside
Type I
Type II
Type III
Type IV
Type VI
Type V
–
OOC
NH
3
COO
–
H
3
N
+
+
FIGURE 11–8 Integral membrane proteins. For known proteins of the
plasma membrane, the spatial relationships of protein domains to the
lipid bilayer fall into six categories. Types I and II have only one trans-
membrane helix; the amino-terminal domain is outside the cell in type
I proteins and inside in type II. Type III proteins have multiple trans-
membrane helices in a single polypeptide. In type IV proteins, trans-
membrane domains of several different polypeptides assemble to form
a channel through the membrane. Type V proteins are held to the
bilayer primarily by covalently linked lipids (see Fig. 11–14), and type
VI proteins have both transmembrane helices and lipid (GPI) anchors.
In this figure, and in figures throughout the book, we represent
transmembrane protein segments in their most likely conformations:
as H9251 helices of six to seven turns. Sometimes these helices are shown
simply as cylinders. As relatively few membrane protein structures
have been deduced by x-ray crystallography, our representation of the
extramembrane domains is arbitrary and not necessarily to scale.
8885d_c11_369-420 2/7/04 6:58 AM Page 375 mac76 mac76:385_reb:
which hydrophobic residues are buried within the
protein core and hydrophilic residues are on the
surface (recall the structures of myoglobin and hemo-
globin, for example). In Chapter 19 we will encounter
several complex membrane proteins having multiple
transmembrane helical segments in which hydrophobic
chains are positioned to interact with the lipid bilayer.
The Topology of an Integral Membrane Protein
Can Be Predicted from Its Sequence
Determination of the three-dimensional structure of a
membrane protein, or its topology, is generally much
more difficult than determining its amino acid sequence,
which can be accomplished by sequencing the protein
or its gene. Thousands of sequences are known for mem-
brane proteins, but relatively few three-dimensional
structures have been established by crystallography or
NMR spectroscopy. The presence of unbroken sequences
of more than 20 hydrophobic residues in a membrane
protein is commonly taken as evidence that these se-
quences traverse the lipid bilayer, acting as hydropho-
bic anchors or forming transmembrane channels. Virtu-
ally all integral proteins have at least one such sequence.
Application of this logic to entire genomic sequences
leads to the conclusion that in many species, 10% to
20% of all proteins are integral membrane proteins.
What can we predict about the secondary structure
of the membrane-spanning portions of integral proteins?
An H9251-helical sequence of 20 to 25 residues is just long
enough to span the thickness (30 ?) of the lipid bilayer
(recall that the length of an H9251 helix is 1.5 ? (0.15 nm)
per amino acid residue). A polypeptide chain sur-
rounded by lipids, having no water molecules with which
to hydrogen-bond, will tend to form H9251 helices or H9252
sheets, in which intrachain hydrogen bonding is maxi-
mized. If the side chains of all amino acids in a helix are
nonpolar, hydrophobic interactions with the surround-
ing lipids further stabilize the helix.
Several simple methods of analyzing amino acid se-
quences yield reasonably accurate predictions of sec-
ondary structure for transmembrane proteins. The rel-
ative polarity of each amino acid has been determined
experimentally by measuring the free-energy change ac-
companying the movement of that amino acid side chain
from a hydrophobic solvent into water. This free energy
of transfer ranges from very exergonic for charged or
polar residues to very endergonic for amino acids with
aromatic or aliphatic hydrocarbon side chains. The
overall hydrophobicity of a sequence of amino acids is
estimated by summing the free energies of transfer for
Chapter 11 Biological Membranes and Transport376
FIGURE 11–9 Bacteriorhodopsin, a membrane-spanning protein.
(PDB ID 2AT9) The single polypeptide chain folds into seven hy-
drophobic H9251 helices, each of which traverses the lipid bilayer roughly
perpendicular to the plane of the membrane. The seven transmem-
brane helices are clustered, and the space around and between them
is filled with the acyl chains of membrane lipids. The light-absorbing
pigment retinal (see Fig. 10–21) is buried deep in the membrane in
contact with several of the helical segments (not shown). The helices
are colored to correspond with the hydropathy plot in Figure 11–11b.
Amino
terminus
Carboxyl
terminus
Inside
Outside
Inside
Outside
FIGURE 11–10 Three-dimensional structure of the photosynthetic
reaction center of Rhodopseudomonas viridis, a purple bacterium.
This was the first integral membrane protein to have its atomic struc-
ture determined by x-ray diffraction methods (PDB ID 1PRC). Eleven
H9251-helical segments from three of the four subunits span the lipid bi-
layer, forming a cylinder 45 ? (4.5 nm) long; hydrophobic residues on
the exterior of the cylinder interact with lipids of the bilayer. In this
ribbon representation, residues that are part of the transmembrane he-
lices are shown in yellow. The prosthetic groups (light-absorbing pig-
ments and electron carriers; see Fig. 19–45) are red.
8885d_c11_369-420 2/7/04 6:58 AM Page 376 mac76 mac76:385_reb:
the residues in the sequence, which yields a hydropa-
thy index for that region (see Table 3–1). To scan a
polypeptide sequence for potential membrane-spanning
segments, an investigator calculates the hydropathy in-
dex for successive segments (called windows) of a given
size, from 7 to 20 residues. For a window of seven
residues, for example, the indices for residues 1 to 7, 2
to 8, 3 to 9, and so on, are plotted as in Figure 11–11
(plotted for the middle residue in each window—
residue 4 for residues 1 to 7, for example). A region with
more than 20 residues of high hydropathy index is
presumed to be a transmembrane segment. When the
sequences of membrane proteins of known three-
dimensional structure are scanned in this way, we find
a reasonably good correspondence between predicted
and known membrane-spanning segments. Hydropathy
analysis predicts a single hydrophobic helix for gly-
cophorin (Fig. 11–11a) and seven transmembrane
segments for bacteriorhodopsin (Fig. 11–11b)—in
agreement with experimental studies.
On the basis of their amino acid sequences and hy-
dropathy plots, many of the transport proteins de-
scribed in this chapter are believed to have multiple
membrane-spanning helical regions—that is, they are
type III or type IV integral proteins (Fig. 11–8). When
predictions are consistent with chemical studies of
protein localization (such as those described above for
glycophorin and bacteriorhodopsin), the assumption
that hydrophobic regions correspond to membrane-
spanning domains is much better justified.
A further remarkable feature of many transmem-
brane proteins of known structure is the presence of Tyr
and Trp residues at the interface between lipid and
water (Fig. 11–12). The side chains of these residues
apparently serve as membrane interface anchors, able
to interact simultaneously with the central lipid phase
and the aqueous phases on either side of the membrane.
11.1 The Composition and Architecture of Membranes 377
FIGURE 11–11 Hydropathy plots. Hydropathy index (see Table 3–1)
is plotted against residue number for two integral membrane proteins.
The hydropathy index for each amino acid residue in a sequence of
defined length (called the window) is used to calculate the average
hydropathy for the residues in that window. The horizontal axis shows
the residue number in the middle of the window. (a) Glycophorin
from human erythrocytes has a single hydrophobic sequence between
residues 75 and 93 (yellow); compare this with Figure 11–7. (b) Bac-
teriorhodopsin, known from independent physical studies to have
seven transmembrane helices (see Fig. 11–9), has seven hydrophobic
regions. Note, however, that the hydropathy plot is ambiguous in the
region of segments 6 and 7. Physical studies have confirmed that this
region has two transmembrane segments.
Hydrophobic
Hydrophilic
H110023
0
3
50 100 150
50 100 150
200
200
250
25010
1
2
3
4
5
6
7
10
Hydropathy index
(b) Bacteriorhodopsin
Residue number
Hydropathy index
(a) Glycophorin
Hydrophobic
Hydrophilic
H110023
0
3
0 50 100
0 50 100
130
130
Residue number
K
+
channel Maltoporin Outer membrane
phospholipase A
OmpX Phosphoporin E
FIGURE 11–12 Tyr and Trp residues of membrane proteins cluster-
ing at the water-lipid interface. The detailed structures of these five
integral membrane proteins are known from crystallographic studies.
The K
H11001
channel (PDB ID 1BL8) is from the bacterium Streptomyces
lividans (see Fig. 11–48); maltoporin (PDB ID 1AF6), outer membrane
phospholipase A (PDB ID 1QD5), OmpX (PDB ID 1QJ9), and phos-
phoporin E (PDB ID 1PHO) are proteins of the outer membrane of
E. coli. Residues of Tyr (orange) and Trp (red) are found predominantly
where the nonpolar region of acyl chains meets the polar head group
region. Charged residues (Lys, Arg, Glu, Asp) are shown in blue; they
are found almost exclusively in the aqueous phases.
8885d_c11_369-420 2/7/04 6:58 AM Page 377 mac76 mac76:385_reb:
FepA OmpLA Maltoporin
TolC
Top view
a-Hemolysin
toxin
The hydrophobic domains of some integral mem-
brane proteins penetrate only one leaflet of the bilayer.
Cyclooxygenase, the target of aspirin action, is an ex-
ample; its hydrophobic helices do not span the whole
membrane but interact strongly with the acyl groups on
one side of the bilayer (see Box 21–2, Fig. 1a).
Not all integral membrane proteins are composed
of transmembrane H9251 helices. Another structural motif
common in membrane proteins is the H9252 barrel (see Fig.
4–20d), in which 20 or more transmembrane segments
form H9252 sheets that line a cylinder (Fig. 11–13). The same
factors that favor H9251-helix formation in the hydrophobic
interior of a lipid bilayer also stabilize H9252 barrels. When
no water molecules are available to hydrogen-bond with
the carbonyl oxygen and nitrogen of the peptide bond,
maximal intrachain hydrogen bonding gives the most
stable conformation. Planar H9252 sheets do not maximize
these interactions and are generally not found in the
membrane interior; H9252 barrels do allow all possible
hydrogen bonds and are apparently common among
membrane proteins. Porins, proteins that allow certain
polar solutes to cross the outer membrane of gram-
negative bacteria such as E. coli, have many-stranded
H9252 barrels lining the polar transmembrane passage.
A polypeptide is more extended in the H9252 confor-
mation than in an H9251 helix; just seven to nine residues of
H9252 conformation are needed to span a membrane. Recall
that in the H9252 conformation, alternating side chains
project above and below the sheet (see Fig. 4–7). In H9252
strands of membrane proteins, every second residue in
the membrane-spanning segment is hydrophobic and in-
teracts with the lipid bilayer; aromatic side chains are
commonly found at the lipid-protein interface. The
other residues may or may not be hydrophilic. The hy-
dropathy plot is not useful in predicting transmembrane
segments for proteins with H9252 barrel motifs, but as the
database of known H9252 barrel motifs increases, sequence-
based predictions of transmembrane H9252 conformations
have become feasible. For example, a number of outer
membrane proteins of gram-negative bacteria (Fig.
11–13) have been correctly predicted, by sequence
analysis, to contain H9252 barrels.
Covalently Attached Lipids Anchor Some
Membrane Proteins
Some membrane proteins contain one or more cova-
lently linked lipids of several types: long-chain fatty
acids, isoprenoids, sterols, or glycosylated derivatives of
phosphatidylinositol, GPI (Fig. 11–14). The attached
lipid provides a hydrophobic anchor that inserts into the
lipid bilayer and holds the protein at the membrane sur-
face. The strength of the hydrophobic interaction be-
tween a bilayer and a single hydrocarbon chain linked
to a protein is barely enough to anchor the protein se-
curely, but many proteins have more than one attached
Chapter 11 Biological Membranes and Transport378
FIGURE 11–13 Membrane proteins with H9252-barrel structure. Five ex-
amples are shown, viewed in the plane of the membrane; The first
four are from the E. coli outer membrane. FepA (PDB ID 1FEP), in-
volved in iron uptake, has 22 membrane-spanning H9252 strands. OmpLA
(derived from PDB ID 1QD5), a phospholipase, is a 12-stranded H9252
barrel that exists as a dimer in the membrane. Maltoporin (derived
from PDB ID 1MAL), a maltose transporter, is a trimer, each monomer
constructed of 16 H9252 strands. TolC (PDB ID 1EK9), another transporter,
has three separate subunits, each contributing four H9252 strands in this
12-stranded barrel. The Staphylococcus aureus H9251-hemolysin toxin
(PDB ID 7AHL; top view below) is composed of seven identical sub-
units, each contributing one hairpin-shaped pair of H9252 strands to the
14-stranded barrel.
8885d_c11_369-420 2/7/04 6:58 AM Page 378 mac76 mac76:385_reb:
lipid moiety. Other interactions, such as ionic attractions
between positively charged Lys residues in the protein
and negatively charged lipid head groups, probably con-
tribute to the stability of the attachment. The associa-
tion of these lipid-linked proteins with the membrane is
certainly weaker than that for integral membrane
proteins and is, in at least some cases, reversible. But
treatment with alkaline carbonate does not release
GPI-linked proteins, which are therefore, by the work-
ing definition, integral proteins.
Beyond merely anchoring a protein to the mem-
brane, the attached lipid may have a specific role. In the
plasma membrane, proteins with GPI anchors are ex-
clusively on the outer face and are confined within clus-
ters, as we shall see below, whereas other types of lipid-
linked proteins (with farnesyl or geranylgeranyl groups
attached; Fig. 11–14) are exclusively on the inner face.
Attachment of a specific lipid to a newly synthesized
membrane protein therefore has a targeting function,
directing the protein to its correct membrane location.
11.1 The Composition and Architecture of Membranes 379
Inside Outside
Palmitoyl group on
internal Cys (or Ser)
COO
H11002
CH
2
N
H
O
O
C
O
O
NH
C
N-Myristoyl group on
amino-terminal Gly
Farnesyl (or geranylgeranyl)
group on
carboxyl-terminal Cys
GPI anchor on
carboxyl terminus
CH
2
CH
3
O
CH
O
COO
H11002
NH
3
CH
2
S
O
C
C
C
C
S
Cys
H11001
O Inositol O GlcNAc
CH
2
CH
2
CH
2
CH
2
C
O
NH
Man
O CH
2
O
O
H11002
O
P
O
H11002
O
O
O
P
Man
Man
Man
H11001
NH
3
H11001
NH
3
FIGURE 11–14 Lipid-linked membrane proteins. Covalently
attached lipids anchor membrane proteins to the lipid bilayer.
A palmitoyl group is shown attached by thioester linkage to a
Cys residue; an N-myristoyl group is generally attached to an
amino-terminal Gly; the farnesyl and geranylgeranyl groups
attached to carboxyl-terminal Cys residues are isoprenoids of
15 and 20 carbons, respectively. These three lipid-protein
assemblies are found only on the inner face of the plasma
membrane. Glycosyl phosphatidylinositol (GPI) anchors are
derivatives of phosphatidylinositol in which the inositol bears
a short oligosaccharide covalently joined to the carboxyl-
terminal residue of a protein through phosphoethanolamine.
GPI-linked proteins are always on the extracellular face of the
plasma membrane.
8885d_c11_369-420 2/7/04 6:58 AM Page 379 mac76 mac76:385_reb:
SUMMARY 11.1 The Composition and Architecture
of Membranes
■ Biological membranes define cellular boundaries,
divide cells into discrete compartments, organize
complex reaction sequences, and act in signal
reception and energy transformations.
■ Membranes are composed of lipids and proteins
in varying combinations particular to each
species, cell type, and organelle. The fluid
mosaic model describes features common to all
biological membranes. The lipid bilayer is the
basic structural unit. Fatty acyl chains of
phospholipids and the steroid nucleus of sterols
are oriented toward the interior of the bilayer;
their hydrophobic interactions stabilize the
bilayer but give it flexibility.
■ Peripheral proteins are loosely associated with
the membrane through electrostatic interactions
and hydrogen bonds or by covalently attached
lipid anchors. Integral proteins associate firmly
with membranes by hydrophobic interactions
between the lipid bilayer and their nonpolar
amino acid side chains, which are oriented
toward the outside of the protein molecule.
■ Some membrane proteins span the lipid
bilayer several times, with hydrophobic
sequences of about 20 amino acid residues
forming transmembrane H9251 helices. Detection of
such hydrophobic sequences in proteins can be
used to predict their secondary structure and
transmembrane disposition. Multistranded
H9252 barrels are also common in integral
membrane proteins. Tyr and Trp residues of
transmembrane proteins are commonly found
at the lipid-water interface.
■ The lipids and proteins of membranes are
inserted into the bilayer with specific sidedness;
thus membranes are structurally and function-
ally asymmetric. Many membrane proteins
contain covalently attached oligosaccharides.
Plasma membrane glycoproteins are always
oriented with the carbohydrate-bearing domain
on the extracellular surface.
11.2 Membrane Dynamics
One remarkable feature of all biological membranes is
their flexibility—their ability to change shape without
losing their integrity and becoming leaky. The basis for
this property is the noncovalent interactions among lipids
in the bilayer and the motions allowed to individual
lipids because they are not covalently anchored to one
another. We turn now to the dynamics of membranes:
the motions that occur and the transient structures al-
lowed by these motions.
Acyl Groups in the Bilayer Interior Are Ordered
to Varying Degrees
Although the lipid bilayer structure is quite stable, its
individual phospholipid and sterol molecules have some
freedom of motion (Fig. 11–15). The structure and flex-
ibility of the lipid bilayer depend on temperature and
on the kinds of lipids present. At relatively low temper-
atures, the lipids in a bilayer form a semisolid gel phase,
in which all types of motion of individual lipid molecules
are strongly constrained; the bilayer is paracrystalline
(Fig. 11–15a). At relatively high temperatures, individual
hydrocarbon chains of fatty acids are in constant motion
produced by rotation about the carbon–carbon bonds of
the long acyl side chains. In this liquid-disordered
state, or fluid state (Fig. 11–15b), the interior of the
bilayer is more fluid than solid and the bilayer is like a
sea of constantly moving lipid. At intermediate temper-
atures, the lipids exist in a liquid-ordered state; there
is less thermal motion in the acyl chains of the lipid bi-
layer, but lateral movement in the plane of the bilayer
still takes place. These differences in bilayer state are
easily observed in liposomes composed of a single lipid,
Chapter 11 Biological Membranes and Transport380
Heat produces thermal
motion of side chains
(gel → fluid transition)
(a) Paracrystalline state (gel)
(b) Fluid state
FIGURE 11–15 Two states of bilayer lipids. (a) In the paracrystalline
state, or gel phase, polar head groups are uniformly arrayed at the
surface, and the acyl chains are nearly motionless and packed with
regular geometry; (b) in the liquid disordered state, or fluid state, acyl
chains undergo much thermal motion and have no regular organiza-
tion. Intermediate between these extremes is the liquid-ordered state,
in which individual phospholipid molecules can diffuse laterally but
the acyl groups remain extended and more or less ordered.
8885d_c11_380 2/11/04 12:13 PM Page 380 mac76 mac76:385_reb:
but biological membranes contain many lipids with a va-
riety of fatty acyl chains and thus do not show sharp
phase changes with temperature.
At temperatures in the physiological range (about
20 to 40 H11034C), long-chain saturated fatty acids (such as
16:0 and 18:0) pack well into a liquid-ordered array, but
the kinks in unsaturated fatty acids (see Fig. 10–1) in-
terfere with this packing, favoring the liquid-disordered
state. Shorter-chain fatty acyl groups have the same ef-
fect. The sterol content of a membrane (which varies
greatly with organism and organelle; Table 11–1) is an-
other important determinant of lipid state. The rigid pla-
nar structure of the steroid nucleus, inserted between
fatty acyl side chains, reduces the freedom of neigh-
boring fatty acyl chains to move by rotation about their
carbon–carbon bonds, forcing acyl chains into their fully
extended conformation. The presence of sterols there-
fore reduces the fluidity in the core of the bilayer, thus
favoring the liquid-ordered phase, and increases the
thickness of the lipid leaflet (as described below).
Cells regulate their lipid composition to achieve a
constant membrane fluidity under various growth
conditions. For example, bacteria synthesize more un-
saturated fatty acids and fewer saturated ones when cul-
tured at low temperatures than when cultured at higher
temperatures (Table 11–2). As a result of this adjust-
ment in lipid composition, membranes of bacteria
cultured at high or low temperatures have about the
same degree of fluidity.
Transbilayer Movement of Lipids Requires Catalysis
At physiological temperature, transbilayer—or “flip-
flop”—diffusion of a lipid molecule from one leaflet of
the bilayer to the other (Fig. 11–16a) occurs very slowly
if at all in most membranes. Transbilayer movement
requires that a polar or charged head group leave its
11.2 Membrane Dynamics 381
Percentage of total fatty acids
*
10 H11034C 20 H11034C 30 H11034C 40 H11034C
Myristic acid (14:0) 4 4 4 8
Palmitic acid (16:0) 18 25 29 48
Palmitoleic acid (16:1) 26 24 23 9
Oleic acid (18:1) 38 34 30 12
Hydroxymyristic acid 13 10 10 8
Ratio of unsaturated to saturated
?
2.9 2.0 1.6 0.38
Source: Data from Marr, A.G. & Ingraham, J.L. (1962) Effect of temperature on the composition of fatty acids in Escherichia coli. J. Bacteriol.
84, 1260.
*
The exact fatty acid composition depends not only on growth temperature but on growth stage and growth medium composition.
?
Ratios calculated as the total percentage of 16:1 plus 18:1 divided by the total percentage of 14:0 plus 16:0. Hydroxymyristic acid was
omitted from this calculation.
TABLE 11–2 Fatty Acid Composition of E. coli Cells Cultured at Different
Temperatures
Flippase
(a) Uncatalyzed transverse
(“flip-flop”) diffusion
(b) Transverse diffusion
catalyzed by flippase
(c) Uncatalyzed lateral
diffusion
fast
(t in seconds)
very fast
(1 mm/s)
very slow
(t in days)
1
2
1
2
FIGURE 11–16 Motion of single phospholipids in a bilayer. (a) Move-
ment from one leaflet to the other is very slow, unless (b) catalyzed
by a flippase; in contrast, lateral diffusion within the leaflet (c) is very
rapid and requires no protein catalysis.
8885d_c11_369-420 2/7/04 6:58 AM Page 381 mac76 mac76:385_reb:
Cell
Fluorescent
probe on
lipids
React cell with
fluorescent probe
to label lipids
View surface with
fluorescence
microscope
Measure rate
of fluorescence
return
With time,
unbleached
phospholipids
diffuse into
bleached area
Intense laser
beam bleaches
small area
aqueous environment and move into the hydrophobic
interior of the bilayer, a process with a large, positive
free-energy change. There are, however, situations in
which such movement is essential. For example, during
synthesis of the bacterial plasma membrane, phospho-
lipids are produced on the inside surface of the mem-
brane and must undergo flip-flop diffusion to enter the
outer leaflet of the bilayer. Similar transbilayer diffusion
must also take place in eukaryotic cells as membrane
lipids synthesized in one organelle move from the inner
to the outer leaflet and into other organelles. A family
of proteins, the flippases (Fig. 11–16b), facilitates flip-
flop diffusion, providing a transmembrane path that is
energetically more favorable and much faster than the
uncatalyzed movement.
Lipids and Proteins Diffuse Laterally in the Bilayer
Individual lipid molecules can move laterally in the plane
of the membrane by changing places with neighboring
lipid molecules (Fig. 11–16c). A molecule in one mono-
layer, or leaflet, of the lipid bilayer—the outer leaflet of
the erythrocyte plasma membrane, for example—can
diffuse laterally so fast that it circumnavigates the ery-
throcyte in seconds. This rapid lateral diffusion within
the plane of the bilayer tends to randomize the posi-
tions of individual molecules in a few seconds.
Lateral diffusion can be shown experimentally by
attaching fluorescent probes to the head groups of lipids
and using fluorescence microscopy to follow the probes
over time (Fig. 11–17). In one technique, a small region
(5 H9262m
2
) of a cell surface with fluorescence-tagged lipids
is bleached by intense laser radiation so that the irradi-
ated patch no longer fluoresces when viewed in the
much dimmer light of the fluorescence microscope.
However, within milliseconds, the region recovers its fluo-
rescence as unbleached lipid molecules diffuse into the
bleached patch and bleached lipid molecules diffuse
away from it. The rate of f luorescence recovery after
photobleaching, or FRAP, is a measure of the rate of
lateral diffusion of the lipids. Using the FRAP technique,
Chapter 11 Biological Membranes and Transport382
FIGURE 11–17 Measurement of lateral diffusion rates of lipids by
fluorescence recovery after photobleaching (FRAP). The lipids in the
outer leaflet of the plasma membrane are labeled by reaction with a
membrane-impermeant fluorescent probe (red), so the surface is uni-
formly labeled when viewed with a fluorescence microscope. A small
area is bleached by irradiation with an intense laser beam, leaving
that area nonfluorescent. With the passage of time, labeled lipid mol-
ecules diffuse into the bleached region, and it again becomes fluo-
rescent. From the time course of fluorescence return to this area, the
diffusion coefficient for the labeled lipid is determined. The rates are
typically high; a lipid moving at this speed could circumnavigate
E. coli in one second. (The FRAP method can also be used to meas-
ure the lateral diffusion of membrane proteins.)
researchers have shown that some membrane lipids dif-
fuse laterally by up to 1 H9262m/s.
Another technique, single particle tracking, allows
one to follow the movement of a single lipid molecule
in the plasma membrane on a much shorter time scale.
Results from these studies confirm the rapid lateral
diffusion within small, discrete regions of the cell sur-
8885d_c11_369-420 2/7/04 6:58 AM Page 382 mac76 mac76:385_reb:
face and show that movement from one such region to
a nearby region is inhibited; lipids behave as though
corralled by fences that they can occasionally jump
(Fig. 11–18).
Many membrane proteins seem to be afloat in a sea
of lipids. Like membrane lipids, these proteins are free
to diffuse laterally in the plane of the bilayer and are in
constant motion, as shown by the FRAP technique with
fluorescence-tagged surface proteins. Some membrane
proteins associate to form large aggregates (“patches”)
on the surface of a cell or organelle in which individual
protein molecules do not move relative to one another;
for example, acetylcholine receptors (see Fig. 11–51)
form dense patches on neuron plasma membranes at
synapses. Other membrane proteins are anchored to
internal structures that prevent their free diffusion. In
the erythrocyte membrane, both glycophorin and the
chloride-bicarbonate exchanger (p. 395) are tethered
to spectrin, a filamentous cytoskeletal protein (Fig.
11–19). One possible explanation for the pattern of lat-
eral diffusion of lipid molecules shown in Figure 11–18
is that membrane proteins immobilized by their associ-
ation with spectrin are the “fences” that define the re-
gions of relatively unrestricted lipid motion.
Sphingolipids and Cholesterol Cluster Together
in Membrane Rafts
We have seen that diffusion of membrane lipids from
one bilayer leaflet into the other is very slow unless cat-
alyzed, and that the different lipid species of the plasma
membrane are asymmetrically distributed in the two
leaflets of the bilayer (Fig. 11–5). Even within a single
leaflet, the lipid distribution is not random. Glycosphin-
golipids (cerebrosides and gangliosides), which typically
contain long-chain saturated fatty acids, form transient
clusters in the outer leaflet that largely exclude glycero-
phospholipids, which typically contain one unsaturated
fatty acyl group and a shorter saturated fatty acyl group.
The long, saturated acyl groups of sphingolipids can
form more compact, more stable associations with the
long ring system of cholesterol than can the shorter, often
unsaturated, chains of phospholipids. The cholesterol-
sphingolipid microdomains in the outer monolayer of
the plasma membrane, visible with atomic-force mi-
croscopy (Box 11–1), are slightly thicker and more
ordered (less fluid) than neighboring microdomains
rich in phospholipids (Fig. 11–20) and are more difficult
11.2 Membrane Dynamics 383
1 μm
Finish
Start
Chloride-bicarbonate
exchange proteins
Glycophorin
Plasma
membrane
Ankyrin
Spectrin
Junctional complex
(actin)
Path of single
lipid molecule
Outside
Inside
FIGURE 11–19 Restricted motion of the erythrocyte chloride-
bicarbonate exchanger and glycophorin. The proteins span the mem-
brane and are tethered to spectrin, a cytoskeletal protein, by another
protein, ankyrin, limiting their lateral mobilities. Ankyrin is anchored
in the membrane by a covalently bound palmitoyl side chain (see Fig.
11–14). Spectrin, a long, filamentous protein, is cross-linked at junc-
tional complexes containing actin. A network of cross-linked spectrin
molecules attached to the cytoplasmic face of the plasma membrane
stabilizes the membrane against deformation. This network of an-
chored membrane proteins may be the “corral” suggested by the ex-
periment shown in Figure 11–18; the lipid tracks shown here are con-
fined to subregions defined by the tethered membrane proteins.
FIGURE 11–18 Hop diffusion of individual lipid molecules. The
motion of a single fluorescent lipid molecule in a cell surface is
recorded on video by fluorescence microscopy, with a time resolu-
tion of 25 μs (equivalent to 40,000 frames/s). The track shown here
represents a molecule followed for 56 ms (a total of 2,250 frames);
the trace begins in the purple area and continues through blue, green,
and orange. The pattern of movement indicates rapid diffusion within
a confined region (about 250 nm in diameter, shown by a single color),
with occasional hops into an adjoining region. This finding suggests
that the lipids are corralled by molecular fences that they occasion-
ally jump.
8885d_c11_383 2/11/04 12:25 PM Page 383 mac76 mac76:385_reb:
to dissolve with nonionic detergents; they behave like
liquid-ordered sphingolipid rafts adrift in a sea of liq-
uid-disordered phospholipids.
These lipid rafts are remarkably enriched in two
classes of integral membrane proteins: those anchored
to the membrane by two covalently attached long-chain
saturated fatty acids (two palmitoyl groups or a pal-
mitoyl and a myristoyl group) and GPI-anchored proteins
(Fig. 11–14). Presumably these lipid anchors, like the
acyl chains of sphingolipids, form more stable associa-
tions with the cholesterol and long acyl groups in rafts
than with the surrounding phospholipids. (It is notable
Chapter 11 Biological Membranes and Transport384
BOX 11–1 WORKING IN BIOCHEMISTRY
Atomic Force Microscopy to Visualize
Membrane Proteins
In atomic force microscopy (AFM), the sharp tip of a
microscopic probe attached to a flexible cantilever is
drawn across an uneven surface such as a membrane
(Fig. 1). Electrostatic and van der Waals interactions
between the tip and the sample produce a force that
moves the probe up and down (in the z dimension)
as it encounters hills and valleys in the sample. A laser
beam reflected from the cantilever detects motions of
as little as 1 ?. In one type of atomic force microscope,
the force on the probe is held constant (relative to a
standard force, on the order of piconewtons) by a
feedback circuit that causes the platform holding the
sample to rise or fall to keep the force constant. A se-
ries of scans in the x and y dimensions (the plane of
the membrane) yields a three-dimensional contour
map of the surface with resolution near the atomic
scale—0.1 nm in the vertical dimension, 0.5 to 1.0 nm
in the lateral dimensions. The membrane rafts shown
in Figure 11–20b were visualized by this technique.
In favorable cases, AFM can be used to study
single membrane protein molecules. Single mole-
cules of bacteriorhodopsin in the purple membranes
of the bacterium Halobacterium salinarum (see
Fig. 11–9) are seen as highly regular structures (Fig.
2a). When a number of images of individual units are
superimposed with the help of a computer, the real
parts of the image reinforce each other and the noise
in individual images is averaged out, yielding a high-
resolution image of the protein (inset in Fig. 2a).
AFM of purified E. coli aquaporin, reconstituted into
lipid bilayers and viewed as if from the outside of a
cell, shows the fine details of the protein’s periplas-
mic domains (Fig. 2b). And AFM reveals that F
o
, the
proton-driven rotor of the chloroplast ATP synthase
(p. 742), is composed of many subunits (14 in Fig.
2c) arranged in a circle.
Laser
Cantilever
Laser light
detector (detects
cantilever deflection)
Platform moves to maintain
constant pressure on cantilever
tip. Excursions in the z dimension
are plotted as a function of x, y.
x y
z
Sample
+
–
10 nm 2 nm
FIGURE 2
FIGURE 1
(a) (b) (c)
8885d_c11_384 2/11/04 12:13 PM Page 384 mac76 mac76:385_reb:
that other lipid-linked proteins, those with covalently
attached isoprenyl groups such as farnesyl, are not
preferentially associated with the outer leaflet of
sphingolipid/cholesterol rafts (Fig. 11–20a).) The “raft”
and “sea” domains of the plasma membrane are not
rigidly separated; membrane proteins can move into and
out of lipid rafts on a time scale of seconds. But in the
shorter time scale (microseconds) more relevant to
many membrane-mediated biochemical processes, many
of these proteins reside primarily in a raft.
We can estimate the fraction of the cell surface
occupied by rafts from the fraction of the plasma mem-
11.2 Membrane Dynamics 385
brane that resists detergent solubilization, which can be
as high as 50% in some cases: the rafts cover half of the
ocean (Fig. 11–20b). Indirect measurements in cultured
fibroblasts suggest a diameter of roughly 50 nm for an
individual raft, which corresponds to a patch containing
a few thousand sphingolipids and perhaps 10 to 50
membrane proteins. Because most cells express more
than 50 different kinds of plasma membrane proteins, it
is likely that a single raft contains only a subset of mem-
brane proteins and that this segregation of membrane
proteins is functionally significant. For a process that
involves interaction of two membrane proteins, their
presence in a single raft would hugely increase the
likelihood of their collision. Certain membrane recep-
tors and signaling proteins, for example, appear to be
segregated together in membrane rafts. Experiments
show that signaling through these proteins can be dis-
rupted by manipulations that deplete the plasma mem-
brane of cholesterol and destroy lipid rafts.
Caveolins Define a Special Class of Membrane Rafts
Caveolin is an integral membrane protein with two
globular domains connected by a hairpin-shaped
hydrophobic domain, which binds the protein to the cy-
toplasmic leaflet of the plasma membrane. Three palmi-
toyl groups attached to the carboxyl-terminal globular
domain further anchor it to the membrane. Caveolin
(actually, a family of related caveolins) binds cholesterol
in the membrane, and the presence of caveolin forces
the associated lipid bilayer to curve inward, forming
caveolae (“little caves”) in the surface of the cell (Fig.
11–21). Caveolae are unusual rafts: they involve both
leaflets of the bilayer—the cytoplasmic leaflet, from
which the caveolin globular domains project, and the ex-
oplasmic leaflet, a typical sphingolipid/cholesterol raft
with associated GPI-anchored proteins. Caveolae are im-
plicated in a variety of cellular functions, including mem-
brane trafficking within cells and the transduction of
external signals into cellular responses. The receptors
for insulin and other growth factors, as well as certain
GTP-binding proteins and protein kinases associated
with transmembrane signaling, appear to be localized in
rafts and perhaps in caveolae. We discuss some possi-
ble roles of rafts in signaling in Chapter 12.
Certain Integral Proteins Mediate Cell-Cell
Interactions and Adhesion
Several families of integral proteins in the plasma mem-
brane provide specific points of attachment between
cells, or between a cell and extracellular matrix proteins.
Integrins are heterodimeric proteins (two unlike sub-
units, H9251 and H9252) anchored to the plasma membrane by a
single hydrophobic transmembrane helix in each sub-
unit (Fig. 11–22; see also Fig. 7–30). The large extra-
cellular domains of the H9251 and H9252 subunits combine to
form a specific binding site for extracellular proteins
Prenylated
protein
Caveolin
Cholesterol
Raft, enriched in
sphingolipids, cholesterol
GPI-linked
protein
Doubly
acylated
protein
Inside
Outside
(a)
Acyl groups
(palmitoyl,
myristoyl)
FIGURE 11–20 Microdomains (rafts) in the plasma membrane.
(a) Stable associations of sphingolipids and cholesterol in the outer
leaflet produce a microdomain, slightly thicker than other membrane
regions, that is enriched with specific types of membrane proteins.
GPI-linked proteins are commonly found in the outer leaflet of such
rafts, and proteins with one or several covalently attached long-chain
acyl groups are common in the inner leaflet. Caveolin is especially
common in inwardly curved rafts called caveolae (see Fig. 11–21).
Proteins with attached prenyl groups (such as Ras; see Fig. 12–6) tend
to be excluded from rafts. (b) The greater thickness of raft regions can
be visualized by atomic force microscopy (see Box 11–1). In this view
of a membrane region, we can see the rafts protruding from a lipid
bilayer ocean; in the rafts, sharp peaks represent GPI-linked proteins.
Note that these peaks are found almost exclusively in rafts.
(b)
8885d_c11_369-420 2/7/04 6:58 AM Page 385 mac76 mac76:385_reb:
such as collagen and fibronectin. As there are 18 dif-
ferent H9251 subunits and at least 8 different H9252 subunits, a
wide variety of specificities may be generated from var-
ious combinations of H9251 and H9252. One common determinant
of integrin binding in several extracellular partners of
integrins is the sequence Arg–Gly–Asp (RGD).
Integrins are not merely adhesives; they serve as
receptors and signal transducers, conveying in-
formation across the plasma membrane in both direc-
tions. Integrins regulate many processes, including
platelet aggregation at the site of a wound, tissue repair,
the activity of immune cells, and the invasion of tissue
by a tumor. Mutation in an integrin gene encoding the
H9252 subunit known as CD18 is the cause of leukocyte ad-
hesion deficiency in humans, a rare genetic disease in
which leukocytes fail to pass out of blood vessels to
reach sites of infection (see Fig. 7–33). Infants with a
severe defect in CD18 commonly die of infections be-
fore the age of two. ■
At least three other families of plasma membrane
proteins are also involved in surface adhesion (Fig.
11–22). Cadherins undergo homophilic (“with same
kind”) interactions with identical cadherins in an adja-
cent cell. Immunoglobulin-like proteins can undergo
either homophilic interactions with their identical coun-
terparts on another cell or heterophilic interactions with
an integrin on a neighboring cell. Selectins have ex-
tracellular domains that, in the presence of Ca
2H11001
, bind
specific polysaccharides on the surface of an adjacent
cell. Selectins are present primarily in the various types
of blood cells and in the endothelial cells that line blood
vessels (see Fig. 7–33). They are an essential part of the
blood-clotting process.
Chapter 11 Biological Membranes and Transport386
Caveola
Inside
Outside
Plasma
membrane
Caveolin dimer
(six fatty acyl
moieties)
FIGURE 11–21 Caveolin forces inward curvature in membranes. The
protein caveolin has a central hydrophobic domain and three long-
chain acyl groups on each monomeric unit, which hold the molecule
to the inside of the plasma membrane. When a number of caveolin
dimers are concentrated in a small region (a raft), they force a curva-
ture in the lipid bilayer, forming a caveola.
FIGURE 11–22 Four examples of integral
protein types that function in cell-cell
interactions. Integrins consist of H9251 and H9252
transmembrane polypeptides; their extra-
cellular domains combine to form binding
sites for divalent metal ions and proteins
of the extracellular matrix (such as
collagen and fibronectin) or for specific
surface proteins of other cells. Cadherin
has four extracellular Ca
2H11001
-binding
domains, the most distal of which
contains the site that binds to cadherin on
another cell surface. N-CAM (neuronal
cell adhesion molecule) is one of a family
of immunoglobulin-like proteins that
mediate Ca
2H11001
-independent interactions
with surface proteins of nearby cells.
Selectins bind tightly to carbohydrate
moieties in neighboring cells; this binding
is Ca
2H11001
-dependent.
Outside
Inside
Plasma
membrane
Ligand-binding
region
Adhesive
domain
Immunoglobulin-like
domains
SelectinN-CAMCadherinIntegrin
Ca
2+2H11001
Ca
2+
Ca
2+
Ca
2+
Ca
2+
2H11001
Ca
2+2H11001
2H11001
2H11001
2H11001
S
S
S
S
S
S
S
S
S
S
b a
Lectin domain
(binds carbohydrates)
8885d_c11_369-420 2/7/04 6:58 AM Page 386 mac76 mac76:385_reb:
Integral proteins play a role in many other cellular
processes. They serve as transporters and ion channels
(discussed in Section 11.3) and as receptors for hor-
mones, neurotransmitters, and growth factors (Chap-
ter 12). They are central to oxidative phosphorylation
and photosynthesis (Chapter 19) and to cell-cell and
antigen-cell recognition in the immune system (Chap-
ter 5). Integral proteins are also important players in
the membrane fusion that accompanies exocytosis, en-
docytosis, and the entry of many types of viruses into
host cells.
Membrane Fusion Is Central to Many
Biological Processes
A remarkable feature of the biological membrane is its
ability to undergo fusion with another membrane with-
out losing its continuity. Although membranes are sta-
ble, they are by no means static. Within the eukaryotic
endomembrane system (which includes the nuclear
membrane, endoplasmic reticulum, Golgi, and various
small vesicles), the membranous compartments con-
stantly reorganize. Vesicles bud from the endoplasmic
reticulum to carry newly synthesized lipids and proteins
to other organelles and to the plasma membrane. Exo-
cytosis, endocytosis, cell division, fusion of egg and
sperm cells, and entry of a membrane-enveloped virus
into its host cell all involve membrane reorganization in
which the fundamental operation is fusion of two mem-
brane segments without loss of continuity (Fig. 11–23).
Specific fusion of two membranes requires that (1)
they recognize each other; (2) their surfaces become
closely apposed, which requires the removal of water mol-
ecules normally associated with the polar head groups
of lipids; (3) their bilayer structures become locally dis-
rupted, resulting in fusion of the outer leaflet of each
membrane (hemifusion); and (4) their bilayers fuse to
form a single continuous bilayer. Receptor mediated en-
docytosis, or regulated secretion, also requires that (5)
the fusion process is triggered at the appropriate time or
in response to a specific signal. Integral proteins called
fusion proteins mediate these events, bringing about
specific recognition and a transient local distortion of the
bilayer structure that favors membrane fusion. (Note that
these fusion proteins are unrelated to the products of two
fused genes, also called fusion proteins, discussed in
Chapter 9.)
Two cases of membrane fusion are especially well
studied: the entry into a host cell of an enveloped virus
such as influenza virus, and the release of neurotrans-
mitters by exocytosis. Both processes involve com-
plexes of fusion proteins that undergo dramatic confor-
mational changes.
The influenza virus is surrounded by a membrane
containing, among other proteins, many molecules of
the hemagglutination (HA) protein (named for its abil-
ity to cause erythrocytes to clump together). The virus
enters a host cell by inducing endocytosis, which en-
closes the virus in an endosome, a small membrane
vesicle with a pH of about 5 (Fig. 11–24). At this pH, a
conformational change in the HA protein occurs, ex-
posing a sequence within the HA protein called the
fusion peptide and enabling the protein to penetrate
the endosomal membrane. The endosomal membrane
and the viral membrane are now connected through the
HA protein. Next, the HA protein bends at its middle to
form a hairpin shape, bringing its two ends together.
This pulls the two membranes into close apposition and
causes fusion of the viral membrane and the endosomal
membrane. The HA protein functions as a trimer (Fig.
11–24). In its low-pH form, three HA domains at the
closed end of the hairpin twist about each other to form
a stable, coiled structure. The fusion process involves
an intermediate stage (hemifusion) in which the outer
leaflet of the viral membrane is fused with the inner
leaflet of the endosomal membrane, while the other two
leaflets maintain their continuity. At the point of hemi-
fusion, the lipid bilayer must be temporarily disorgan-
ized, presumably caused by the HA fusion peptide
11.2 Membrane Dynamics 387
Budding of vesicles
from Golgi complex
Fusion of endosome
and lysosome
Viral infection
Fusion
of sperm and egg
Fusion of small
vacuoles (plants)
Separation of two
plasma membranes
at cell division
Exocytosis
Endocytosis
FIGURE 11–23 Membrane fusion. The fusion of two membranes is
central to a variety of cellular processes involving both organelles and
the plasma membrane.
8885d_c11_369-420 2/7/04 6:58 AM Page 387 mac76 mac76:385_reb:
Cytosol
Secretory
vesicle
Neurotransmitter molecules
v-SNARE
t-SNARE
SNAP 25
Plasma membrane
v-SNARE and t-SNARE bind to
each other, zipping up from the
amino termini and drawing the
two membranes together.
Zipping causes curvature and
lateral tension on bilayers, favoring
hemifusion between outer leaflets
and causing formation of an
energetically unfavorable void space.
Inner leaflets of both membranes
come into contact.
Complete fusion creates a fusion
pore.
Pore widens; vesicle contents
are released outside cell.
Neurotransmitter-filled vesicle
approaches plasma membrane.
Unstable
void space
Chapter 11 Biological Membranes and Transport388
FIGURE 11–24 Fusion induced by the hemagglutinin (HA) protein
during viral infection. HA protein is exposed on the membrane sur-
face of the influenza virus. When the virus moves from the neutral pH
of the interstitial fluid to the low-pH compartment (endosome) in the
host cell, HA undergoes dramatic shape changes that mediate fusion
of the viral and endosomal membranes, releasing the viral contents
into the cytoplasm.
Host cell
Virus
Endosome
HA protein in pH 7 form
has fusion peptides buried.
HA protein
(trimer)
Fusion
peptide
HA
hairpins
Low pH of endosome triggers
extension of HA fusion peptides,
which insert into endosomal
membrane.
HA folds into hairpins, drawing
viral and endosomal membranes
together.
HA fusion peptide creates local
disruption of bilayer, and hemifusion
occurs; outer monolayer of virus
fuses with inner monolayer of
endosome.
Complete fusion allows viral
contents to enter cytoplasm.
Virus binds sialic acid
receptors on host surface.
Virus triggers endocytosis;
becomes enclosed in an
endosome.
FIGURE 11–25 Fusion during neurotransmitter release at a synapse.
The membrane of the secretory vesicle contains the v-SNARE synap-
tobrevin (red). The target (plasma) membrane contains the t-SNAREs
syntaxin (blue) and SNAP25 (violet). When a local increase in [Ca
2H11001
]
signals release of neurotransmitter, the v-SNARE, SNAP25, and
t-SNARE interact, forming a coiled bundle of four H9251 helices, pulling
the two membranes together and disrupting the bilayer locally, which
leads to membrane fusion and neurotransmitter release.
8885d_c11_369-420 2/7/04 6:58 AM Page 388 mac76 mac76:385_reb:
domains. Complete fusion results in release of the viral
contents into the host cell cytoplasm.
Neurotransmitters are released at synapses when
intracellular vesicles loaded with neurotransmitter
fuse with the plasma membrane. This process involves
a family of proteins called SNARES (Fig. 11–25).
SNAREs in the cytoplasmic face of the intracellular
vesicles are called v-SNAREs; those in the target
membranes with which the vesicles fuse (the plasma
membrane during exocytosis) are t-SNAREs. Two
other proteins, SNAP25 and NSF, are also involved.
During fusion, v- and t-SNAREs bind to each other and
undergo a structural change that produces a bundle
of long thin rods made up of helices from both v- and
t-SNARES and two helices from SNAP25 (Fig. 11–25).
The two SNAREs initially interact at their ends, then
zip up into the bundle of helices. This structural
change pulls the two membranes into contact and ini-
tiates the fusion of their lipid bilayers.
The complex of SNAREs and SNAP25 is the target
of the powerful Clostridium botulinum toxin, a pro-
tease that cleaves specific bonds in these proteins, pre-
venting neurotransmission and causing the death of the
organism. Because of its very high specificity for these
proteins, purified botulinum toxin has served as a pow-
erful tool for dissecting the mechanism of neurotrans-
mitter release in vivo and in vitro.
SUMMARY 11.2 Membrane Dynamics
■ Lipids in a biological membrane can exist in
liquid-ordered or liquid-disordered states; in
the latter state, thermal motion of acyl chains
makes the interior of the bilayer fluid. Fluidity
is affected by temperature, fatty acid
composition, and sterol content.
■ Flip-flop diffusion of lipids between the inner
and outer leaflets of a membrane is very slow
except when specifically catalyzed by flippases.
■ Lipids and proteins can diffuse laterally within
the plane of the membrane, but this mobility is
limited by interactions of membrane proteins
with internal cytoskeletal structures and
interactions of lipids with lipid rafts. One class
of lipid rafts consists of sphingolipids and
cholesterol with a subset of membrane proteins
that are GPI-linked or attached to several
long-chain fatty acyl moieties.
■ Caveolin is an integral membrane protein that
associates with the inner leaflet of the plasma
membrane, forcing it to curve inward to form
caveolae, probably involved in membrane
transport and signaling.
■ Integrins are transmembrane proteins of the
plasma membrane that act both to attach cells
to each other and to carry messages between
the extracellular matrix and the cytoplasm.
■ Specific proteins mediate the fusion of two
membranes, which accompanies processes such
as viral invasion and endocytosis and
exocytosis.
11.3 Solute Transport across Membranes
Every living cell must acquire from its surroundings the
raw materials for biosynthesis and for energy produc-
tion, and must release to its environment the byprod-
ucts of metabolism. A few nonpolar compounds can
dissolve in the lipid bilayer and cross the membrane
unassisted, but for polar or charged compounds or ions,
a membrane protein is essential for transmembrane
movement. In some cases a membrane protein simply
facilitates the diffusion of a solute down its concentra-
tion gradient, but transport often occurs against a gra-
dient of concentration, electrical charge, or both, in
which case solutes must be “pumped” in a process that
requires energy (Fig. 11–26). The energy may come
directly from ATP hydrolysis or may be supplied in the
form of movement of another solute down its electro-
chemical gradient with enough energy to carry another
solute up its gradient. Ions may also move across mem-
branes via ion channels formed by proteins, or they may
be carried across by ionophores, small molecules that
mask the charge of the ions and allow them to diffuse
through the lipid bilayer. With very few exceptions, the
traffic of small molecules across the plasma membrane
is mediated by proteins such as transmembrane chan-
nels, carriers, or pumps. Within the eukaryotic cell, dif-
ferent compartments have different concentrations of
metabolic intermediates and products and of ions, and
these, too, must move across intracellular membranes
in tightly regulated, protein-mediated processes.
Passive Transport Is Facilitated
by Membrane Proteins
When two aqueous compartments containing unequal
concentrations of a soluble compound or ion are sepa-
rated by a permeable divider (membrane), the solute
moves by simple diffusion from the region of higher
concentration, through the membrane, to the region of
lower concentration, until the two compartments have
equal solute concentrations (Fig. 11–27a). When ions of
opposite charge are separated by a permeable mem-
brane, there is a transmembrane electrical gradient, a
membrane potential, V
m
(expressed in volts or milli-
volts). This membrane potential produces a force op-
posing ion movements that increase V
m
and driving ion
movements that reduce V
m
(Fig. 11–27b). Thus the di-
rection in which a charged solute tends to move spon-
taneously across a membrane depends on both the
11.3 Solute Transport across Membranes 389
8885d_c11_369-420 2/7/04 6:58 AM Page 389 mac76 mac76:385_reb:
Chapter 11 Biological Membranes and Transport390
S
in
S
in
S
out
S
out
S
in
S
in
Ion
Ion
Ion
Ion
Ion
Ion
S
out
S
out
ATP
Facilitated diffusion
(down electrochemical
gradient)
Simple diffusion
(nonpolar compounds only,
down concentration
gradient)
Primary active
transport (against
electrochemical
gradient)
Secondary
active transport
(against electrochemical
gradient, driven by ion
moving down its gradient)
Ion channel
(down
electrochemical
gradient; may
be gated by a
ligand or ion)
Ionophore-
mediated
ion transport
(down
electrochemical
gradient)
ADP + P
i
FIGURE 11–26 Summary of transport types.
FIGURE 11–27 Movement of solutes across a permeable membrane.
(a) Net movement of electrically neutral solutes is toward the side of
lower solute concentration until equilibrium is achieved. The solute
concentrations on the left and right sides of the membrane are desig-
nated C
1
and C
2
. The rate of transmembrane movement (indicated by
the large arrows) is proportional to the concentration gradient, C
1
/C
2
.
(b) Net movement of electrically charged solutes is dictated by a com-
bination of the electrical potential (V
m
) and the chemical concentra-
tion difference across the membrane; net ion movement continues un-
til this electrochemical potential reaches zero.
C
1
>> C
2
Before equilibrium
Net flux
C
1
C
2
C
1
= C
2
At equilibrium
No net flux
(a)
C
1
C
2
V
m
> 0
Before equilibrium
V
m
= 0
At equilibrium
(b)
8885d_c11_369-420 2/7/04 6:58 AM Page 390 mac76 mac76:385_reb:
chemical gradient (the difference in solute concentra-
tion) and the electrical gradient (V
m
) across the mem-
brane. Together, these two factors are referred to as the
electrochemical gradient or electrochemical po-
tential. This behavior of solutes is in accord with the
second law of thermodynamics: molecules tend to spon-
taneously assume the distribution of greatest random-
ness and lowest energy.
To pass through a lipid bilayer, a polar or charged
solute must first give up its interactions with the water
molecules in its hydration shell, then diffuse about 3 nm
(30 ?) through a solvent (lipid) in which it is poorly
soluble (Fig. 11–28). The energy used to strip away the
hydration shell and to move the polar compound from
water into and through lipid is regained as the com-
pound leaves the membrane on the other side and is re-
hydrated. However, the intermediate stage of trans-
membrane passage is a high-energy state comparable to
the transition state in an enzyme-catalyzed chemical re-
action. In both cases, an activation barrier must be over-
come to reach the intermediate stage (Fig. 11–28; com-
pare with Fig. 6–3). The energy of activation (H9004G
?
) for
translocation of a polar solute across the bilayer is so
large that pure lipid bilayers are virtually impermeable
to polar and charged species over periods of time rele-
vant to cell growth and division.
Membrane proteins lower the activation energy for
transport of polar compounds and ions by providing an
alternative path through the bilayer for specific solutes.
Proteins that bring about this facilitated diffusion, or
passive transport, are not enzymes in the usual sense;
their “substrates” are moved from one compartment to
another, but are not chemically altered. Membrane pro-
teins that speed the movement of a solute across a mem-
brane by facilitating diffusion are called transporters
or permeases.
Like enzymes, transporters bind their substrates
with stereochemical specificity through multiple weak,
noncovalent interactions. The negative free-energy
change associated with these weak interactions,
H9004G
binding
, counterbalances the positive free-energy
change that accompanies loss of the water of hydration
from the substrate, H9004G
dehydration
, thereby lowering H9004G
?
for transmembrane passage (Fig. 11–28). Transporters
span the lipid bilayer several times, forming a trans-
membrane channel lined with hydrophilic amino acid
side chains. The channel provides an alternative path
for a specific substrate to move across the lipid bilayer
without its having to dissolve in the bilayer, further low-
ering H9004G
?
for transmembrane diffusion. The result is an
increase of several orders of magnitude in the rate of
transmembrane passage of the substrate.
Transporters Can Be Grouped into Superfamilies
Based on Their Structures
We know from genomic studies that transporters con-
stitute a significant fraction of all proteins encoded in
the genomes of both simple and complex organisms.
There are probably a thousand or more different trans-
porters in the human genome. A few hundred trans-
porters from various species have been studied with bio-
chemical, genetic, and electrophysiological tools, but
investigators have determined the three-dimensional
structures for only a handful of these. Examination of
the many transporter genes reveals obvious sequence
similarities among subsets of transporters. And as ex-
perience has shown, similar amino acid sequences in
proteins generally reflect similar three-dimensional
structures and, often, similar mechanisms of action. It
is reasonable to hope that by determining the structure
and mechanism of action of at least one member of each
transporter family, we can learn much about the other
members of the family—about their structures, sub-
strate specificities, transport rates, and mechanisms of
energy coupling. A phylogenetic tree in which proteins
are grouped together based on sequence homologies has
the potential to tell us much about the transport prop-
erties of individual proteins on that tree. When this
11.3 Solute Transport across Membranes 391
Free energy,
G
?G
simple
diffusion
?G
?
transport
Simple diffusion
without transporter
Diffusion
with transporter
Transporter
Hydrated
solute
(b)
(a)
?
FIGURE 11–28 Energy changes accompanying passage of a hydro-
philic solute through the lipid bilayer of a biological membrane. (a) In
simple diffusion, removal of the hydration shell is highly endergonic,
and the energy of activation (H9004G
?
) for diffusion through the bilayer is
very high. (b) A transporter protein reduces the H9004G
?
for transmem-
brane diffusion of the solute. It does this by forming noncovalent in-
teractions with the dehydrated solute to replace the hydrogen bond-
ing with water and by providing a hydrophilic transmembrane
passageway.
8885d_c11_369-420 2/7/04 6:58 AM Page 391 mac76 mac76:385_reb:
phylogeny is combined with knowledge of structure,
specificity, or mechanism, we have a very useful and
relatively simple representation of the huge group of
transporters (Table 11–3).
Transporters can usefully be classified into super-
families, whose members have considerable similarity of
sequence and might therefore be expected to share
structural and functional properties. There are two very
broad categories of transporters: carriers and channels
(Fig. 11–29). Carriers bind their substrates with high
stereospecificity, catalyze transport at rates well below
the limits of free diffusion, and are saturable in the same
sense as are enzymes: there is some substrate concen-
tration above which further increases will not produce
a greater rate of activity. Channels generally allow
transmembrane movement at rates several orders of
magnitude greater than those typical of carriers, rates
approaching the limit of unhindered diffusion. Channels
typically show less stereospecificity than carriers and
are usually not saturable. Most channels are oligomeric
complexes of several, often identical, subunits, whereas
many carriers function as monomeric proteins. The clas-
sification as carrier or channel is the broadest distinc-
tion among transporters. Within each of these categories
Chapter 11 Biological Membranes and Transport392
TABLE 11–3 The Transporter Classification (TC) System
1.A. H9251 Helix type channels
1.A.1. Voltage-gated ion channel VIC superfamily
Voltage-gated K
H11001
channel
1.A.3. Ryanodine/IP
3
receptor Ca
2H11001
channel
1.A.8. Major intrinsic protein family
Aquaporins
1.A.9. Ligand-gated ion channel (LIC) of neurotransmitter receptors
Acetylcholine receptor/channel
1.B. H9252 Barrel porins
1.B.1. General bacterial porin (GBP) family
1.C. Pore-forming toxins
1.C.7. Diphtheria toxin family
1.C.18. Mellitin family (bee venoms)
2.A. Porters: uniporters, symporters, and antiporters
2.A.1. Major facilitator superfamily (MFS)
Lactose transporter/permease of E. coli
2.A.1.1. Sugar porter family
GLUT1 glucose transporter of erythrocyte
2.A.1.9. P
i
-H
H11001
symporter
2.A.12. ATP-ADP antiporter (AAA) family
2.A.13. C
4
-dicarboxylate uptake (Dcu) family
2.A.21. Solute-Na
H11001
symporter (SSS) family
Na
H11001
-glucose symporter in epithelial cells
2.A.73. HCO
3
H11002
transporters
HCO
3
H11002
-Cl
H11002
antiporter
2.B. Nonribosomally synthesized porters
2.B.1. Valinomycin carrier family
Valinomycin
3.A. Diphosphate bond hydrolysis–driven transporters (use PP
i
, not ATP)
3.A.1. ATP-binding cassette (ABC) superfamily
CFTR Cl
H11002
channel; multidrug transporter MDR1
3.A.2. H
H11001
- or Na
H11001
-translocating F-type, V-type, A-type ATPase superfamily
F
O
F
1
ATPase proton pump; V
O
V
1
ATPase; A
O
A
1
ATPase
3.A.3. P-type ATPase superfamily
Na
H11001
K
H11001
ATPase antiporter; SERCA Ca
2H11001
pump
Note: The three broad groups correspond to groups 1, 2, and 3 in Figure 11-29. The individual transporters listed here (screened in yellow)
are discussed in this chapter.
8885d_c11_369-420 2/7/04 6:58 AM Page 392 mac76 mac76:385_reb:
are superfamilies of various types, defined not only by
their primary sequences but by their secondary struc-
tures. Some channels are constructed primarily of hel-
ical transmembrane segments, others have H9252-barrel
structures (Table 11–3). Among the carriers, some sim-
ply facilitate diffusion down a concentration gradient;
they are the uniporter superfamily. Others (active trans-
porters) can drive substrates across the membrane
against a concentration gradient, some using energy
provided directly by a chemical reaction (primary ac-
tive transporters) and some coupling uphill transport of
one substrate with the downhill transport of another
(secondary active transporters). We now consider some
well-studied representatives of the main transporter su-
perfamilies. You will encounter some of these trans-
porters again in later chapters in the context of the
metabolic pathways in which they participate.
The Glucose Transporter of Erythrocytes Mediates
Passive Transport
Energy-yielding metabolism in erythrocytes depends on
a constant supply of glucose from the blood plasma,
where the glucose concentration is maintained at about
5 mM. Glucose enters the erythrocyte by facilitated dif-
fusion via a specific glucose transporter, at a rate about
50,000 times greater than the uncatalyzed diffusion rate.
The glucose transporter of erythrocytes (called GLUT1
to distinguish it from related glucose transporters in
other tissues) is a type III integral protein (M
r
~45,000)
with 12 hydrophobic segments, each of which is believed
to form a membrane-spanning helix. The detailed struc-
ture of GLUT1 is not yet known, but one plausible model
suggests that the side-by-side assembly of several he-
lices produces a transmembrane channel lined with hy-
drophilic residues that can hydrogen-bond with glucose
as it moves through the channel (Fig. 11–30).
The process of glucose transport can be described
by analogy with an enzymatic reaction in which the “sub-
strate” is glucose outside the cell (S
out
), the “product”
is glucose inside (S
in
), and the “enzyme” is the trans-
porter, T. When the rate of glucose uptake is measured
11.3 Solute Transport across Membranes 393
Transporters
Carriers Channels
Secondary
active
transporters
Uniporters
Primary
active
transporters
1
2 3
FIGURE 11–29 Classification of transporters. The numbers here cor-
respond to the main subdivisions in Table 11–3.
Outside
Inside
+
NH
3
COO
–
Hydrophobic
Polar
Charged
(a)
1
Ser
Leu
Val
Thr
Asn
Ile
Phe
2
3
4
5
6
7
(b)
Ser Leu Val Thr Asn Phe Ile
(c)
Glc
FIGURE 11–30 Proposed structure of GLUT1. (a) Transmembrane he-
lices are represented as oblique (angled) rows of three or four amino
acid residues, each row depicting one turn of the H9251 helix. Nine of the
12 helices contain three or more polar or charged amino acid residues,
often separated by several hydrophobic residues. (b) A helical wheel
diagram shows the distribution of polar and nonpolar residues on the
surface of a helical segment. The helix is diagrammed as though ob-
served along its axis from the amino terminus. Adjacent residues in
the linear sequence are connected with arrows, and each residue is
placed around the wheel in the position it occupies in the helix; re-
call that 3.6 residues are required to make one complete turn of the
H9251 helix. In this example, the polar residues (blue) are on one side of
the helix and the hydrophobic residues (yellow) on the other. This is,
by definition, an amphipathic helix. (c) Side-by-side association of five
or six amphipathic helices, each with its polar face oriented toward
the central cavity, can produce a transmembrane channel lined with
polar and charged residues. This channel provides many opportuni-
ties for hydrogen bonding with glucose as it moves through the trans-
porter. The three-dimensional structure of GLUT1 has not yet been de-
termined by x-ray crystallography, but researchers expect that the
hydrophilic transmembrane channels of this and many other trans-
porters and ion channels will resemble this model.
8885d_c11_369-420 2/7/04 6:58 AM Page 393 mac76 mac76:385_reb:
as a function of external glucose concentration (Fig.
11–31), the resulting plot is hyperbolic; at high
external glucose concentrations the rate of uptake ap-
proaches V
max
. Formally, such a transport process can
be described by the equations
in which k
1
, k
H110021
, and so forth, are the forward and re-
verse rate constants for each step; T
2
is the transporter
conformation that faces out, and T
2
the one that faces
in. The steps are summarized in Figure 11–32.
The rate equations for this process can be derived
exactly as for enzyme-catalyzed reactions (Chapter 6),
yielding an expression analogous to the Michaelis-
Menten equation:
V
0
H11005
V
max
[S]
out
H5007H5007
K
t
H11001 [S]
out
S
out
S
in
S
out
H11001 T
1
H11001 T
2
T
1
k
1
k
H110021
?
S
in
T
2
?
k
3
k
H110023
k
4
k
H110024
k
2
k
H110022
in which V
0
is the initial velocity of accumulation of glu-
cose inside the cell when its concentration in the
surrounding medium is [S]
out
, and K
t
(K
transport
) is a
constant analogous to the Michaelis constant, a combi-
nation of rate constants that is characteristic of each
transport system. This equation describes the initial
velocity, the rate observed when [S]
in
H11005 0. As is the case
for enzyme-catalyzed reactions, the slope-intercept
form of the equation describes a linear plot of 1/V
0
against 1/[S]
out
, from which we can obtain values of K
t
and V
max
(Fig. 11–31b). When [S] H11005 K
t
, the rate of up-
take is
1
?
2
V
max
; the transport process is half-saturated.
The concentration of blood glucose, 4.5 to 5 mM, is about
Chapter 11 Biological Membranes and Transport394
Extracellular glucose
concentration, [S]
out
(mM)
Initial velocity of glucose
entry
,
V
0
(
m
M
/min)
V
max
V
max
K
t
(a)
1
2
1
[S]
out
1
mM
K
t
1
V
max
1
( )
H11002
1
H9262
M
/min
1
V
0
(
)
(b)
FIGURE 11–31 Kinetics of glucose transport into erythrocytes. (a) The
initial rate of glucose entry into an erythrocyte, V
0
, depends upon the
initial concentration of glucose on the outside, [S]
out
. (b) Double-
reciprocal plot of the data in (a). The kinetics of facilitated diffusion
is analogous to the kinetics of an enzyme-catalyzed reaction. Com-
pare these plots with Figure 6–11, and Figure 1 in Box 6–1. Note that
K
t
is analogous to K
m
, the Michaelis constant.
D-Glucose
Inside
T
1
T
2
T
1
T
2
T
1
Outside
1
2
3
4
FIGURE 11–32 Model of glucose transport into erythrocytes by
GLUT1. The transporter exists in two conformations: T
1
, with the
glucose-binding site exposed on the outer surface of the plasma mem-
brane, and T
2
, with the binding site exposed on the inner surface. Glu-
cose transport occurs in four steps. 1 Glucose in blood plasma binds
to a stereospecific site on T
1
; this lowers the activation energy for 2
a conformational change from S
out
H11554 T
1
to S
in
H11554 T
2
, effecting the trans-
membrane passage of the glucose. 3 Glucose is now released from
T
2
into the cytoplasm, and 4 the transporter returns to the T
1
confor-
mation, ready to transport another glucose molecule.
8885d_c11_394 2/11/04 12:13 PM Page 394 mac76 mac76:385_reb:
three times K
t
, which ensures that GLUT1 is nearly sat-
urated with substrate and operates near V
max
.
Because no chemical bonds are made or broken in
the conversion of S
out
to S
in
, neither “substrate” nor
“product” is intrinsically more stable, and the process
of entry is therefore fully reversible. As [S]
in
approaches
[S]
out
, the rates of entry and exit become equal. Such a
system is therefore incapable of accumulating the sub-
strate (glucose) within a cell at concentrations above
that in the surrounding medium; it simply achieves equi-
libration of glucose on the two sides of the membrane
much faster than would occur in the absence of a spe-
cific transporter. GLUT1 is specific for D-glucose, hav-
ing a measured K
t
of 1.5 mM. For the close analogs D-
mannose and D-galactose, which differ only in the
position of one hydroxyl group, the values of K
t
are 20
and 30 mM, respectively; and for L-glucose, K
t
exceeds
3,000 mM. Thus GLUT1 shows the three hallmarks of
passive transport: high rates of diffusion down a
concentration gradient, saturability, and specificity.
Twelve glucose transporters are encoded in the hu-
man genome, each with unique kinetic properties, pat-
terns of tissue distribution, and function (Table 11–4).
In liver, GLUT2 transports glucose out of hepatocytes
when liver glycogen is broken down to replenish blood
glucose. GLUT2 has a K
t
of about 66 mM and can there-
fore respond to increased levels of intracellular glucose
(produced by glycogen breakdown) by increasing out-
ward transport. Skeletal muscle and adipose tissue have
yet another glucose transporter, GLUT4 (K
t
H11005 5 mM),
which is distinguished by its stimulation by insulin: its
activity increases when release of insulin signals a high
blood glucose concentration, thus increasing the rate of
glucose uptake into muscle and adipose tissue (Box
11–2 describes some malfunctions of this transporter).
The Chloride-Bicarbonate Exchanger Catalyzes
Electroneutral Cotransport of Anions across the
Plasma Membrane
The erythrocyte contains another facilitated diffusion
system, an anion exchanger that is essential in CO
2
transport to the lungs from tissues such as skeletal mus-
cle and liver. Waste CO
2
released from respiring tissues
into the blood plasma enters the erythrocyte, where it
is converted to bicarbonate (HCO
3
H11002
) by the enzyme car-
bonic anhydrase. (Recall that HCO
3
H11002
is the primary
buffer of blood pH; see Box 2–4). The HCO
3
H11002
reenters
the blood plasma for transport to the lungs (Fig. 11–33).
Because HCO
3
H11002
is much more soluble in blood plasma
than is CO
2
, this roundabout route increases the ca-
pacity of the blood to carry carbon dioxide from the tis-
sues to the lungs. In the lungs, HCO
3
H11002
reenters the ery-
throcyte and is converted to CO
2
, which is eventually
released into the lung space and exhaled. To be effec-
tive, this shuttle requires very rapid movement of HCO
3
H11002
across the erythrocyte membrane.
The chloride-bicarbonate exchanger, also called
the anion exchange (AE) protein, increases the
permeability of the erythrocyte membrane to HCO
3
H11002
more than a millionfold. Like the glucose transporter, it
is an integral protein that probably spans the membrane
at least 12 times. This protein mediates the simultane-
ous movement of two anions: for each HCO
3
H11002
ion that
moves in one direction, one Cl
H11002
ion moves in the op-
posite direction (Fig. 11–33), with no net transfer of
charge; the exchange is electroneutral. The coupling
of Cl
H11002
and HCO
3
H11002
movements is obligatory; in the ab-
sence of chloride, bicarbonate transport stops. In this
respect, the anion exchanger is typical of all systems,
called cotransport systems, that simultaneously carry
11.3 Solute Transport across Membranes 395
TABLE 11–4 Glucose Transporters in the Human Genome
Transporter Tissue(s) where expressed Gene Role
*
GLUT1 Ubiquitous SLC2A1 Basal glucose uptake
GLUT2 Liver, pancreatic islets, intestine SLC2A2 In liver, removal of excess glucose from
blood; in pancreas, regulation of insulin release
GLUT3 Brain (neuronal) SLC2A3 Basal glucose uptake
GLUT4 Muscle, fat, heart SLC2A4 Activity increased by insulin
GLUT5 Intestine, testis, kidney, sperm SLC2A5 Primarily fructose transport
GLUT6 Spleen, leukocytes, brain SLC2A6 Possibly no transporter function
GLUT7 Liver microsomes SLC2A7 —
GLUT8 Testis, blastocyst, brain SLC2A8 —
GLUT9 Liver, kidney SLC2A9 —
GLUT10 Liver, pancreas SLC2A10 —
GLUT11 Heart, skeletal muscle SLC2A11 —
GLUT12 Skeletal muscle, adipose, small intestine SLC2A12 —
*
Dash indicates role uncertain.
8885d_c11_369-420 2/7/04 6:58 AM Page 395 mac76 mac76:385_reb:
BOX 11–2 BIOCHEMISTRY IN MEDICINE
Defective Glucose and Water Transport in
Two Forms of Diabetes
When ingestion of a carbohydrate-rich meal causes
blood glucose to exceed the usual concentration be-
tween meals (about 5 mM), excess glucose is taken up
by the myocytes of cardiac and skeletal muscle (which
store it as glycogen) and by adipocytes (which convert
it to triacylglycerols). Glucose uptake into myocytes
and adipocytes is mediated by the glucose transporter
GLUT4. Between meals, some GLUT4 is present in the
plasma membrane, but most is sequestered in the
membranes of small intracellular vesicles (Fig. 1). In-
sulin released from the pancreas in response to high
blood glucose triggers the movement of these intra-
cellular vesicles to the plasma membrane, where they
fuse, thus exposing GLUT4 molecules on the outer sur-
face of the cell (see Fig. 12–8). With more GLUT4 mol-
ecules in action, the rate of glucose uptake increases
15-fold or more. When blood glucose levels return to
normal, insulin release slows and most GLUT4 mole-
cules are removed from the plasma membrane and
stored in vesicles.
In type I (juvenile onset) diabetes mellitus, the in-
ability to release insulin (and thus to mobilize glucose
transporters) results in low rates of glucose uptake
into muscle and adipose tissue. One consequence is
a prolonged period of high blood glucose after a
carbohydrate-rich meal. This condition is the basis for
the glucose tolerance test used to diagnose diabetes
(Chapter 23).
The water permeability of epithelial cells lining
the renal collecting duct in the kidney is due to the
presence of an aquaporin (AQP-2) in their apical
plasma membranes (facing the lumen of the duct).
Antidiuretic hormone (ADH) regulates the retention
of water by mobilizing AQP-2 molecules stored in
vesicle membranes within the epithelial cells, much as
insulin mobilizes GLUT4 in muscle and adipose tissue.
When the vesicles fuse with the epithelial cell plasma
membrane, water permeability greatly increases and
more water is reabsorbed from the collecting duct and
returned to the blood. When the ADH level drops,
AQP-2 is resequestered within vesicles, reducing
water retention. In the relatively rare human disease
diabetes insipidus, a genetic defect in AQP-2 leads
to impaired water reabsorption by the kidney. The
result is excretion of copious volumes of very dilute
urine.
FIGURE 1 Regulation by insulin of glucose transport by GLUT4 into a myocyte.
When insulin level drops,
glucose transporters are
removed from the plasma
membrane by endocytosis,
forming small vesicles.
2
3
1
5
4
Glucose transporters
“stored” within cell in
membrane vesicles.
Patches of the endosome enriched with
glucose transporters bud off to become
small vesicles, ready to return to the
surface when insulin levels rise again.
The smaller
vesicles fuse with
larger endosome.
Glucose
transporter
Plasma
membrane
Insulin receptor
Insulin
When insulin interacts with its receptor, vesicles
move to surface and fuse with the plasma
membrane, increasing the number of glucose
transporters in the plasma membrane.
8885d_c11_396 2/11/04 12:14 PM Page 396 mac76 mac76:385_reb:
two solutes across a membrane. When, as in this case,
the two substrates move in opposite directions, the
process is antiport. In symport, two substrates are
moved simultaneously in the same direction. As we
noted earlier, transporters that carry only one substrate,
such as the erythrocyte glucose transporter, are uni-
port systems (Fig. 11–34).
The human genome has genes for three closely
related chloride-bicarbonate exchangers, all with the
same predicted transmembrane topology. Erythrocytes
contain the AE1 transporter, AE2 is prominent in liver,
and AE3 is present in plasma membranes of the brain,
heart, and retina. Similar anion exchangers are also
found in plants and microorganisms.
Active Transport Results in Solute Movement against
a Concentration or Electrochemical Gradient
In passive transport, the transported species always
moves down its electrochemical gradient and is not ac-
cumulated above the equilibrium concentration. Active
transport, by contrast, results in the accumulation of a
solute above the equilibrium point. Active transport is
thermodynamically unfavorable (endergonic) and takes
place only when coupled (directly or indirectly) to an
exergonic process such as the absorption of sunlight, an
oxidation reaction, the breakdown of ATP, or the con-
comitant flow of some other chemical species down its
electrochemical gradient. In primary active trans-
port, solute accumulation is coupled directly to an ex-
ergonic chemical reaction, such as conversion of ATP to
ADP H11001 P
i
(Fig. 11–35). Secondary active transport
occurs when endergonic (uphill) transport of one solute
is coupled to the exergonic (downhill) flow of a differ-
ent solute that was originally pumped uphill by primary
active transport.
The amount of energy needed for the transport of
a solute against a gradient can be calculated from the
initial concentration gradient. The general equation for
the free-energy change in the chemical process that con-
verts S to P is
H9004G H11005 H9004GH11032H11034 H11001 RT ln [P]/[S] (11–1)
where R is the gas constant, 8.315 J/mol H11554 K, and T is
the absolute temperature. When the “reaction” is simply
11.3 Solute Transport across Membranes 397
Carbon dioxide produced
by catabolism enters
erythrocyte.
Bicarbonate
dissolves in
blood plasma.
Carbon dioxide leaves
erythrocyte and is
exhaled.
CO
2
H11001 H
2
O
CO
2
H11001 H
2
O
HH11001 Cl
H11002 H11002H11001
HCO
3
HH11001 ClHCO
3
Bicarbonate enters
erythrocyte from
blood plasma.
CO
2
H11002H11002
H11002H11002H11001
H11002H11002
CO
2
Chloride-bicarbonate
exchange protein
HCO
3
Cl
HCO
3
Cl
In respiring tissues
In lungs
carbonic anhydrase
carbonic anhydrase
S
Uniport Symport Antiport
Cotransport
S
1
S
2
S
2
S
1
FIGURE 11–33 Chloride-bicarbonate exchanger of the erythrocyte
membrane. This cotransport system allows the entry and exit of HCO
3
H11002
without changes in the transmembrane electrical potential. Its role is
to increase the CO
2
-carrying capacity of the blood.
FIGURE 11–34 Three general classes of transport systems. Trans-
porters differ in the number of solutes (substrates) transported and the
direction in which each is transported. Examples of all three types of
transporters are discussed in the text. Note that this classification tells
us nothing about whether these are energy-requiring (active transport)
or energy-independent (passive transport) processes.
FIGURE 11–35 Two types of active transport. (a) In primary active
transport, the energy released by ATP hydrolysis drives solute move-
ment against an electrochemical gradient. (b) In secondary active
transport, a gradient of ion X (often Na
H11001
) has been established by
primary active transport. Movement of X down its electrochemical gra-
dient now provides the energy to drive cotransport of a second solute
(S) against its electrochemical gradient.
(a) Primary active
transport
(b) Secondary active
transport
ATP
ADP H11001P
i
ATP
ADP H11001P
i
X
X
X
X
X
X
X
X
X
X
X
X
X
X
XX
X
X
X
X
X
X
X
S
S
S
S
S
S
S
X
S
X
X
8885d_c11_369-420 2/7/04 6:58 AM Page 397 mac76 mac76:385_reb:
transport of a solute from a region where its concen-
tration is C
1
to a region where its concentration is C
2
,
no bonds are made or broken and the standard free-en-
ergy change, H9004GH11032H11034, is zero. The free-energy change for
transport, H9004G
t
, is then
H9004G
t
H11005 RT ln H5007
C
C
2
1
H5007 (11–2)
If there is a tenfold difference in concentration between
two compartments, the cost of moving 1 mol of an un-
charged solute at 25 H11034C across a membrane separating
the compartments is therefore
H9004G
t
H11005 (8.315 J/mol H11554 K)(298 K)(ln 10/1) H11005 5,700 J/mol
H11005 5.7 kJ/mol
Equation 11–2 holds for all uncharged solutes.
When the solute is an ion, its movement without an
accompanying counterion results in the endergonic sep-
aration of positive and negative charges, producing an
electrical potential; such a transport process is said to
be electrogenic. The energetic cost of moving an ion
depends on the electrochemical potential (p. 391), the
sum of the chemical and electrical gradients:
H9004G
t
H11005 RT ln
H20898
H5007
C
C
2
1
H5007
H20899
H11001 Z H9004H9274 (11–3)
where Z is the charge on the ion, is the Faraday con-
stant (96,480 J/V H11554 mol), and H9004H9274 is the transmembrane
electrical potential (in volts). Eukaryotic cells typically
have electrical potentials across their plasma mem-
branes of about 0.05 to 0.1 V (with the inside negative
relative to the outside), so the second term of Equation
11–3 can make a significant contribution to the total
free-energy change for transporting an ion. Most cells
maintain more than tenfold differences in ion concen-
trations across their plasma or intracellular membranes,
and for many cells and tissues active transport is there-
fore a major energy-consuming process.
The mechanism of active transport is of fundamen-
tal importance in biology. As we shall see in Chapter 19,
the formation of ATP in mitochondria and chloroplasts
occurs by a mechanism that is essentially ATP-driven
ion transport operating in reverse. The energy made
available by the spontaneous flow of protons across a
membrane is calculable from Equation 11–3; remember
that H9004G for flow down an electrochemical gradient has
a negative value, and H9004G for transport of ions against
an electrochemical gradient has a positive value.
P-Type ATPases Undergo Phosphorylation during
Their Catalytic Cycles
The family of active transporters called P-type ATPases
are ATP-driven cation transporters that are reversibly
phosphorylated by ATP as part of the transport cycle;
phosphorylation forces a conformational change that is
central to moving the cation across the membrane. All
P-type transport ATPases have similarities in amino acid
sequence, especially near the Asp residue that under-
goes phosphorylation, and all are sensitive to inhibition
by the phosphate analog vanadate.
Each P-type ATPase transporter is an integral protein
with ten predicted membrane-spanning regions in a sin-
gle polypeptide; some also have a second subunit. The
P-type transporters are very widely distributed. In ani-
mal tissues, the Na
H11001
K
H11001
ATPase (an antiporter for Na
H11001
and K
H11001
) and the Ca
2H11001
ATPase (a uniporter for Ca
2H11001
)
are ubiquitous P-type ATPases that maintain differences
in the ionic composition of the cytosol and the extra-
cellular medium. Parietal cells in the lining of the mam-
malian stomach have a P-type ATPase that pumps H
H11001
and K
H11001
across the plasma membrane, thereby acidify-
ing the stomach contents. In vascular plants, a P-type
ATPase pumps protons out of the cell, establishing an
electrochemical difference of as much as 2 pH units and
250 mV across the plasma membrane. A similar P-type
ATPase in the bread mold Neurospora pumps protons
out of cells to establish an inside-negative membrane
potential, which is used to drive the uptake of substrates
and ions from the surrounding medium by secondary
active transport. Bacteria use P-type ATPases to pump
out toxic heavy metal ions such as Cd
2H11001
and Cu
2H11001
.
In virtually every animal cell type, the concentra-
tion of Na
H11001
is lower in the cell than in the surrounding
medium, and the concentration of K
H11001
is higher (Fig.
11–36). This imbalance is maintained by a primary ac-
tive transport system in the
plasma membrane. The en-
zyme Na
H11545
K
H11545
ATPase, dis-
covered by Jens Skou in 1957,
couples breakdown of ATP to
the simultaneous movement
of both Na
H11001
and K
H11001
against
their electrochemical gradi-
ents. For each molecule of
ATP converted to ADP and P
i
,
the transporter moves two K
H11001
ions inward and three Na
H11001
ions outward across the
plasma membrane. The Na
H11001
K
H11001
ATPase is an integral
protein with two subunits (M
r
~50,000 and ~110,000),
both of which span the membrane.
The detailed mechanism by which ATP hydrolysis
is coupled to transport awaits determination of the pro-
tein’s three-dimensional structure, but a current model
(Fig. 11–37) proposes that the ATPase cycles between
two forms, a phosphorylated form (designated P-Enz
II
)
with high affinity for K
H11001
and low affinity for Na
H11001
, and a
dephosphorylated form (Enz
I
) with high affinity for Na
H11001
Vanadate
V
O
H11002
OH
O
Phosphate
P
O
H11002
OH
OO
H11002
O
H11002
Chapter 11 Biological Membranes and Transport398
Jens Skou
8885d_c11_369-420 2/7/04 6:58 AM Page 398 mac76 mac76:385_reb:
and low affinity for K
H11001
. The conversion of ATP to ADP
and P
i
takes place in two steps catalyzed by the enzyme,
involving formation then hydrolysis of the phospho-
enzyme:
(1) ATP H11001 Enz
I
88n ADP H11001 P-Enz
II
(2) P-Enz
II
H11001 H
2
O 88n Enz
I
H11001 P
i
Sum: ATP H11001 H
2
O 88n ADP H11001 P
i
Because three Na
H11001
ions move outward for every two K
H11001
ions that move inward, the process is electrogenic—it
creates a net separation of charge across the membrane.
The result is a transmembrane potential of H1100250 to H1100270
mV (inside negative relative to outside), which is char-
acteristic of most animal cells and essential to the con-
duction of action potentials in neurons. The central role
of the Na
H11001
K
H11001
ATPase is reflected in the energy invested
in this single reaction: about 25% of the total energy
consumption of a human at rest!
The steroid derivative ouabain (pronounced
wah’-bane; from waa bayyo, Somali for “arrow
poison”) is a potent and specific inhibitor of the Na
H11001
K
H11001
ATPase. Oubain binds preferentially to the form of the
enzyme that is open to the extracellular side, locking in
two Na
H11001
ions and preventing the changes of conforma-
tion necessary to ion transport. Another very potent
toxin, palytoxin (produced by a coral on the Hawaiian
shoreline), also targets the Na
H11001
K
H11001
ATPase, but it binds
to the protein so as to lock it into a position in which
the ion-binding sites are permanently accessible from
both sides, converting the transporter into a nonspecific
ion channel. This allows exit of K
H11001
from cells and de-
flates the (essential) ion gradient across the plasma
membrane, which accounts for the high toxicity of this
compound.
OH
CH
3
O
H
H
H
HH
OH OH
O
Ouabain
OH
OH
HO
HO
HC CH
2
OC
O
CH
2
H
3
C
C
OH
11.3 Solute Transport across Membranes 399
Membrane potential H11005
H1100250 to H1100270 mV
H11001
H11001H11001H11001 H11001H11001H11001
H11002H11002H11002 H11002H11002H11002
H11001H11001H11001 H11001H11001H11001H11001
H11002H11002H11002 H11002H11002H11002H11002
H11001
H11001
H11001
H11001
H11002
H11002
H11002
H11002
H11002
H11002
H11002
H11002
H11002
H11002
H11001
H11001
H11001
H11001
H11001
Extracellular fluid
or blood plasma
3 Na
H11001
Na
H11001
K
H11001
ATPase
2
K
H11001
Cytosol [K
H11001
] H11005 140 mM
[Na
H11001
] H11005 12 mM
[K
H11001
] H11005 4 mM
[Na
H11001
] H11005 145 mM
ATP ADP H11001 P
i
FIGURE 11–36 Na
H11545
K
H11545
ATPase. In animal cells, this active transport
system is primarily responsible for setting and maintaining the intra-
cellular concentrations of Na
H11001
and K
H11001
and for generating the trans-
membrane electrical potential. It does this by moving three Na
H11001
out
of the cell for every two K
H11001
it moves in. The electrical potential is cen-
tral to electrical signaling in neurons, and the gradient of Na
H11001
is used
to drive the uphill cotransport of solutes in many cell types.
FIGURE 11–37 Postulated mechanism of Na
H11545
and K
H11545
transport by
the Na
H11545
K
H11545
ATPase.
Transporter binds 3 Na
H11001
from the inside of the
cell.
Enz
I
P–Enz
II
P–Enz
II
Enz
I
Phosphorylation
favors P–Enz
II
.
Transporter
releases 3 Na
H11001
to the outside
and binds 2 K
H11001
from the outside
of the cell.
Dephosphorylation
favors Enz
I
.
Transporter releases
2 K
H11001
to the inside.
OutsideInside
P
i
2 K
H11001
2 K
H11001
3 Na
H11001
P
P
ATP
ADP
3 Na
H11001
8885d_c11_399 2/11/04 12:14 PM Page 399 mac76 mac76:385_reb:
Ouabain and another steroid derivative, digitoxi-
genin, are the active ingredients of digitalis, an extract
of the leaves of the foxglove plant. (Ouabain is found in
lower concentrations in a number of other plants, pre-
sumably serving to discourage herbivores.) Digitalis has
been used to treat congestive heart failure since its in-
troduction for that purpose (treatment of “dropsy”) by
the British physician William Withering in 1785. It
strengthens heart muscle contractions without increas-
ing the heart rate and thus increases the efficiency of
the heart. Digitalis inhibits the efflux of Na
H11001
, raising the
intracellular [Na
H11001
] enough to activate a Na
H11001
-Ca
2H11001
antiporter in cardiac muscle. The increased influx of
Ca
2H11001
through this antiporter produces elevated cytoso-
lic [Ca
2H11001
], which strengthens the contractions of the
heart. The potency of ouabain in animals led to the sug-
gestion (50 years ago) that this plant product might act
by mimicking a normal regulator of the Na
H11001
K
H11001
ATPase
produced in animals, and it now appears that this may
be so. Ouabain itself has been isolated from bovine ad-
renal glands and has been detected in the blood plasma
and hypothalamus of mammals. ■
P-Type Ca
2H11545
Pumps Maintain a Low Concentration
of Calcium in the Cytosol
The cytosolic concentration of free Ca
2H11001
is generally at
or below 100 nM, far lower than that in the surrounding
medium, whether pond water or blood plasma. The ubiq-
uitous occurrence of inorganic phosphates (P
i
and PP
i
)
at millimolar concentrations in the cytosol necessitates
a low cytosolic Ca
2H11001
concentration, because inorganic
phosphate combines with calcium to form relatively in-
soluble calcium phosphates. Calcium ions are pumped
out of the cytosol by a P-type ATPase, the plasma mem-
brane Ca
2H11545
pump. Another P-type Ca
2H11001
pump in the
endoplasmic reticulum moves Ca
2H11001
into the ER lumen,
a compartment separate from the cytosol. In myocytes,
Ca
2H11001
is normally sequestered in a specialized form of
endoplasmic reticulum called the sarcoplasmic reticu-
lum. The sarcoplasmic and endoplasmic reticulum
calcium (SERCA) pumps are closely related in struc-
ture and mechanism, and both are inhibited by the
tumor-promoting agent thapsigargin, which does not af-
fect the plasma membrane Ca
2H11001
pump.
The plasma membrane Ca
2H11001
pump and SERCA
pumps are integral proteins that cycle between phos-
phorylated and dephosphorylated conformations in a
mechanism similar to that for Na
H11001
K
H11001
ATPase (Fig.
11–37). Phosphorylation favors a conformation with a
high-affinity Ca
2H11001
-binding site exposed on the cyto-
plasmic side, and dephosphorylation favors one with a
low-affinity Ca
2H11001
-binding site on the lumenal side. By
this mechanism, the energy released by hydrolysis of
ATP during one phosphorylation-dephosphorylation cy-
cle drives Ca
2H11001
across the membrane against a large
electrochemical gradient.
The Ca
2H11001
pump of the sarcoplasmic reticulum,
which comprises 80% of the protein in that membrane,
consists of a single polypeptide (M
r
~100,000) that
spans the membrane ten times and has three cytoplas-
mic domains formed by loops that connect the trans-
membrane helices (Fig. 11–38). The two Ca
2H11001
-binding
sites are located near the middle of the membrane bi-
Chapter 11 Biological Membranes and Transport400
ATP
binding
site
N domain
A domain
Phosphorylation
site (Asp
351
)
P domain
Ca
2+
binding
sites
Ca
2+
Cytoplasm
ER lumen
90°
20°
FIGURE 11–38 Structure of the Ca
2H11545
pump of sarcoplasmic reticu-
lum. (PDB ID 1EUL) Ten transmembrane helices surround the path for
Ca
2H11001
movement through the membrane. Two of the helices are inter-
rupted near the middle of the bilayer, and their nonhelical regions
form the binding sites for two Ca
2H11001
ions (green). The carboxylate
groups of an Asp residue in one helix and a Glu residue in another
are central to the Ca
2H11001
-binding sites. Three globular domains extend
from the cytoplasmic side: the N (nucleotide-binding) domain has the
binding site for ATP; the P (phosphorylation) domain contains the
Asp
351
residue (blue) that undergoes reversible phosphorylation, and
the A (actuator) domain somehow mediates the structural changes that
alter the Ca
2H11001
affinity of the Ca
2H11001
-binding site and its exposure to
cytoplasm or lumen. Note the long distance between the phosphoryla-
tion site and the Ca
2H11001
-binding site. There is strong evidence that dur-
ing one transport cycle, the N domain tips about 20H11034 to the right, bring-
ing the ATP site close to Asp
351
, and that during each catalytic cycle
the A domain twists by about 90H11034 around the normal (perpendicular)
to the membrane. These conformational changes must expose the
Ca
2H11001
-binding site first on one side of the membrane, then on the other,
changing the Ca
2H11001
affinity of the site from high on the cytoplasmic
side to lower on the lumenal side. A complete understanding of the
coupling between phosphorylation and Ca
2H11001
transport awaits deter-
mination of all the conformations involved in the cycle.
8885d_c11_369-420 2/7/04 6:58 AM Page 400 mac76 mac76:385_reb:
layer, 40 to 50 ? from the phosphorylated Asp residue
characteristic of all P-type ATPases, so the effects of
Asp phosphorylation are not direct. They must be me-
diated by conformational changes that alter the affinity
for Ca
2H11001
and open a path for Ca
2H11001
release on the lu-
menal side of the membrane.
The amino acid sequences of the SERCA pumps and
the Na
H11001
K
H11001
ATPase share 30% identity and 65% se-
quence similarity, and their topology relative to the
membrane is also the same. Thus it seems likely that
the Na
H11001
K
H11001
ATPase structure is similar to that of the
SERCA pumps and that all P-type ATPase transporters
share the same basic structure.
F-Type ATPases Are Reversible, ATP-Driven
Proton Pumps
The F-type ATPase active transporters play a central
role in energy-conserving reactions in mitochondria,
bacteria, and chloroplasts; we discuss that role in detail
in our description of oxidative phosphorylation and pho-
tophosphorylation in Chapter 19. The F-type ATPases
catalyze the uphill transmembrane passage of protons
driven by ATP hydrolysis (“F-type” originated in the
identification of these ATPases as energy-coupling fac-
tors). The F
o
integral membrane protein complex (Fig.
11–39; subscript o denoting its inhibition by the drug
oligomycin) provides a transmembrane pore for protons,
and the peripheral protein F
1
(subscript 1 indicating
that it was the first of several factors isolated from mi-
tochondria) is a molecular machine that uses the energy
of ATP to drive protons uphill (into a region of higher
H
H11001
concentration). The F
o
F
1
organization of proton-
pumping transporters must have developed very early
in evolution. Eubacteria such as E. coli use an F
o
F
1
ATPase complex in their plasma membrane to pump
protons outward, and archaebacteria have a closely
homologous proton pump, the A
o
A
1
ATPase.
The reaction catalyzed by F-type ATPases is re-
versible, so a proton gradient can supply the energy to
drive the reverse reaction, ATP synthesis (Fig. 11–40).
When functioning in this direction, the F-type ATPases
are more appropriately named ATP synthases. ATP
synthases are central to ATP production in mitochon-
dria during oxidative phosphorylation and in chloro-
plasts during photophosphorylation, as well as in eu-
bacteria and archaebacteria. The proton gradient
needed to drive ATP synthesis is produced by other
types of proton pumps powered by substrate oxidation
or sunlight. As noted above, we return to a detailed de-
scription of these processes in Chapter 19.
V-type ATPases, a class of proton-transporting
ATPases structurally (and possibly mechanistically) re-
lated to the F-type ATPases, are responsible for acidi-
fying intracellular compartments in many organisms
(thus V for vacuolar). Proton pumps of this type main-
tain the vacuoles of fungi and higher plants at a pH be-
tween 3 and 6, well below that of the surrounding cy-
tosol (pH 7.5). V-type ATPases are also responsible for
the acidification of lysosomes, endosomes, the Golgi
complex, and secretory vesicles in animal cells. All V-
type ATPases have a similar complex structure, with an
integral (transmembrane) domain (V
o
) that serves as a
proton channel and a peripheral domain (V
1
) that con-
tains the ATP-binding site and the ATPase activity. The
mechanism by which V-type ATPases couple ATP hy-
drolysis to the uphill transport of protons is not under-
stood in detail.
11.3 Solute Transport across Membranes 401
H9252
H9251
H9251
H9280
H9251
H9252
H9252
H9253
H9254
b
2
c
12
H
+
H
+
a
ADP + P
i
ATP
F
1
F
o
(f)
FIGURE 11–39 Structure of the F
o
F
1
ATPase/ATP synthase. F-type
ATPases have a peripheral domain, F
1
, consisting of three H9251 subunits,
three H9252 subunits, one H9254 subunit (purple), and a central shaft (the H9253 sub-
unit, green). The integral portion of F-type ATPases, F
o
(yellow), has
multiple copies of c, one a, and two b subunits. F
o
provides a trans-
membrane channel through which about four protons are pumped
(red arrows) for each ATP hydrolyzed on the H9252 subunits of F
1
. The
remarkable mechanism by which these two events are coupled is de-
scribed in detail in Chapter 19. It involves rotation of F
o
relative to F
1
(black arrow). The structures of V
o
V
1
and A
o
A
1
are essentially similar
to that of F
o
F
1
, and the mechanisms are probably similar, too.
ATP ADP H11001P
i
H
H11001
H
H11001
ATP
synthase
Proton
pump
H
H11001
FIGURE 11–40 Reversibility of F-type ATPases. An ATP-driven proton
transporter also can catalyze ATP synthesis (red arrows) as protons flow
down their electrochemical gradient. This is the central reaction in the
processes of oxidative phosphorylation and photophosphorylation,
both described in detail in Chapter 19.
8885d_c11_369-420 2/7/04 6:58 AM Page 401 mac76 mac76:385_reb:
ABC Transporters Use ATP to Drive the Active
Transport of a Wide Variety of Substrates
ABC transporters (Fig. 11–41) constitute a large
family of ATP-dependent transporters that pump
amino acids, peptides, proteins, metal ions, various
lipids, bile salts, and many hydrophobic compounds,
including drugs, out of cells against a concentration
gradient. One ABC transporter in humans, the multi-
drug transporter (MDR1), is responsible for the
striking resistance of certain tumors to some generally
effective antitumor drugs. MDR1 has a broad substrate
specificity for hydrophobic compounds, including, for
example, the chemotherapeutic drugs adriamycin,
doxorubicin, and vinblastine. By pumping these drugs
out of the cell, the transporter prevents their accu-
mulation within a tumor and thus blocks their thera-
peutic effects. MDR1 is an integral membrane protein
(M
r
170,000) with 12 transmembrane segments and
two ATP-binding domains (“cassettes”), which give the
family its name: ATP-binding cassette transporters.
All ABC transporters have two nucleotide-binding
domains (NBDs) and two transmembrane domains (Fig.
11–41). In some cases, all these domains are in a single
long polypeptide; other ABC transporters have two sub-
units, each contributing an NBD and a domain with six
(or in some cases ten) transmembrane helices. Although
many of the ABC transporters are in the plasma mem-
brane, some types are also found in the endoplasmic
reticulum and in the membranes of mitochondria and
lysosomes. Most ABC transporters act as pumps, but at
least some members of the superfamily act as ion chan-
nels that are opened and closed by ATP hydrolysis. The
CFTR transporter (Box 11–3) is a Cl
H11002
channel operated
by ATP hydrolysis.
The NBDs of all ABC proteins are similar in sequence
and presumably in three-dimensional structure; they are
the conserved molecular motor that can be coupled to a
wide variety of pumps and channels. When coupled with
a pump, the ATP-driven motor moves solutes against a
concentration gradient; when coupled with an ion chan-
nel, the motor opens and closes the channel using ATP
as energy source. The stoichiometry of ABC pumps is
about one ATP hydrolyzed per molecule of substrate
transported, but neither the mechanism of coupling nor
the site of substrate binding are known.
Some ABC transporters have very high speci-
ficity for a single substrate; others are more
promiscuous. The human genome contains at least 48
genes that encode ABC transporters, many of which are
involved in maintaining the lipid bilayer and in trans-
porting sterols, sterol derivatives, and fatty acids
throughout the body. The flippases that move mem-
brane lipids from one leaflet of the bilayer to the other
are ABC transporters, and the cellular machinery for ex-
porting excess cholesterol includes an ABC transporter.
Mutations in the genes that encode some of these pro-
teins contribute to several genetic diseases, including
cystic fibrosis (Box 11–3), Tangier disease (p. 827), reti-
nal degeneration, anemia, and liver failure.
ABC transporters are also present in simpler ani-
mals and in plants and microorganisms. Yeast has 31
genes that encode ABC transporters, Drosophila has
56, and E. coli has 80, representing 2% of its entire
genome. The presence of ABC transporters that confer
antibiotic resistance in pathogenic microbes
(Pseudomonas aeruginosa, Staphylococcus aureus,
Candida albicans, Neisseria gonorrhoeae, and Plas-
modium falciparum) is a serious public health con-
cern and makes these transporters attractive targets for
drug design. ■
Ion Gradients Provide the Energy for Secondary
Active Transport
The ion gradients formed by primary transport of Na
H11001
or H
H11001
can in turn provide the driving force for cotrans-
port of other solutes. Many cell types contain transport
Chapter 11 Biological Membranes and Transport402
FIGURE 11–41 Structures of two ABC trans-
porters of E. coli. (a) The lipid A flippase
MsbA (PDB ID 1JSQ) and (b) the vitamin B
12
importer BtuCD (PDB ID 1L7V). Both struc-
tures are homodimers. The two nucleotide-
binding domains (NBDs, in red) extend into
the cytoplasm. In (b), residues involved in
ATP binding and hydrolysis are shown as ball-
and-stick structures. Each monomer of MsbA
has six transmembrane helical segments
(blue), and each monomer of BtuCD has ten.
NBDs
Cytoplasm
Extracellular
space
(a) MsbA (b) BtuCD
NBDs
8885d_c11_402 2/11/04 12:47 PM Page 402 mac76 mac76:385_reb:
systems that couple the spontaneous, downhill flow of
these ions to the simultaneous uphill pumping of an-
other ion, sugar, or amino acid (Table 11–5). The lac-
tose transporter (lactose permease) of E. coli is
the well-studied prototype for proton-driven cotrans-
porters. This protein consists of a single polypeptide
chain (417 residues) that functions as a monomer to
transport one proton and one lactose molecule into the
11.3 Solute Transport across Membranes 403
BOX 11–3 BIOCHEMISTRY IN MEDICINE
A Defective Ion Channel in Cystic Fibrosis
Cystic fibrosis (CF) is a serious and relatively com-
mon hereditary disease of humans. About 5% of white
Americans are carriers, having one defective and one
normal copy of the gene. Only individuals with two de-
fective copies show the severe symptoms of the dis-
ease: obstruction of the gastrointestinal and respira-
tory tracts, commonly leading to bacterial infection of
the airways and death due to respiratory insufficiency
before the age of 30. In CF, the thin layer of mucus
that normally coats the internal surfaces of the lungs
is abnormally thick, obstructing air flow and providing
a haven for pathogenic bacteria, particularly Staphy-
lococcus aureus and Pseudomonas aeruginosa.
The defective gene in CF patients was discov-
ered in 1989. It encodes a membrane protein called
cystic fibrosis transmembrane conductance regula-
tor, or CFTR. Hydropathy analysis predicted that
CFTR has 12 transmembrane helices and is struc-
turally related to the multidrug (MDR1) transporters
of drug-resistant tumors (Fig. 1). The normal CFTR
protein proved to be an ion channel specific for
Cl
H11002
ions. The Cl
H11002
channel activity increases greatly
when phosphoryl groups are transferred from ATP
to several side chains of the protein, catalyzed by
cAMP-dependent protein kinase (Chapter 12). The
mutation responsible for CF in 70% of cases results
in deletion of a Phe residue at position 508, with the
effect that the mutant protein is not correctly folded
and inserted in the plasma membrane. Other muta-
tions yield a protein that is inserted properly but can-
not be activated by phosphorylation. In each case, the
fundamental problem is a nonfunctional Cl
H11002
channel
in the epithelial cells that line the airways (Fig. 2),
the digestive tract, and exocrine glands (pancreas,
sweat glands, bile ducts, and vas deferens).
Normally, epithelial cells that line the inner sur-
face of the lungs secrete a substance that traps and
kills bacteria, and the cilia on the epithelial cells con-
stantly sweep away the resulting debris. When CFTR
is defective or missing, this process is less efficient,
and frequent infections by bacteria such as S. aureus
and P. aeruginosa progressively damage the lungs
and reduce respiratory efficiency.
FIGURE 1 Topology of the cystic fibrosis transmembrane conduc-
tance regulator, CFTR. It has 12 transmembrane helices, and three
functionally significant domains extend from the cytoplasmic sur-
face: NBD
1
and NBD
2
are nucleotide-binding domains to which
ATP binds, and a regulatory domain (R domain) is the site of phos-
phorylation by cAMP-dependent protein kinase. Oligosaccharide
chains are attached to several residues on the outer surface of the
segment between helices 7 and 8. The most commonly occurring
mutation leading to CF is the deletion of Phe
508
, in the NBD
1
do-
main. The structure of CFTR is very similar to that of the multidrug
transporter of tumors, described in the text.
FIGURE 2 Mucus lining the surface of the lungs traps bacteria. In
healthy lungs, these bacteria are killed and swept away by the ac-
tion of cilia. In CF, the bactericidal activity is impaired, resulting in
recurring infections and progressive damage to the lungs.
Oligosaccharide
chains of
glycoprotein
Outside
Inside
R domain
Phe
508
COO
–
NH
3
NBD
1
NBD
2
+
8885d_c11_369-420 2/7/04 6:58 AM Page 403 mac76 mac76:385_reb:
cell, with the net accumulation of lactose (Fig. 11–42).
E. coli normally produces a gradient of protons and
charge across its plasma membrane by oxidizing fuels
and using the energy of oxidation to pump protons
outward. (This mechanism is discussed in detail in
Chapter 19.) The lipid bilayer is impermeable to pro-
tons, but the lactose transporter provides a route for
proton reentry, and lactose is simultaneously carried
into the cell by symport. The endergonic accumulation
of lactose is thereby coupled to the exergonic flow of
protons into the cell, with a negative overall free-energy
change.
The lactose transporter is one member of the
major facilitator superfamily (MFS) of trans-
porters, which comprises 28 families. Almost all proteins
in this superfamily have 12 transmembrane domains
(the few exceptions have 14). The proteins share rela-
tively little sequence homology, but the similarity of
their secondary structures and topology suggests a
common tertiary structure. The crystallographic solu-
tion of the E. coli lactose transporter by Ron Kaback
and So Iwata in 2003 may provide a glimpse of this gen-
eral structure (Fig. 11–43a). The protein has 12 trans-
membrane helices, and connecting loops that protrude
into the cytoplasm or the periplasmic space. All six
amino-terminal and six carboxyl-terminal helices form
very similar domains, to produce a structure with a
rough twofold symmetry. In the crystallized form of the
protein, a large aqueous cavity is exposed on the cyto-
plasmic side of the membrane. The substrate-binding
site is in this cavity, more or less in the middle of the
membrane. The side of the transporter facing outward
(the periplasmic face) is closed tightly, with no channel
big enough for lactose to enter. The proposed mecha-
Chapter 11 Biological Membranes and Transport404
TABLE 11–5 Cotransport Systems Driven by Gradients of Na
H11545
or H
H11545
Transported solute Cotransported solute
Organism/tissue/cell type (moving against its gradient) (moving down its gradient) Type of transport
E. coli Lactose H
H11001
Symport
Proline H
H11001
Symport
Dicarboxylic acids H
H11001
Symport
Intestine, kidney (vertebrates) Glucose Na
H11001
Symport
Amino acids Na
H11001
Symport
Vertebrate cells (many types) Ca
2H11001
Na
H11001
Antiport
Higher plants K
H11001
H
H11001
Antiport
Fungi (Neurospora)K
H11001
H
H11001
Antiport
Lactose
(outside)
Lactose
transporter
Proton pump
(inhibited by CN
H11002
)
Lactose
(inside)
H
H11001
H
H11001
H
H11001
H
H11001
H
H11001
H
H11001
H
H11001
H
H11001
H
H11001
H
H11001
H
H11001
(a)
H11001H11001
H11001
H11001
H11001
H11001
H11002
H11002
H11002
H11002
H11002
H11002
Fuel
CO
2
[Lactose]
inside
Time
(b)
[Lactose]
medium
Active
transport
+CN
–
, or mutation
at Glu
325
or Arg
302
Efflux
CN
H11002
inhibition of
fuel oxidation
FIGURE 11–42 Lactose uptake in E. coli. (a) The primary transport
of H
H11001
out of the cell, driven by the oxidation of a variety of fuels, es-
tablishes both a proton gradient and an electrical potential (inside neg-
ative) across the membrane. Secondary active transport of lactose into
the cell involves symport of H
H11001
and lactose by the lactose transporter.
The uptake of lactose against its concentration gradient is entirely de-
pendent on this inflow of H
H11001
, driven by the electrochemical gradient.
(b) When the energy-yielding oxidation reactions of metabolism are
blocked by cyanide (CN
H11002
), the lactose transporter allows equilibra-
tion of lactose inside and outside the cell via passive transport. Mu-
tations that affect Glu
325
or Arg
302
have the same effect as cyanide.
The dashed line represents the concentration of lactose in the sur-
rounding medium.
8885d_c11_369-420 2/7/04 6:58 AM Page 404 mac76 mac76:385_reb:
nism for transmembrane passage of the substrate (Fig.
11–43b) involves a rocking motion between the two
domains, driven by substrate binding and proton
movement, alternately exposing the substrate-binding
domain to the cytoplasm and to the periplasm. This
so-called rocking banana model is similar to that
shown in Figure 11–32 for GLUT1.
How is proton movement into the cell coupled with
lactose uptake? Extensive genetic studies of the lactose
transporter have established that of the 417 residues in
the protein, only 6 are absolutely essential for cotrans-
port of H
H11001
and lactose—some for lactose binding, oth-
ers for proton transport. Mutation in either of two
residues (Glu
325
and Arg
302
; Fig. 11–43) results in a pro-
tein still able to catalyze facilitated diffusion of lactose
but incapable of coupling H
H11001
flow to uphill lactose trans-
port. A similar effect is seen in wild-type (unmutated)
cells when their ability to generate a proton gradient is
blocked with CN
H11002
: the transporter carries out facilitated
diffusion normally, but it cannot pump lactose against a
concentration gradient (Fig. 11–42b). The balance be-
tween the two conformations of the lactose transporter
is affected by changes in charge pairing between side
chains.
In intestinal epithelial cells, glucose and certain
amino acids are accumulated by symport with Na
H11001
,
down the Na
H11001
gradient established by the Na
H11001
K
H11001
ATPase of the plasma membrane (Fig. 11–44). The api-
cal surface of the intestinal epithelial cell is covered with
microvilli, long thin projections of the plasma membrane
11.3 Solute Transport across Membranes 405
Cytoplasm
Periplasmic
space
(a) (b)
FIGURE 11–43 Structure of the lactose transporter (lactose perme-
ase) of E. coli. (a) Ribbon representation viewed parallel to the plane
of the membrane shows the 12 transmembrane helices arranged in
two nearly symmetrical domains shown in different shades of blue. In
the form of the protein for which the crystal structure was determined,
the substrate sugar (red) is bound near the middle of the membrane
where it is exposed to the cytoplasm (derived from PDB ID 1PV7). (b)
The structural changes postulated to take place during one transport
cycle. The two halves of the transporter undergo a large, reversible
conformational change in which the two domains tilt relative to each
other, exposing the substrate-binding site first to the periplasm (struc-
ture on the right), where lactose is picked up, then to the cytoplasm
(left), where the lactose is released. The interconversion of the two
forms is driven by changes in the pairing of charged (protonatable)
side chains such as those of Glu
325
and Arg
302
(green), which is af-
fected by the transmembrane proton gradient.
Apical
surface
Microvilli
Intestinal
lumen
Blood
Na
H11001
K
H11001
ATPase
Basal
surface
Glucose
Glucose
Glucose uniporter
GLUT2 (facilitates
downhill efflux)
Epithelial cell
2 Na
H11001
Na
H11001
-
glucose
symporter
(driven by high
extracellular [Na
H11001
])
2 K
H11001
3 Na
H11001
FIGURE 11–44 Glucose transport in intestinal
epithelial cells. Glucose is cotransported with Na
H11001
across the apical plasma membrane into the epithelial
cell. It moves through the cell to the basal surface,
where it passes into the blood via GLUT2, a passive
glucose transporter. The Na
H11001
K
H11001
ATPase continues to
pump Na
H11001
outward to maintain the Na
H11001
gradient that
drives glucose uptake.
8885d_c11_405 2/11/04 2:06 PM Page 405 mac76 mac76:385_reb:
that greatly increase the surface area exposed to the
intestinal contents. Na
H11545
-glucose symporters in the
apical plasma membrane take up glucose from the in-
testine in a process driven by the downhill flow of Na
H11001
:
2Na
H11001
out
H11001 glucose
out
88n 2Na
H11001
in
H11001 glucose
in
The energy required for this process comes from two
sources: the greater concentration of Na
H11001
outside than
inside (the chemical potential) and the transmembrane
potential (the electrical potential), which is inside-
negative and therefore draws Na
H11001
inward. The electro-
chemical potential of Na
H11001
is
where n H11005 2, the number of Na
H11001
ions cotransported
with each glucose molecule. Given the typical membrane
potential of H1100250 mV, an intracellular [Na
H11001
] of 12 mM,
and an extracellular [Na
H11001
] of 145 mM, the energy, H9004G,
made available as two Na
H11001
ions reenter the cell is 22.5 kJ,
enough to pump glucose against a large concentration
gradient:
H9004G
t
H11005H1100222.5 kJ H11005 RT ln
and thus
H33360 9,000
That is, the cotransporter can pump glucose inward
until its concentration within the epithelial cell is about
9,000 times that in the intestine. As glucose is pumped
from the intestine into the epithelial cell at the apical
surface, it is simultaneously moved from the cell into
the blood by passive transport through a glucose trans-
porter (GLUT2) in the basal surface (Fig. 11–44). The
crucial role of Na
H11001
in symport and antiport systems such
as these requires the continued outward pumping of
Na
H11001
to maintain the transmembrane Na
H11001
gradient.
[Glucose]
in
H5007H5007
[Glucose]
out
[glucose]
in
H5007H5007
[glucose]
out
H9004G H11005 RT ln
[NaH11001]
in
H11001 n H9004E
[Na
H11001
]
out
Because of the essential role of ion gradients in
active transport and energy conservation, compounds
that collapse ion gradients across cellular membranes
are effective poisons, and those that are specific for in-
fectious microorganisms can serve as antibiotics. One
such substance is valinomycin, a small cyclic peptide
that neutralizes the K
H11001
charge by surrounding it with
six carbonyl oxygens (Fig. 11–45). The hydrophobic
peptide then acts as a shuttle, carrying K
H11001
across mem-
branes down its concentration gradient and deflating
that gradient. Compounds that shuttle ions across mem-
branes in this way are called ionophores (“ion bear-
ers”). Both valinomycin and monensin (a Na
H11001
-carrying
ionophore) are antibiotics; they kill microbial cells by
disrupting secondary transport processes and energy-
conserving reactions.
Aquaporins Form Hydrophilic Transmembrane
Channels for the Passage of Water
A family of integral proteins discovered by Peter Agre,
the aquaporins (AQPs), provide channels for rapid
movement of water molecules
across all plasma membranes
(Table 11–6 lists a few exam-
ples). Ten aquaporins are
known in humans, each with
its specialized role. Erythro-
cytes, which swell or shrink
rapidly in response to abrupt
changes in extracellular os-
molarity as blood travels
through the renal medulla,
have a high density of aqua-
porin in their plasma mem-
branes (2 H11003 10
5
copies of
AQP-1 per cell). In the nephron (the functional unit of
the kidney), the plasma membranes of proximal
renal tubule cells have five different aquaporin types.
Chapter 11 Biological Membranes and Transport406
FIGURE 11–45 Valinomycin, a peptide ionophore that binds K
H11545
. In
this image, the surface contours are shown as a transparent mesh,
through which a stick structure of the peptide and a K
H11001
atom (green)
are visible. The oxygen atoms (red) that bind K
H11001
are part of a central
hydrophilic cavity. Hydrophobic amino acid side chains (yellow) coat
the outside of the molecule. Because the exterior of the K
H11001
-
valinomycin complex is hydrophobic, the complex readily diffuses
through membranes, carrying K
H11001
down its concentration gradient. The
resulting dissipation of the transmembrane ion gradient kills microbial
cells, making valinomycin a potent antibiotic.
Peter Agre
8885d_c11_406 2/11/04 12:47 PM Page 406 mac76 mac76:385_reb:
These cells reabsorb water during urine formation, a
process for which water movement across membranes
is essential (Box 11–3). The plant Arabidopsis
thaliana has 38 genes that encode various types of
aquaporins, reflecting the critical roles of water move-
ment in plant physiology. Changes in turgor pressure,
for example, require rapid movement of water across a
membrane.
Water molecules flow through an AQP-1 channel at
the rate of about 10
9
s
H110021
. For comparison, the highest
known turnover number for an enzyme is that for
catalase, 4 H11003 10
7
s
H110021
, and many enzymes have turnover
numbers between 1 s
H110021
and 10
4
s
H110021
(see Table 6–7).
The low activation energy for passage of water through
aquaporin channels (H9004G
?
H11021 15 kJ/mol) suggests that
water moves through the channels in a continuous
stream, in the direction dictated by the osmotic gradi-
ent. (For a discussion of osmosis, see p. 57.) It is
essential that aquaporins not allow passage of protons
(hydronium ions, H
3
O
H11001
), which would collapse mem-
brane electrochemical potentials. And they do not. What
is the basis for this extraordinary selectivity?
We find an answer in the structure of AQP-1, as de-
termined by x-ray diffraction analysis (Fig. 11–46).
AQP-1 has four monomers (each M
r
28,000) associated
in a tetramer, each monomer forming a transmembrane
pore with a diameter (2 to 3 ?) sufficient to allow pas-
sage of water molecules in single file. Each monomer
consists of six transmembrane helical segments and two
shorter helices, each of which contains the sequence
Asn–Pro–Ala (NPA). The NPA-containing short helices
extend toward the middle of the bilayer from opposite
11.3 Solute Transport across Membranes 407
(b)
(a) (c)
(d)
FIGURE 11–46 Structure of an aquaporin, AQP-1. The protein is a
tetramer of identical monomeric units, each of which forms a trans-
membrane pore (derived from PBD ID 1J4N). (a) Surface model viewed
perpendicular to the plane of the membrane. The protein contains four
pores, one in each subunit. (The opening at the junction of the sub-
units is not a pore.) (b) An AQP-1 tetramer, viewed in the plane of the
membrane. The helices of each subunit cluster around a central trans-
membrane pore. In each monomer, two short helical loops, one
between helices 2 and 3 and the other between 5 and 6, contain the
Asn–Pro–Ala (NPA) sequences found in all aquaporins, and form part
of the water channel. (c) Surface representation of a single subunit,
viewed in the plane of the membrane. The near side of the AQP-1
monomer has been cut away to reveal the channel running from top
to bottom. The series of water molecules (orange spheres) shows the
likely path of water molecules through the aquaporin channel, as pre-
dicted by molecular dynamics simulations in which investigators use
the properties of water and aquaporin to calculate the lowest energy
states. Hydrophilic atoms that provide selective interactions with wa-
ter in the channel are colored red. A Phe residue (Phe
58
) at the con-
striction is shown in blue. (d) A view down the channel, showing the
constriction region of the specificity pore, which lets only a molecule
as small as water pass. The side chains of Phe
58
, His
182
, Cys
191
, and
Arg
197
create this constriction.
8885d_c11_407 2/11/04 12:48 PM Page 407 mac76 mac76:385_reb:
sides, with their NPA regions overlapping in the middle
of the membrane to form part of the specificity filter—
the structure that allows only water to pass.
The residues that line the channel of each AQP-1
monomer are generally nonpolar, but carbonyl oxygens
in the peptide backbone, projecting into the narrow part
of the channel at intervals, can form hydrogen bonds
with individual water molecules as they pass through;
the two Asn residues (Asn
76
and Asn
192
) in the NPA
loops also hydrogen-bond with the water. The structure
does not admit closely spaced water molecules that
might form a chain to allow proton hopping (see Fig.
2–14), which would effectively move protons across the
membrane. Critical Arg and His residues and electric
dipoles formed by the short helices of the NPA loops
provide positive charges in positions that repel any pro-
tons that might leak through the pore.
Ion-Selective Channels Allow Rapid Movement
of Ions across Membranes
Ion-selective channels—first recognized in neurons
and now known to be present in the plasma membranes
of all cells, as well as in the intracellular membranes of
eukaryotes—provide another mechanism for moving in-
organic ions across membranes. Ion channels, together
with ion pumps such as the Na
H11001
K
H11001
ATPase, determine
a plasma membrane’s permeability to specific ions and
regulate the cytosolic concentration of ions and the
membrane potential. In neurons, very rapid changes in
the activity of ion channels cause the changes in mem-
brane potential (the action potentials) that carry signals
from one end of a neuron to the other. In myocytes,
rapid opening of Ca
2H11001
channels in the sarcoplasmic
reticulum releases the Ca
2H11001
that triggers muscle con-
traction. We discuss the signaling functions of ion chan-
nels in Chapter 12. Here we describe the structural ba-
sis for ion-channel function, using as examples a
bacterial K
H11001
channel, the neuronal Na
H11001
channel, and
the acetylcholine receptor ion channel.
Ion channels are distinguished from ion trans-
porters in at least three ways. First, the rate of flux
through channels can be several orders of magnitude
greater than the turnover number for a transporter—
10
7
to 10
8
ions/s for an ion channel, near the theoreti-
cal maximum for unrestricted diffusion. Second, ion
channels are not saturable: rates do not approach a max-
imum at high substrate concentration. Third, they are
“gated”—opened or closed in response to some cellular
event. In ligand-gated channels (which are generally
oligomeric), binding of an extracellular or intracellular
small molecule forces an allosteric transition in the pro-
tein, which opens or closes the channel. In voltage-
gated ion channels, a change in transmembrane elec-
trical potential (V
m
) causes a charged protein domain
to move relative to the membrane, opening or closing
the ion channel. Both types of gating can be very fast.
A channel typically opens in a fraction of a millisecond
and may remain open for only milliseconds, making
these molecular devices effective for very fast signal
transmission in the nervous system.
Ion-Channel Function Is Measured Electrically
Because a single ion channel typically remains open for
only a few milliseconds, monitoring this process is be-
Chapter 11 Biological Membranes and Transport408
TABLE 11–6 Aquaporins
Aquaporin Roles and/or location
AQP-1 Fluid reabsorption in proximal renal tubule; secretion of
aqueous humor in eye and cerebrospinal fluid in central
nervous system; water homeostasis in lung
AQP-2 Water permeability in renal collecting duct
(mutations produce nephrogenic diabetes insipidus)
AQP-3 Water retention in renal collecting duct
AQP-4 Cerebrospinal fluid reabsorption in central nervous system;
regulation of brain edema
AQP-5 Fluid secretion in salivary glands, lachrymal glands, and alveolar
epithelium of lung
AQP-6 Kidney
AQP-7 Renal proximal tubule, intestine
AQP-8 Liver, pancreas, colon, placenta
AQP-9 Liver, leukocytes
TIP Regulation of turgor pressure in plant tonoplast
PIP Plant plasma membrane
AQY Yeast plasma membrane
8885d_c11_369-420 2/7/04 6:58 AM Page 408 mac76 mac76:385_reb:
yond the limit of most biochemical measurements. Ion
fluxes must therefore be measured electrically, either as
changes in V
m
(in the millivolt range) or as electric cur-
rents I (in the microampere or picoampere range), us-
ing microelectrodes and appropriate amplifiers. In
patch-clamping, a technique developed by Erwin Neher
and Bert Sakmann in 1976, very small currents are
measured through a tiny region of the membrane sur-
face containing only one or a few ion-channel molecules
(Fig. 11–47). The researcher can measure the size and
duration of the current that flows during one opening
of an ion channel and can determine how often a chan-
nel opens and how that frequency is affected by trans-
membrane potential, regulatory ligands, toxins, and
other agents. Patch-clamp studies have revealed that as
many as 10
4
ions can move through a single ion chan-
nel in 1 ms. Such an ion flux represents a huge ampli-
fication of the initial signal; for example, only two acetyl-
choline molecules are needed to open an acetylcholine
receptor channel (as described below).
The Structure of a K
H11545
Channel Reveals the Basis
for Its Specificity
The structure of a potassium channel from the bac-
terium Streptomyces lividans, determined crystallo-
graphically by Roderick MacKinnon in 1998, provides
much insight into the way ion channels work. This bac-
terial ion channel is related in sequence to all other
known K
H11001
channels and serves as the prototype for such
channels, including the volt-
age-gated K
H11001
channel of neu-
rons. Among the members of
this protein family, the simi-
larities in sequence are great-
est in the “pore region,” which
contains the ion selectivity fil-
ter that allows K
H11001
(radius 1.33
?) to pass 10,000 times more
readily than Na
H11001
(radius 0.95
?)—at a rate (about 10
8
ions/s) approaching the theo-
retical limit for unrestricted
diffusion.
The K
H11001
channel consists of four identical subunits
that span the membrane and form a cone within a cone
surrounding the ion channel, with the wide end of the
double cone facing the extracellular space (Fig. 11–48).
11.3 Solute Transport across Membranes 409
Electronics to hold transmembrane
potential (V
m
) constant and measure
current flowing across membrane
Micropipette applied tightly
to plasma membrane
Patch of membrane
pulled from cell
Time
Inw
ard
current
50ms
10pA
Patch of membrane
placed in aqueous
solution
Channel
Electrodes
Micropipette
FIGURE 11–47 Electrical measurements of ion-channel function. The
“activity” of an ion channel is estimated by measuring the flow of ions
through it, using the patch-clamp technique. A finely drawn-out pipette
(micropipette) is pressed against the cell surface, and negative pres-
sure in the pipette forms a pressure seal between pipette and mem-
brane. As the pipette is pulled away from the cell, it pulls off a tiny
patch of membrane (which may contain one or a few ion channels).
After placing the pipette and attached patch in an aqueous solution,
the researcher can measure channel activity as the electric current that
flows between the contents of the pipette and the aqueous solution.
In practice, a circuit is set up that “clamps” the transmembrane po-
tential at a given value and measures the current that must flow to
maintain this voltage. With highly sensitive current detectors, re-
searchers can measure the current flowing through a single ion chan-
nel, typically a few picoamperes. The trace showing the current as a
function of time (in milliseconds) reveals how fast the channel opens
and closes, how frequently it opens, and how long it stays open.
Clamping the V
m
at different values permits determination of the ef-
fect of membrane potential on these parameters of channel function.
Erwin Neher Bert Sakmann
Roderick MacKinnon
8885d_c11_369-420 2/7/04 6:58 AM Page 409 mac76 mac76:385_reb:
Each subunit has two transmembrane H9251 helices as well
as a third, shorter helix that contributes to the pore re-
gion. The outer cone is formed by one of the trans-
membrane helices of each subunit. The inner cone,
formed by the other four transmembrane helices, sur-
rounds the ion channel and cradles the ion selectivity
filter.
Both the ion specificity and the high flux through
the channel are understandable from what we know of
the channel’s structure. At the inner and outer plasma
membrane surfaces, the entryways to the channel have
several negatively charged amino acid residues, which
presumably increase the local concentration of cations
such as K
H11001
and Na
H11001
. The ion path through the mem-
brane begins (on the inner surface) as a wide, water-
filled channel in which the ion can retain its hydration
sphere. Further stabilization is provided by the short H9251
helices in the pore region of each subunit, with the par-
tial negative charges of their electric dipoles pointed at
K
H11001
in the channel. About two-thirds of the way through
the membrane, this channel narrows in the region of the
selectivity filter, forcing the ion to give up its hydrating
water molecules. Carbonyl oxygen atoms in the back-
bone of the selectivity filter replace the water molecules
in the hydration sphere, forming a series of perfect co-
ordination shells through which the K
H11001
moves. This fa-
vorable interaction with the filter is not possible for Na
H11001
,
which is too small to make contact with all the poten-
tial oxygen ligands. The preferential stabilization of K
H11001
is the basis for the ion selectivity of the filter, and mu-
tations that change residues in this part of the protein
eliminate the channel’s ion selectivity.
There are four potential K
H11001
-binding sites along the
selectivity filter, each composed of an oxygen “cage”
that provides ligands for the K
H11001
ions (Fig. 11–49). In
the crystal structure, two K
H11001
ions are visible within the
selectivity filter, about 7.5 ? apart, and two water mol-
ecules occupy the unfilled positions. K
H11001
ions pass
through the filter in single file; their mutual electrostatic
repulsion most likely just balances the interaction of
each ion with the selectivity filter and keeps them mov-
ing. Movement of the two K
H11001
ions is concerted: first they
occupy positions 1 and 3, then they hop to positions 2
and 4 (Fig. 11–48c). The energetic difference between
these two configurations (1, 3 and 2, 4) is very small;
energetically, the selectivity pore is not a series of hills
and valleys but a flat surface, which is ideal for rapid
ion movement through the channel. The structure of the
channel appears to have been optimized during evolu-
tion to give maximal flow rates and high specificity.
The Neuronal Na
H11545
Channel Is a Voltage-Gated
Ion Channel
Sodium ion channels in the plasma membranes of neu-
rons and of myocytes of heart and skeletal muscle sense
Chapter 11 Biological Membranes and Transport410
(a)
Outside
Inside
(b)
Backbone carbonyl
oxygens form cage that
fits K
+
precisely,
replacing waters of
hydration sphere
Alternating K
+
sites
(blue or green) occupied
Helix dipole
stabilizes K
+
Large
water-filled
vestibule allows
hydration of K
+
K
+
with hydrating
water molecules
Extracellular
space
Cytosol
– –
++
FIGURE 11–48 Structure and function of the K
H11545
channel of Strep-
tomyces lividans. (PDB ID 1BL8) (a) Viewed in the plane of the mem-
brane, the channel consists of eight transmembrane helices (two from
each of the four identical subunits), forming a cone with its wide end
toward the extracellular space. The inner helices of the cone (lighter
colored) line the transmembrane channel, and the outer helices in-
teract with the lipid bilayer. Short segments of each subunit converge
in the open end of the cone to make a selectivity filter. (b) This view
perpendicular to the plane of the membrane shows the four subunits
arranged around a central channel just wide enough for a single K
H11001
ion to pass. (c) Diagram of a K
H11001
channel in cross section, showing
the structural features critical to function. (See also Fig. 11–49.) (c)
8885d_c11_369-420 2/7/04 6:58 AM Page 410 mac76 mac76:385_reb:
electrical gradients across the membrane and respond
by opening or closing. These voltage-gated ion channels
are typically very selective for Na
H11001
over other monova-
lent or divalent cations (by factors of 100 or more) and
have a very high flux rate (H1102210
7
ions/s). Normally (in
the resting state) in the closed conformation, Na
H11001
chan-
nels are opened—activated—by a reduction in the
transmembrane electrical potential, then they undergo
very rapid inactivation. Within milliseconds of the open-
ing, the channel closes and remains inactive for many
milliseconds. Activation followed by inactivation of Na
H11001
channels is the basis for signaling by neurons (see Fig.
12–5).
The essential component of a Na
H11001
channel is a sin-
gle, large polypeptide (1,840 amino acid residues) or-
ganized into four domains clustered around a central
channel (Fig. 11–50a, b), providing a path for Na
H11001
through the membrane. The path is made Na
H11001
-specific
by a “pore region” composed of the segments between
transmembrane helices 5 and 6 of each domain, which
fold into the channel. Helix 4 of each domain has a high
density of positively charged residues; this segment is
believed to move within the membrane in response to
changes in the transmembrane voltage, from the “rest-
ing” potential of about H1100260 mV (inside negative) to
about H1100130 mV. The movement of helix 4 triggers open-
ing of the channel, and this is the basis for voltage gat-
ing (Fig. 11–50c).
Inactivation of the channel is thought to occur by a
ball-and-chain mechanism. A protein domain on the cy-
tosolic surface of the Na
H11001
channel, the inactivation gate
(the ball), is tethered to the channel by a short segment
of the polypeptide (the chain) (Fig. 11–50b). This do-
main is free to move about when the channel is closed,
but when it opens, a site on the inner face of the chan-
nel becomes available for the tethered ball to bind,
blocking the channel. The length of the tether appears
to determine how long an ion channel stays open; the
longer the tether, the longer the open period. Inactiva-
tion of other ion channels may proceed by a similar
mechanism.
The Acetylcholine Receptor Is a Ligand-Gated
Ion Channel
Another very well-studied ion channel is the nicotinic
acetylcholine receptor, essential in the passage of an
electrical signal from a motor neuron to a muscle fiber
at the neuromuscular junction (signaling the muscle to
contract). (Nicotinic receptors were originally distin-
guished from muscarinic receptors by the sensitivity of
the former to nicotine, the latter to the mushroom al-
kaloid muscarine. They are structurally and functionally
different.) Acetylcholine released by the motor neuron
diffuses a few micrometers to the plasma membrane of
a myocyte, where it binds to the acetylcholine receptor.
This forces a conformational change in the receptor,
causing its ion channel to open. The resulting inward
movement of positive charges depolarizes the plasma
membrane, triggering contraction. The acetylcholine re-
ceptor allows Na
H11001
, Ca
2H11001
, and K
H11001
to pass through with
equal ease, but other cations and all anions are unable
to pass. Movement of Na
H11001
through an acetylcholine re-
ceptor ion channel is unsaturable (its rate is linear with
respect to extracellular [Na
H11001
]) and very fast—about
2 H11003 10
7
ions/s under physiological conditions.
This receptor channel is typical of many other ion
channels that produce or respond to electrical signals:
it has a “gate” that opens in response to stimulation by
a signal molecule (in this case acetylcholine) and an in-
trinsic timing mechanism that closes the gate after a
split second. Thus the acetylcholine signal is transient—
an essential feature of electrical signal conduction. We
understand the structural changes underlying gating in
the acetylcholine receptor, but not the exact mechanism
of “desensitization”—of closing the gate even in the con-
tinued presence of acetylcholine.
CH
2
CH
3
H11001
N
Acetylcholine
O
CH
2
C
CH
3
CH
3
O
CH
3
11.3 Solute Transport across Membranes 411
FIGURE 11–49 K
H11545
binding sites in the selectivity pore of the K
H11545
channel. (PDB ID 1J95) Carbonyl oxygens (red) of the peptide back-
bone in the selectivity filter protrude into the channel, interacting with
and stabilizing a K
H11001
ion passing through. These ligands are perfectly
positioned to interact with each of four K
H11001
ions, but not with the
smaller Na
H11001
ions. This preferential interaction with K
H11001
is the basis for
the ion selectivity. The mutual repulsion between K
H11001
ions results in
occupation of only two of the four K
H11001
sites at a time (both green or
both blue) and counteracts the tendency for a lone K
H11001
to stay bound
in one site. The combined effect of K
H11001
binding to carbonyl oxygens
and repulsion between K
H11001
ions ensures that an ion keeps moving,
changing positions within 10 to 100 ns, and that there are no large
energy barriers to ion flow along the path through the membrane.
8885d_c11_369-420 2/7/04 6:58 AM Page 411 mac76 mac76:385_reb:
Chapter 11 Biological Membranes and Transport412
Inside
Outside
1
2345
6
COO
H11002
Domain
Inactivation gate Voltage sensor
Selectivity filter
(pore region)
Activation
gate
I II III IV
NH
3
H11001
(a)
Outside
Inside
H11001H11001H11001H11001H11001
H11002H11002H11002H11002H11002
Outside
Inside
Activation
gate
Membrane polarized,
channel closed
Aqueous ion
channel
Membrane depolarized,
channel open
Voltage
sensor
Na
H11001
Na
H11001
H11001
H11001
H11001
H11001
H11001
H11001
H11001
H11001
H11001
H11001
H11001 H11001
H11001 H11001
H11001 H11001
H11001 H11001
H11001 H11001
(c)
Selectivity
filter (pore)
II
III
IV
Activation
gate
Voltage
sensor
Tether
1
2 3
4
56
Inactivation
gate (open)
I
(b)
FIGURE 11–50 Voltage-gated Na
H11545
channels of neurons. Sodium
channels of different tissues and organisms have a variety of subunits,
but only the principal subunit (H9251) is essential. (a) The H9251 subunit is a
large protein with four homologous domains (I to IV), each contain-
ing six transmembrane helices (1 to 6). Helix 4 in each domain (blue)
is the voltage sensor; helix 6 (orange) is thought to be the activation
gate. The segments between helices 5 and 6, the pore region (red),
form the selectivity filter, and the segment connecting domains III and
IV (green) is the inactivation gate. (b) The four domains are wrapped
about a central transmembrane channel lined with polar amino acid
residues. The segments linking helices 5 and 6 (red) in each domain
come together near the extracellular surface to form the selectivity fil-
ter, which is conserved in all Na
H11001
channels. The filter gives the chan-
nel its ability to discriminate between Na
H11001
and other ions of similar
size. The inactivation gate (green) closes (dotted lines) soon after the
activation gate opens. (c) The voltage-sensing mechanism involves
movement of helix 4 (blue) perpendicular to the plane of the mem-
brane in response to a change in transmembrane potential. As shown
at the top, the strong positive charge on helix 4 allows it to be pulled
inward in response to the inside-negative membrane potential (V
m
).
Depolarization lessens this pull, and helix 4 relaxes by moving out-
ward (bottom). This movement is communicated to the activation gate
(orange), inducing conformational changes that open the channel in
response to depolarization.
8885d_c11_369-420 2/7/04 6:58 AM Page 412 mac76 mac76:385_reb:
The nicotinic acetylcholine receptor has five sub-
units: single copies of subunits H9252, H9253, and H9254, and two
identical H9251 subunits each with an acetylcholine-binding
site. All five subunits are related in sequence and terti-
ary structure, each having four transmembrane helical
segments (M1 to M4) (Fig. 11–51a). The five subunits
surround a central pore, which is lined with their M2
helices. The pore is about 20 ? wide in the parts of the
channel that protrude on the cytoplasmic and extra-
cellular surfaces, but narrows as it passes through the
11.3 Solute Transport across Membranes 413
COO
H11002
M1 M3
M4
M2
COO
H11002
M1
M2
M3
M4
M1
M4
M2
M3
Inside
Outside
Acetylcholine
binding sites
M2 amphipathic helices
surround channel
(a)
Subunit folds into four
transmembrane a helices
NH
3
H11001
H11001
a Subunit
(b,g,d are homologous) bd
g
aa
NH
3
Closed
2 Acetylcholine
(b)
Open
Bulky hydrophobic
Leu side chains of
M2 helices close
the channel.
Binding of two acetylcholine
molecules causes twisting
of the M2 helices.
M2 helices now have
smaller, polar residues
lining the channel.
FIGURE 11–51 Structure of the acetylcholine
receptor ion channel. (a) Each of the five subunits
(H9251
2
H9252H9253H9254) has four transmembrane helices, M1 to M4.
The M2 helices are amphipathic; the others have
mainly hydrophobic residues. The five subunits are
arranged around a central transmembrane channel,
which is lined with the polar sides of the M2
helices. At the top and bottom of the channel are
rings of negatively charged amino acid residues. (b)
This top view of a cross section through the center
of the M2 helices shows five Leu side chains (one
from each M2 helix) protruding into the channel,
constricting it to a diameter too small to allow
passage of ions such as Ca
2H11001
, Na
H11001
, and K
H11001
. When
both acetylcholine receptor sites (one on each H9251
subunit) are occupied, a conformational change
occurs. As the M2 helices twist slightly, the five Leu
residues (yellow) rotate away from the channel and
are replaced by smaller, polar residues (blue). This
gating mechanism opens the channel, allowing the
passage of Ca
2H11001
, Na
H11001
, or K
H11001
.
8885d_c11_369-420 2/7/04 6:58 AM Page 413 mac76 mac76:385_reb:
lipid bilayer. Near the center of the bilayer is a ring of
bulky hydrophobic side chains of Leu residues in the M2
helices, positioned so close together that they prevent
ions from passing through the channel. Allosteric con-
formational changes induced by acetylcholine binding
to the two H9251 subunits include a slight twisting of the M2
Chapter 11 Biological Membranes and Transport414
TABLE 11–7 Transport Systems Described Elsewhere in This Text
Transport system and location Figure number Role
Adenine nucleotide antiporter of mitochondrial 19–26 Imports substrate ADP for oxidative
inner membrane phosphorylation, and exports product ATP
Acyl-carnitine/carnitine transporter of mitochondrial 17–6 Imports fatty acids into matrix for H9252 oxidation
inner membrane
P
i
-H
H11001
symporter of mitochondrial inner membrane 19–26 Supplies P
i
for oxidative phosphorylation
Malate–H9251-ketoglutarate transporter of mitochondrial 19–27 Shuttles reducing equivalents (as malate) from
inner membrane matrix to cytosol
Glutamate-aspartate transporter of mitochondrial 19–27 Completes shuttling begun by
inner membrane malate–H9251-ketoglutarate shuttle
Citrate transporter of mitochondrial inner membrane 21–10 Provides cytosolic citrate as source of acetyl-CoA
for lipid synthesis
Pyruvate transporter of mitochondrial inner 21–10 Is part of mechanism for shuttling citrate from
membrane matrix to cytosol
Fatty acid transporter of myocyte plasma 17–3 Imports fatty acids for fuel
membrane
Complex I, III, and IV proton transporters of 19–15 Acts as energy-conserving mechanism in oxidative
mitochondrial inner membrane phosphorylation, converting electron flow into
proton gradient
Thermogenin (uncoupler protein), a proton pore of 19–30, 23–22 Allows dissipation of proton gradient in
mitochondrial inner membrane mitochondria as means of thermogenesis and/or
disposal of excess fuel
Cytochrome bf complex, a proton transporter of 19–50, 19–54 Acts as proton pump, driven by electron flow
chloroplast thylakoid through the Z scheme; source of proton gradient
for photosynthetic ATP synthesis
Bacteriorhodopsin, a light-driven proton pump 19–59 Is light-driven source of proton gradient for ATP
synthesis in halophilic bacterium
F
o
F
1
ATPase/ATP synthase of mitochondrial inner 19–58 Interconverts energy of proton gradient and ATP
membrane, chloroplast thylakoid, and bacterial during oxidative phosphorylation and
plasma membrane photophosphorylation
P
i
–triose phosphate antiporter of chloroplast inner 20–15, 20–16 Exports photosynthetic product from stroma;
membrane imports P
i
for ATP synthesis
Bacterial protein transporter 27–39 Exports secreted proteins through plasma
membrane
Protein translocase of ER 27–33 Transports into ER proteins destined for plasma
membrane, secretion, or organelles
Nuclear pore protein translocase 27–37 Shuttles proteins between nucleus and cytoplasm
LDL receptor in animal cell plasma membrane 21–42 Imports, by receptor-mediated endocytosis, lipid
carrying particles
Glucose transporter of animal cell plasma 12–8 Increases capacity of muscle and adipose tissue to
membrane; regulated by insulin take up excess glucose from blood
IP
3
-gated Ca
2H11001
channel of endoplasmic reticulum 12–19 Allows signaling via changes of cytosolic Ca
2H11001
concentration
cGMP-gated Ca
2H11001
channel of retinal rod and cone 12–32 Allows signaling via rhodopsin linked to cAMP
cells phosphodiesterase in vertebrate eye
Voltage-gated Na
H11001
channel of neuron 12–5 Creates action potentials in neuronal signal
transmission
8885d_c11_369-420 2/7/04 6:58 AM Page 414 mac76 mac76:385_reb:
helices (Fig. 11–51b), which draws these hydrophobic
side chains away from the center of the channel, open-
ing it to the passage of ions.
Based on similarities between the amino acid se-
quences of other ligand-gated ion channels and the
acetylcholine receptor, the receptor channels that re-
spond to the extracellular signals H9253-aminobutyric acid
(GABA), glycine, and serotonin have been classified in
the acetylcholine receptor superfamily, and probably
share three-dimensional structure and gating mecha-
nisms. The GABA
A
and glycine receptors are anion
channels specific for Cl
H11002
or HCO
3
H11002
, whereas the
serotonin receptor, like the acetylcholine receptor, is
cation-specific. The subunits of each of these channels,
like those of the acetylcholine receptor, have four
transmembrane helical segments and form oligomeric
channels.
A second class of ligand-gated ion channels respond
to intracellular ligands: 3H11032,5H11032-cyclic guanosine mono-
nucleotide (cGMP) in the vertebrate eye, cGMP and
cAMP in olfactory neurons, and ATP and inositol 1,4,5-
trisphosphate (IP3) in many cell types. These channels
are composed of multiple subunits, each with six trans-
membrane helical domains. We discuss the signaling
functions of these ion channels in Chapter 12.
Table 11–7 shows a number of transporters not dis-
cussed in this chapter but encountered later in the book
in the context of the paths in which they act.
Defective Ion Channels Can Have Adverse
Physiological Consequences
The importance of ion channels to physiological
processes is clear from the effects of mutations
in specific ion-channel proteins (Table 11–8). Genetic
defects in the voltage-gated Na
H11001
channel of the myocyte
plasma membrane result in diseases in which muscles
are periodically either paralyzed (as in hyperkalemic pe-
riodic paralysis) or stiff (as in paramyotonia congenita).
As noted earlier, cystic fibrosis is the result of a muta-
tion that changes one amino acid in the protein CFTR,
a Cl
H11002
ion channel; the defective process here is not neu-
rotransmission but secretion by various exocrine gland
cells whose activities are tied to Cl
H11002
ion fluxes.
Many naturally occurring toxins act on ion channels,
and the potency of these toxins further illustrates the
importance of normal ion-channel function. Tetro-
dotoxin (produced by the puffer fish, Sphaeroides
rubripes) and saxitoxin (produced by the marine di-
noflagellate Gonyaulax, which causes “red tides”) act
by binding to the voltage-gated Na
H11001
channels of neurons
and preventing normal action potentials. Puffer fish is
an ingredient of the Japanese delicacy fugu, which may
be prepared only by chefs specially trained to separate
11.3 Solute Transport across Membranes 415
Ion channel Affected gene Disease
Na
H11001
(voltage-gated, skeletal muscle) SCN4A Hyperkalemic periodic paralysis (or paramyotonia congenita)
Na
H11001
(voltage-gated, neuronal ) SCN1A Generalized epilepsy with febrile seizures
Na
H11001
(voltage-gated, cardiac muscle) SCN5A Long QT syndrome 3
Ca
2H11001
(neuronal) CACNA1A Familial hemiplegic migraine
Ca
2H11001
(voltage-gated, retina) CACNA1F Congenital stationary night blindness
Ca
2H11001
(polycystin-1) PKD1 Polycystic kidney disease
K
H11001
(neuronal) KCNQ4 Dominant deafness
K
H11001
(voltage-gated, neuronal) KCNQ2 Benign familial neonatal convulsions
Nonspecific cation (cGMP-gated, retinal) CNCG1 Retinitis pigmentosa
Acetylcholine receptor (skeletal muscle) CHRNA1 Congenital myasthenic syndrome
Cl
H11002
CFTR Cystic fibrosis
TABLE 11–8 Some Diseases Resulting from Ion Channel Defects
H
2
N
H
2
N
HN
O
H
HO OH
N
H
N
N
H
O
Saxitoxin
H11001
NH
2
H11001
H
2
N
CH
2
OH
H11001
N
H
H
N
O
O
OH
HO
HO
OH
O
H11002
H
H
H
H
H
H
Tetrodotoxin
8885d_c11_369-420 2/7/04 6:58 AM Page 415 mac76 mac76:385_reb:
succulent morsel from deadly poison. Eating shellfish
that have fed on Gonyaulax can also be fatal; shellfish
are not sensitive to saxitoxin, but they concentrate it in
their muscles, which become highly poisonous to organ-
isms higher up the food chain. The venom of the black
mamba snake contains dendrotoxin, which interferes
with voltage-gated K
H11001
channels. Tubocurarine, the ac-
tive component of curare (used as an arrow poison in
the Amazon), and two other toxins from snake venoms,
cobrotoxin and bungarotoxin, block the acetylcholine re-
ceptor or prevent the opening of its ion channel. By
blocking signals from nerves to muscles, all these toxins
cause paralysis and possibly death. On the positive side,
the extremely high affinity of bungarotoxin for the
acetylcholine receptor (K
d
H11005 10
H1100215
M) has proved use-
ful experimentally: the radiolabeled toxin was used to
quantify the receptor during its purification. ■
SUMMARY 11.3 Solute Transport across
Membranes
■ Movement of polar compounds and ions across
biological membranes requires protein
transporters. Some transporters simply
facilitate passive diffusion across the membrane
from the side with higher concentration to the
side with lower. Others bring about active
movement of solutes against an electrochemical
gradient; such transport must be coupled to a
source of metabolic energy.
■ Carriers, like enzymes, show saturation and
stereospecificity for their substrates. Transport
via these systems may be passive or active.
Primary active transport is driven by ATP or
electron-transfer reactions; secondary active
transport, by coupled flow of two solutes, one
of which (often H
H11001
or Na
H11001
) flows down its
electrochemical gradient as the other is pulled
up its gradient.
■ The GLUT transporters, such as GLUT1 of
erythrocytes, carry glucose into cells by
facilitated diffusion. These transporters are
uniporters, carrying only one substrate.
Symporters permit simultaneous passage of two
substances in the same direction; examples are
the lactose transporter of E. coli, driven by the
energy of a proton gradient (lactose-H
H11001
symport), and the glucose transporter of
intestinal epithelial cells, driven by a Na
H11001
gradient (glucose-Na
H11001
symport). Antiporters
mediate simultaneous passage of two
substances in opposite directions; examples are
the chloride-bicarbonate exchanger of
erythrocytes and the ubiquitous Na
H11001
K
H11001
ATPase.
■ In animal cells, Na
H11001
K
H11001
ATPase maintains the
differences in cytosolic and extracellular
concentrations of Na
H11001
and K
H11001
, and the
resulting Na
H11001
gradient is used as the energy
source for a variety of secondary active
transport processes.
■ The Na
H11001
K
H11001
ATPase of the plasma membrane
and the Ca
2H11001
transporters of the sarcoplasmic
and endoplasmic reticulums (the SERCA
pumps) are examples of P-type ATPases; they
undergo reversible phosphorylation during their
catalytic cycle and are inhibited by the
phosphate analog vanadate. F-type ATPase
proton pumps (ATP synthases) are central to
energy-conserving mechanisms in mitochondria
and chloroplasts. V-type ATPases produce
gradients of protons across some intracellular
membranes, including plant vacuolar
membranes.
■ ABC transporters carry a variety of substrates,
including many drugs, out of cells, using ATP
as energy source.
■ Ionophores are lipid-soluble molecules that
bind specific ions and carry them passively
across membranes, dissipating the energy of
electrochemical ion gradients.
■ Water moves across membranes through
aquaporins.
■ Ion channels provide hydrophilic pores through
which select ions can diffuse, moving down
their electrical or chemical concentration
gradients; they are characteristically
unsaturable and have very high flux rates.
Many ion channels are highly specific for one
ion, and most are gated by either voltage or a
ligand. In bacterial K
H11001
channels, a selectivity
filter provides ligands with the right geometry
to replace the water of hydration of a K
H11001
ion as
it crosses the membrane. Some K
H11001
channels
are voltage gated. The acetylcholine
receptor/channel is gated by acetylcholine,
which triggers subtle conformational changes
that open and close the transmembrane path.
Chapter 11 Biological Membranes and Transport416
O
O
H
H
H
OH
OH
N
CH
2
H
3
CO
OCH
3
2Cl
H11002
CH
2
H
3
CCH
3
CH
3
H11001
N
H11001
D-Tubocurarine chloride
8885d_c11_369-420 2/7/04 6:58 AM Page 416 mac76 mac76:385_reb:
Chapter 11 Further Reading 417
Key Terms
fluid mosaic model
371
micelle 372
bilayer 373
integral proteins 373
peripheral proteins
373
hydropathy index 377
H9252 barrel 378
gel phase 380
liquid-disordered state
380
liquid-ordered state 380
flippases 382
FRAP 382
microdomains 383
rafts 384
caveolin 385
caveolae 385
fusion proteins 387
SNAREs 389
simple diffusion 389
membrane potential
(V
m
) 389
electrochemical
gradient 391
electrochemical
potential 391
facilitated diffusion
391
passive transport 391
transporters 391
carriers 392
channels 392
electroneutral 395
cotransport systems
395
antiport 397
symport 397
uniport 397
active transport 397
electrogenic 398
P-type ATPases 398
SERCA pump 400
F-type ATPases 401
ATP synthase 401
V-type ATPases 401
ABC transporters 402
multidrug transporter
402
ionophores 406
aquaporins (AQPs) 406
ion channel 408
Terms in bold are defined in the glossary.
Further Reading
Composition and Architecture of Membranes
Boon, J.M. & Smith, B.D. (2002) Chemical control of phospho-
lipid distribution across bilayer membranes. Med. Res. Rev. 22,
251–281.
Intermediate-level review of phospholipid asymmetry and
factors that influence it.
Dowhan, W. (1997) Molecular basis for membrane phospholipids
diversity: why are there so many lipids? Annu. Rev. Biochem. 66,
199–232.
Ediden, M. (2002) Lipids on the frontier: a century of cell-
membrane bilayers. Nat. Rev. Mol. Cell Biol. 4, 414–418.
Short review of how the notion of a lipid bilayer membrane was
developed and confirmed.
Haltia, T. & Freire, E. (1995) Forces and factors that contribute
to the structural stability of membrane proteins. Biochim.
Biophys. Acta 1241, 295–322.
Good discussion of the secondary and tertiary structures of
membrane proteins and the factors that stabilize them.
von Heijne, G. (1994) Membrane proteins: from sequence
to structure. Annu. Rev. Biophys. Biomol. Struct. 23,
167–192.
A review of the steps required to predict the structure of an
integral protein from its sequence.
White, S.H., Ladokhin, A.S., Jayasinghe, S., & Hristova, K.
(2001) How membranes shape protein structure. J. Biol. Chem.
276, 32,395–32,398.
Brief, intermediate-level review of the forces that shape
transmembrane helices.
Wimley, W.C. (2003) The versatile H9252 barrel membrane protein.
Curr. Opin. Struct. Biol. 13, 1–8.
Intermediate-level review.
Membrane Dynamics
Brown, D.A. & London, E. (1998) Functions of lipid rafts in
biological membranes. Annu. Rev. Cell Dev. Biol. 14, 111–136.
Chen, Y.A. & Scheller, R.H. (2001) SNARE-mediated membrane
fusion. Nat. Rev. Mol. Cell Biol. 2, 98–106.
Intermediate-level review.
Edidin, M. (2003) The state of lipid rafts: from model membranes
to cells. Annu. Rev. Biophys. Biomol. Struct. 32, 257–283.
Advanced review.
Frye, L.D. & Ediden, M. (1970) The rapid intermixing of cell-
surface antigens after formation of mouse-human heterokaryons.
J. Cell Sci. 7, 319–335.
The classic demonstration of membrane protein mobility.
Mayer, A. (2002) Membrane fusion in eukaryotic cells. Annu.
Rev. Cell Dev. Biol. 18, 289–314.
Advanced review of membrane fusion, with emphasis on the
conserved general features.
Parton, R.G. (2003) Caveolae—from ultrastructure to molecular
mechanisms. Nat. Rev. Mol. Cell Biol. 4, 162–167.
A concise historical review of caveolae, caveolin, and rafts.
Sprong, H., van der Sluijs, P., & van Meer, G. (2001) How
proteins move lipids and lipids move proteins. Nat. Rev. Mol. Cell
Biol. 2, 504–513.
Intermediate-level review.
van Deurs, B., Roepstorff, K., Hommelgaard, A.M., &
Sandvig, K. (2003) Caveolae: anchored, multifunctional platforms
in the lipid ocean. Trends Cell Biol. 13, 92–100.
Vereb, G., Sz?llósi, J., Matcó, J., Nagy, P., Farkas, T., Vigh,
L., Mátyus, L., Waldmann, T.A., & Damjanovich, S. (2003)
Dynamic, yet structured: the cell membrane three decades after
the Singer-Nicolson model. Proc. Natl. Acad. Sci. USA 100,
8053–8058.
Intermediate-level review of membrane structure and dynamics.
Transporters
Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback,
H.R., & Iwata, S. (2003) Structure and mechanism of the lactose
permease of Escherichia coli. Science 301, 610–615.
8885d_c11_369-420 2/7/04 6:58 AM Page 417 mac76 mac76:385_reb:
Chapter 11 Biological Membranes and Transport418
Fujiyoshi, Y., Mitsuoka, K., de Groot, B.L., Philippsen, A.,
Grubmüller, H., Agre, P., & Engel, A. (2002) Structure and
function of water channels. Curr. Opin. Struct. Biol. 12, 509–515.
Jorgensen, P.L., H?kansson, K.O., & Karlish, S.J.D. (2003)
Structure and mechanism of Na,K-ATPase: functional sites and
their interactions. Annu. Rev. Physiol. 65, 817–849.
Kjellbom, P., Larsson, C., Johansson, I., Karlsson, M., &
Johanson, U. (1999) Aquaporins and water homeostasis in plants.
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Intermediate-level review.
Mueckler, M. (1994) Facilitative glucose transporters. Eur. J.
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Saier, M.H., Jr. (2000) A functional-phylogenetic classification
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Schmitt, L. & Tampé, R. (2002) Structure and mechanism of
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Stokes, D.L. & Green, N.M. (2003) Structure and function of the
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Advanced review.
Sui, H., Han, B.-G., Lee, J.K., Walian, P., & Jap, B.K. (2001)
Structural basis of water-specific transport through the AQP1
water channel. Nature 414, 872–878.
High-resolution solution of the aquaporin structure by x-ray
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Ion Channels
Changeux, J.P. (1993) Chemical signaling in the brain. Sci. Am.
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Discussion of structure and function of the acetylcholine
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Doyle, D.A., Cabral, K.M., Pfuetzner, R.A., Kuo, A., Gulbis,
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H11001
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Advanced discussion of the conformational changes induced by
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Clear description of the electrophysiological methods used to
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H11001
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1. Determining the Cross-Sectional Area of a Lipid
Molecule When phospholipids are layered gently onto the
surface of water, they orient at the air-water interface with
their head groups in the water and their hydrophobic tails in
the air. An experimental apparatus (a) has been devised that
reduces the surface area available to a layer of lipids. By meas-
uring the force necessary to push the lipids together, it is pos-
sible to determine when the molecules are packed tightly in
a continuous monolayer; as that area is approached, the force
needed to further reduce the surface area increases sharply
(b). How would you use this apparatus to determine the av-
erage area occupied by a single lipid molecule in the monolayer?
2. Evidence for a Lipid Bilayer In 1925, E. Gorter and
F. Grendel used an apparatus like that described in Problem 1
to determine the surface area of a lipid monolayer formed by
lipids extracted from erythrocytes of several animal species.
They used a microscope to measure the dimensions of indi-
vidual cells, from which they calculated the average surface
area of one erythrocyte. They obtained the data shown in the
Problems
Force applied here
to compress
monolayer
(a)
Force (dyne/cm)
Area (nm
2
/molecule)
(b)
40
30
1.41.00.60.2
20
10
8885d_c11_369-420 2/7/04 6:58 AM Page 418 mac76 mac76:385_reb:
Chapter 11 Problems 419
table. Were these investigators justified in concluding that
“chromocytes [erythrocytes] are covered by a layer of fatty sub-
stances that is two molecules thick” (i.e., a lipid bilayer)?
Total surface
Volume of Number area of lipid Total surface
packed of cells monolayer area of one
Animal cells (mL) (per mm
3
) from cells (m
2
) cell (H9262m
2
)
Dog 40 8,000,000 62 98
Sheep 10 9,900,000 6.0 29.8
Human 1 4,740,000 0.92 99.4
Source: Data from Gorter, E. & Grendel, F. (1925) On bimolecular layers of lipoids on the
chromocytes of the blood. J. Exp. Med. 41, 439–443.
3. Number of Detergent Molecules per Micelle When
a small amount of sodium dodecyl sulfate (SDS;
Na
H11001
CH
3
(CH
2
)
11
OSO
3
H11002
) is dissolved in water, the detergent
ions enter the solution as monomeric species. As more de-
tergent is added, a concentration is reached (the critical mi-
celle concentration) at which the monomers associate to form
micelles. The critical micelle concentration of SDS is 8.2 mM.
The micelles have an average particle weight (the sum of the
molecular weights of the constituent monomers) of 18,000.
Calculate the number of detergent molecules in the average
micelle.
4. Properties of Lipids and Lipid Bilayers Lipid bilay-
ers formed between two aqueous phases have this important
property: they form two-dimensional sheets, the edges of
which close upon each other and undergo self-sealing to form
liposomes.
(a) What properties of lipids are responsible for this
property of bilayers? Explain.
(b) What are the consequences of this property for the
structure of biological membranes?
5. Length of a Fatty Acid Molecule The carbon–carbon
bond distance for single-bonded carbons such as those in a
saturated fatty acyl chain is about 1.5 ?. Estimate the length
of a single molecule of palmitate in its fully extended form.
If two molecules of palmitate were placed end to end, how
would their total length compare with the thickness of the
lipid bilayer in a biological membrane?
6. Temperature Dependence of Lateral Diffusion The
experiment described in Figure 11–17 was performed at
37 H11034C. If the experiment were carried out at 10 H11034C, what ef-
fect would you expect on the rate of diffusion? Why?
7. Synthesis of Gastric Juice: Energetics Gastric juice
(pH 1.5) is produced by pumping HCl from blood plasma
(pH 7.4) into the stomach. Calculate the amount of free en-
ergy required to concentrate the H
H11001
in 1 L of gastric juice at
37 H11034C. Under cellular conditions, how many moles of ATP must
be hydrolyzed to provide this amount of free energy? The
free-energy change for ATP hydrolysis under cellular condi-
tions is about H1100258 kJ/mol (as explained in Chapter 13). Ig-
nore the effects of the transmembrane electrical potential.
8. Energetics of the Na
H11545
K
H11545
ATPase For a typical ver-
tebrate cell with a transmembrane potential of H110020.070 V (in-
side negative), what is the free-energy change for transport-
ing 1 mol of Na
H11001
out of the cell and into the blood at 37 H11034C?
Assume the concentration of Na
H11001
inside the cell is 12 mM,
and that in blood plasma is 145 mM.
9. Action of Ouabain on Kidney Tissue Ouabain specif-
ically inhibits the Na
H11001
K
H11001
ATPase activity of animal tissues but
is not known to inhibit any other enzyme. When ouabain is
added to thin slices of living kidney tissue, it inhibits oxygen
consumption by 66%. Why? What does this observation tell us
about the use of respiratory energy by kidney tissue?
10. Energetics of Symport Suppose that you determined
experimentally that a cellular transport system for glucose,
driven by symport of Na
H11001
, could accumulate glucose to con-
centrations 25 times greater than in the external medium,
while the external [Na
H11001
] was only 10 times greater than the
intracellular [Na
H11001
]. Would this violate the laws of thermody-
namics? If not, how could you explain this observation?
11. Location of a Membrane Protein The following ob-
servations are made on an unknown membrane protein, X. It
can be extracted from disrupted erythrocyte membranes into
a concentrated salt solution, and it can be cleaved into frag-
ments by proteolytic enzymes. Treatment of erythrocytes
with proteolytic enzymes followed by disruption and extrac-
tion of membrane components yields intact X. However, treat-
ment of erythrocyte “ghosts” (which consist of just plasma
membranes, produced by disrupting the cells and washing
out the hemoglobin) with proteolytic enzymes followed by
disruption and extraction yields extensively fragmented X.
What do these observations indicate about the location of X
in the plasma membrane? Do the properties of X resemble
those of an integral or peripheral membrane protein?
12. Membrane Self-sealing Cellular membranes are self-
sealing—if they are punctured or disrupted mechanically,
they quickly and automatically reseal. What properties of
membranes are responsible for this important feature?
13. Lipid Melting Temperatures Membrane lipids in tis-
sue samples obtained from different parts of the leg of a rein-
deer have different fatty acid compositions. Membrane lipids
from tissue near the hooves contain a larger proportion of un-
saturated fatty acids than those from tissue in the upper leg.
What is the significance of this observation?
14. Flip-Flop Diffusion The inner leaflet (monolayer) of
the human erythrocyte membrane consists predominantly of
phosphatidylethanolamine and phosphatidylserine. The outer
leaflet consists predominantly of phosphatidylcholine and
sphingomyelin. Although the phospholipid components of the
membrane can diffuse in the fluid bilayer, this sidedness is
preserved at all times. How?
15. Membrane Permeability At pH 7, tryptophan crosses
a lipid bilayer at about one-thousandth the rate of the closely
related substance indole:
Suggest an explanation for this observation.
N
H
8885d_c11_369-420 2/7/04 6:58 AM Page 419 mac76 mac76:385_reb:
Chapter 11 Biological Membranes and Transport420
16. Water Flow through an Aquaporin Each human
erythrocyte has about 2 H11003 10
5
AQP-1 monomers. If water
molecules flow through the plasma membrane at a rate of
5 H11003 10
8
per AQP-1 tetramer per second, and the volume of
an erythrocyte is 5 H11003 10
H1100211
mL, how rapidly could an ery-
throcyte halve its volume as it encounters the high osmolar-
ity (1 M) in the interstitial fluid of the renal medulla? Assume
that the erythrocyte consists entirely of water.
17. Labeling the Lactose Transporter A bacterial lac-
tose transporter, which is highly specific for its substrate lac-
tose, contains a Cys residue that is essential to its transport
activity. Covalent reaction of N-ethylmaleimide (NEM) with
this Cys residue irreversibly inactivates the transporter. A
high concentration of lactose in the medium prevents inacti-
vation by NEM, presumably by sterically protecting the Cys
residue, which is in or near the lactose-binding site. You know
nothing else about the transporter protein. Suggest an ex-
periment that might allow you to determine the M
r
of the Cys-
containing transporter polypeptide.
18. Predicting Membrane Protein Topology from Se-
quence You have cloned the gene for a human erythrocyte
protein, which you suspect is a membrane protein. From the
nucleotide sequence of the gene, you know the amino acid
sequence. From this sequence alone, how would you evalu-
ate the possibility that the protein is an integral protein? Sup-
pose the protein proves to be an integral protein, either type
I or type II. Suggest biochemical or chemical experiments that
might allow you to determine which type it is.
19. Intestinal Uptake of Leucine You are studying the
uptake of L-leucine by epithelial cells of the mouse intestine.
Measurements of the rate of uptake of L-leucine and several
of its analogs, with and without Na
H11001
in the assay buffer, yield
the results given in the table. What can you conclude about
the properties and mechanism of the leucine transporter?
Would you expect L-leucine uptake to be inhibited by
ouabain?
Uptake in Uptake in
presence of Na
H11001
absence of Na
H11001
Substrate V
max
K
t
(mM)V
max
K
t
(mM)
L-Leucine 420 0.24 23 0.24
D-Leucine 310 4.7 5 4.7
L-Valine 225 0.31 19 0.31
20. Effect of an Ionophore on Active Transport Con-
sider the leucine transporter described in Problem 19. Would
V
max
and/or K
t
change if you added a Na
H11001
ionophore to the
assay solution containing Na
H11001
? Explain.
21. Surface Density of a Membrane Protein E. coli can
be induced to make about 10,000 copies of the lactose trans-
porter (M
r
31,000) per cell. Assume that E. coli is a cylinder
1 H9262m in diameter and 2 H9262m long. What fraction of the plasma
membrane surface is occupied by the lactose transporter mol-
ecules? Explain how you arrived at this conclusion.
Biochemistry on the Internet
22. Membrane Protein Topology The receptor for the
hormone epinephrine in animal cells is an integral membrane
protein (M
r
64,000) that is believed to have seven membrane-
spanning regions.
(a) Show that a protein of this size is capable of span-
ning the membrane seven times.
(b) Given the amino acid sequence of this protein, how
would you predict which regions of the protein form the
membrane-spanning helices?
(c) Go to the Protein Data Bank (www.rcsb.org/pdb).
Use the PDB identifier 1DEP to retrieve the data page for a
portion of the H9252-adrenergic receptor (one type of epineph-
rine receptor) from a turkey. Using Chime to explore the
structure, predict where this portion of the receptor is lo-
cated: within the membrane or at the membrane surface.
Explain.
(d) Retrieve the data for a portion of another receptor,
the acetylcholine receptor of neurons and myocytes, using
the PDB identifier 1A11. As in (c), predict where this por-
tion of the receptor is located and explain your answer.
If you have not used the PDB or Chemscape Chime, you
can find instructions at www.whfreeman.com/lehninger.
8885d_c11_420 2/11/04 12:48 PM Page 420 mac76 mac76:385_reb:
chapter
T
he ability of cells to receive and act on signals from
beyond the plasma membrane is fundamental to life.
Bacterial cells receive constant input from membrane
proteins that act as information receptors, sampling the
surrounding medium for pH, osmotic strength, the avail-
ability of food, oxygen, and light, and the presence of
noxious chemicals, predators, or competitors for food.
These signals elicit appropriate responses, such as mo-
tion toward food or away from toxic substances or the
formation of dormant spores in a nutrient-depleted
medium. In multicellular organisms, cells with different
functions exchange a wide variety of signals. Plant cells
respond to growth hormones and to variations in sun-
light. Animal cells exchange information about the con-
centrations of ions and glucose in extracellular fluids,
the interdependent metabolic activities taking place in
different tissues, and, in an embryo, the correct place-
ment of cells during development. In all these cases, the
signal represents information that is detected by spe-
cific receptors and converted to a cellular response,
which always involves a chemical process. This con-
version of information into a chemical change, signal
transduction, is a universal property of living cells.
The number of different biological signals is large
(Table 12–1), as is the variety of biological responses to
these signals, but organisms use just a few evolutionar-
ily conserved mechanisms to detect extracellular signals
and transduce them into intracellular changes. In this
chapter we examine some examples of the major classes
of signaling mechanisms, looking at how they are inte-
grated in specific biological functions such as the trans-
mission of nerve signals; responses to hormones and
growth factors; the senses of sight, smell, and taste; and
BIOSIGNALING
12.1 Molecular Mechanisms of Signal
Transduction 422
12.2 Gated Ion Channels 425
12.3 Receptor Enzymes 429
12.4 G Protein–Coupled Receptors and Second
Messengers 435
12.5 Multivalent Scaffold Proteins and
Membrane Rafts 443
12.6 Signaling in Microorganisms and Plants 452
12.7 Sensory Transduction in Vision, Olfaction,
and Gustation 456
12.8 Regulation of Transcription by Steroid
Hormones 465
12.9 Regulation of the Cell Cycle
by Protein Kinases 466
12.10 Oncogenes, Tumor Suppressor Genes, and
Programmed Cell Death 471
When I first entered the study of hormone action, some
25 years ago, there was a widespread feeling among
biologists that hormone action could not be studied
meaningfully in the absence of organized cell structure.
However, as I reflected on the history of biochemistry, it
seemed to me there was a real possibility that hormones
might act at the molecular level.
—Earl W. Sutherland, Nobel Address, 1971
12
421
Antigens
Cell surface glycoproteins/
oligosaccharides
Developmental signals
Extracellular matrix components
Growth factors
Hormones
Light
Mechanical touch
Neurotransmitters
Nutrients
Odorants
Pheromones
Tastants
Some Signals to Which Cells Respond
TABLE 12–1
()
8885d_c12_421 2/23/04 9:11 AM Page 421 mac76 mac76:
control of the cell cycle. Often, the end result of a sig-
naling pathway is the phosphorylation of a few specific
target-cell proteins, which changes their activities and
thus the activities of the cell. Throughout our discus-
sion we emphasize the conservation of fundamental
mechanisms for the transduction of biological signals
and the adaptation of these basic mechanisms to a wide
range of signaling pathways.
12.1 Molecular Mechanisms
of Signal Transduction
Signal transductions are remarkably specific and
exquisitely sensitive. Specificity is achieved by precise
molecular complementarity between the signal and re-
ceptor molecules (Fig. 12–1a), mediated by the same
kinds of weak (noncovalent) forces that mediate
enzyme-substrate and antigen-antibody interactions.
Multicellular organisms have an additional level of speci-
ficity, because the receptors for a given signal, or the
intracellular targets of a given signal pathway, are pres-
ent only in certain cell types. Thyrotropin-releasing hor-
mone, for example, triggers responses in the cells of the
anterior pituitary but not in hepatocytes, which lack re-
ceptors for this hormone. Epinephrine alters glycogen
metabolism in hepatocytes but not in erythrocytes; in
this case, both cell types have receptors for the hor-
mone, but whereas hepatocytes contain glycogen and
the glycogen-metabolizing enzyme that is stimulated by
epinephrine, erythrocytes contain neither.
Three factors account for the extraordinary sensi-
tivity of signal transducers: the high affinity of recep-
tors for signal molecules, cooperativity (often but not
always) in the ligand-receptor interaction, and amplifi-
cation of the signal by enzyme cascades. The affinity
between signal (ligand) and receptor can be expressed
as the dissociation constant K
d
, usually 10
H1100210
M or
less—meaning that the receptor detects picomolar
concentrations of a signal molecule. Receptor-ligand in-
teractions are quantified by Scatchard analysis, which
yields a quantitative measure of affinity (K
d
) and
the number of ligand-binding sites in a receptor sam-
ple (Box 12–1).
Cooperativity in receptor-ligand interactions re-
sults in large changes in receptor activation with small
changes in ligand concentration (recall the effect of co-
operativity on oxygen binding to hemoglobin; see Fig.
5–12). Amplification by enzyme cascades results
when an enzyme associated with a signal receptor is ac-
tivated and, in turn, catalyzes the activation of many
molecules of a second enzyme, each of which activates
many molecules of a third enzyme, and so on (Fig.
12–1b). Such cascades can produce amplifications of
several orders of magnitude within milliseconds.
The sensitivity of receptor systems is subject to
modification. When a signal is present continuously,
desensitization of the receptor system results (Fig.
12–1c); when the stimulus falls below a certain thresh-
old, the system again becomes sensitive. Think of what
happens to your visual transduction system when you
walk from bright sunlight into a darkened room or from
darkness into the light.
A final noteworthy feature of signal-transducing
systems is integration (Fig. 12–1d), the ability of the
system to receive multiple signals and produce a uni-
fied response appropriate to the needs of the cell or or-
ganism. Different signaling pathways converse with
Chapter 12 Biosignaling422
Receptor
Response
Signal
Receptor
1
Signal 1
Receptor
2
Signal 2
(c) Desensitization/Adaptation
Receptor activation triggers
a feedback circuit that shuts
off the receptor or removes
it from the cell surface.
(d) Integration
When two signals have
opposite effects on a
metabolic characteristic
such as the concentration
of a second messenger X,
or the membrane potential
V
m
, the regulatory outcome
results from the integrated
input from both receptors.
[X] or V
m
[X] or V
m
Response
Net H9004[X] or V
m
Enzyme
3
33 333333
Enzyme 2 2
Enzyme 1
Signal
2
3
Effect
S
1
S
2
(a) Specificity
Signal molecule fits
binding site on its
complementary receptor;
other signals do not fit.
(b) Amplification
When enzymes activate
enzymes, the number of
affected molecules
increases geometrically
in an enzyme cascade.
Receptor
FIGURE 12–1 Four features of signal-transducing systems.
8885d_c12_422 2/20/04 1:13 PM Page 422 mac76 mac76:385_reb:
BOX 12–1 WORKING IN BIOCHEMISTRY
Scatchard Analysis Quantifies the
Receptor-Ligand Interaction
The cellular actions of a hormone begin when the hor-
mone (ligand, L) binds specifically and tightly to its
protein receptor (R) on or in the target cell. Binding
is mediated by noncovalent interactions (hydrogen-
bonding, hydrophobic, and electrostatic) between
the complementary surfaces of ligand and receptor.
Receptor-ligand interaction brings about a conforma-
tional change that alters the biological activity of the
receptor, which may be an enzyme, an enzyme regu-
lator, an ion channel, or a regulator of gene expression.
Receptor-ligand binding is described by the
equation
R H11001 L 34 RL
Receptor Ligand Receptor-ligand complex
This binding, like that of an enzyme to its substrate,
depends on the concentrations of the interacting com-
ponents and can be described by an equilibrium con-
stant:
k
H110011
R H11001 L 34 RL
Receptor Ligand k
H110021
Receptor-ligand
complex
K
a
H11005H11005H110051/K
d
where K
a
is the association constant and K
d
is the dis-
sociation constant.
Like enzyme-substrate binding, receptor-ligand
binding is saturable. As more ligand is added to a fixed
amount of receptor, an increasing fraction of receptor
molecules is occupied by ligand (Fig. 1a). A rough
measure of receptor-ligand affinity is given by the con-
centration of ligand needed to give half-saturation of
the receptor. Using Scatchard analysis of receptor-
ligand binding, we can estimate both the dissociation
constant K
d
and the number of receptor-binding sites
in a given preparation. When binding has reached
equilibrium, the total number of possible binding sites,
B
max
, equals the number of unoccupied sites, repre-
sented by [R], plus the number of occupied or ligand-
bound sites, [RL]; that is, B
max
H11005 [R] H11001 [RL]. The num-
ber of unbound sites can be expressed in terms of total
sites minus occupied sites: [R] H11005 B
max
H11002 [RL]. The
equilibrium expression can now be written
K
a
H11005
Rearranging to obtain the ratio of receptor-bound lig-
and to free (unbound) ligand, we get
H11005H11005K
a
(B
max
H11002 [RL])
H11005 (B
max
H11002 [RL])
1
H5007
K
d
[RL]
H5007
[L]
[Bound]
H5007
[Free]
[RL]
H5007H5007H5007
[L](B
max
H11002 [RL])
k
H110011
H5007
k
H110021
[RL]
H5007
[R][L]
From this slope-intercept form of the equation, we can
see that a plot of [bound ligand]/[free ligand] versus
[bound ligand] should give a straight line with a slope of
H11002K
a
(H110021/K
d
) and an intercept on the abscissa of B
max
,
the total number of binding sites (Fig. 1b). Hormone-
ligand interactions typically have K
d
values of 10
H110029
to
10
H1100211
M, corresponding to very tight binding.
Scatchard analysis is reliable for the simplest
cases, but as with Lineweaver-Burk plots for enzymes,
when the receptor is an allosteric protein, the plots
deviate from linearity.
Bound hormone
,
[
RL]
Total hormone added, [L] H11001 [RL]
Total binding
Specific binding
Nonspecific binding
(a)
Bound hormone
F
ree hormone
,
[
RL] [L
]
Bound hormone, [RL](b)
Slope H11005 H11002
1
K
d
B
max
FIGURE 1 Scatchard analysis of a receptor-ligand interaction. A
radiolabeled ligand (L)—a hormone, for example—is added at sev-
eral concentrations to a fixed amount of receptor (R), and the frac-
tion of the hormone bound to receptor is determined by separating
the receptor-hormone complex (RL) from free hormone. (a) A plot
of [RL] versus [L] H11001 [RL] (total hormone added) is hyperbolic, rising
toward a maximum for [RL] as the receptor sites become saturated.
To control for nonsaturable, nonspecific binding sites (eicosanoid
hormones bind nonspecifically to the lipid bilayer, for example),
a separate series of binding experiments is also necessary. A large
excess of unlabeled hormone is added along with the dilute so-
lution of labeled hormone. The unlabeled molecules compete with
the labeled molecules for specific binding to the saturable site on
the receptor, but not for the nonspecific binding. The true value for
specific binding is obtained by subtracting nonspecific binding from
total binding. (b) A linear plot of [RL]/[L] versus [RL] gives K
d
and
B
max
for the receptor-hormone complex. Compare these plots with
those of V
0
versus [S] and 1/V
0
versus 1/[S] for an enzyme-substrate
complex (see Fig. 6–12, Box 6–1).
8885d_c12_423 2/20/04 1:13 PM Page 423 mac76 mac76:385_reb:
each other at several levels, generating a wealth of in-
teractions that maintain homeostasis in the cell and the
organism.
We consider here the molecular details of several
representative signal-transduction systems. The trigger
for each system is different, but the general features of
signal transduction are common to all: a signal interacts
with a receptor; the activated receptor interacts with
cellular machinery, producing a second signal or a
change in the activity of a cellular protein; the meta-
bolic activity (broadly defined to include metabolism of
RNA, DNA, and protein) of the target cell undergoes a
change; and finally, the transduction event ends and the
cell returns to its prestimulus state. To illustrate these
general features of signaling systems, we provide
examples of each of six basic signaling mechanisms
(Fig. 12–2).
1. Gated ion channels of the plasma membrane that
open and close (hence the term “gating”) in
response to the binding of chemical ligands or
changes in transmembrane potential. These are
the simplest signal transducers. The acetylcholine
receptor ion channel is an example of this
mechanism (Section 12.2).
2. Receptor enzymes, plasma membrane receptors
that are also enzymes. When one of these
receptors is activated by its extracellular ligand, it
catalyzes the production of an intracellular second
messenger. An example is the insulin receptor
(Section 12.3).
3. Receptor proteins (serpentine receptors) that
indirectly activate (through GTP-binding
proteins, or G proteins) enzymes that generate
intracellular second messengers. This is illustrated
by the H9252-adrenergic receptor system that detects
epinephrine (adrenaline) (Section 12.4).
4. Nuclear receptors (steroid receptors) that, when
bound to their specific ligand (such as the
hormone estrogen), alter the rate at which specific
genes are transcribed and translated into cellular
proteins. Because steroid hormones function
through mechanisms intimately related to the
regulation of gene expression, we consider them
here only briefly (Section 12.8) and defer a
detailed discussion of their action until Chapter 28.
5. Receptors that lack enzymatic activity but attract
and activate cytoplasmic enzymes that act on
downstream proteins, either by directly converting
them to gene-regulating proteins or by activating a
cascade of enzymes that finally activates a gene
regulator. The JAK-STAT system exemplifies the
first mechanism (Section 12.3); and the TLR4
(Toll) signaling system in humans, the second
(Section 12.6).
Chapter 12 Biosignaling424
FIGURE 12–2 Six general types of signal transducers.
S
SS
S
Ion
S
S
S
S
Gated ion channel
Opens or closes in
response to concentration
of signal ligand (S)
or membrane potential.
Receptor enzyme
Ligand binding to
extracellular domain
stimulates enzyme
activity in intracellular
domain.
Plasma
membrane
Nuclear
envelope
DNA
Steroid receptor
Steroid binding to a
nuclear receptor
protein allows the
receptor to regulate
the expression of
specific genes.
Serpentine receptor
External ligand binding
to receptor (R) activates an
intracellular GTP-binding
protein (G), which regulates
an enzyme (Enz) that
generates an intracellular
second messenger, X.
Receptor with no intrinsic
enzyme activity
Interacts with cytosolic
protein kinase, which
activates a gene-regulating
protein (directly or through a
cascade of protein kinases),
changing gene expression.
Kinase
cascade
R
G
Enz
mRNA
Protein
mRNA
DNA
Protein
X
S
S
Adhesion
receptor
Binds molecules
in extracellular
matrix, changes
conformation,
thus altering its
interaction with
cytoskeleton.
8885d_c12_424 2/20/04 1:16 PM Page 424 mac76 mac76:385_reb:
6. Receptors (adhesion receptors) that interact with
macromolecular components of the extracellular
matrix (such as collagen) and convey to the
cytoskeletal system instructions on cell migration
or adherence to the matrix. Integrins (discussed in
Chapter 10) illustrate this general type of
transduction mechanism.
As we shall see, transductions of all six types commonly
require the activation of protein kinases, enzymes that
transfer a phosphoryl group from ATP to a protein side
chain.
SUMMARY 12.1 Molecular Mechanisms
of Signal Transduction
■ All cells have specific and highly sensitive
signal-transducing mechanisms, which have
been conserved during evolution.
■ A wide variety of stimuli, including hormones,
neurotransmitters, and growth factors, act
through specific protein receptors in the
plasma membrane.
■ The receptors bind the signal molecule, amplify
the signal, integrate it with input from other
receptors, and transmit it into the cell. If the
signal persists, receptor desensitization reduces
or ends the response.
■ Eukaryotic cells have six general types of
signaling mechanisms: gated ion channels;
receptor enzymes; membrane proteins that act
through G proteins; nuclear proteins that bind
steroids and act as transcription factors;
membrane proteins that attract and activate
soluble protein kinases; and adhesion receptors
that carry information between the
extracellular matrix and the cytoskeleton.
12.2 Gated Ion Channels
Ion Channels Underlie Electrical Signaling
in Excitable Cells
The excitability of sensory cells, neurons, and myocytes
depends on ion channels, signal transducers that pro-
vide a regulated path for the movement of inorganic ions
such as Na
H11001
, K
H11001
, Ca
2H11001
, and Cl
H11002
across the plasma mem-
brane in response to various stimuli. Recall from Chap-
ter 11 that these ion channels are “gated”; they may be
open or closed, depending on whether the associated
receptor has been activated by the binding of its spe-
cific ligand (a neurotransmitter, for example) or by a
change in the transmembrane electrical potential, V
m
.
The Na
H11001
K
H11001
ATPase creates a charge imbalance across
the plasma membrane by carrying 3 Na
H11001
out of the cell
for every 2 K
H11001
carried in (Fig. 12–3a), making the in-
side negative relative to the outside. The membrane is
said to be polarized. By convention, V
m
is negative when
the inside of the cell is negative relative to the outside.
For a typical animal cell, V
m
H11005H1100260 to H1100270 mV.
Because ion channels generally allow passage of ei-
ther anions or cations but not both, ion flux through a
channel causes a redistribution of charge on the two
sides of the membrane, changing V
m
. Influx of a posi-
tively charged ion such as Na
H11001
, or efflux of a negatively
charged ion such as Cl
H11002
, depolarizes the membrane and
brings V
m
closer to zero. Conversely, efflux of K
H11001
hy-
perpolarizes the membrane and V
m
becomes more neg-
ative. These ion fluxes through channels are passive, in
contrast to active transport by the Na
H11001
K
H11001
ATPase.
The direction of spontaneous ion flow across a
polarized membrane is dictated by the electrochemical
12.2 Gated Ion Channels 425
High
High
High
Low
Low
Low
Low
High
The electrogenic Na
+
K
+
ATPase establishes the
membrane potential.
Ions tend to move down
their electrochemical
gradient across the
polarized membrane.
ATP ADP
H11545
P
i
Na
H11545
K
H11545
ATPase
2 K
H11545
[Na
H11545
]
[K
H11545
]
[Ca
2H11545
]
[Cl
H11546
]
3 Na
H11545
H11545H11545
H11546H11546
H11545
H11546
H11545H11545
H11546H11546
H11545
H11546
H11546
H11545
H11546
H11545
H11546
H11545
H11546
H11545
H11546
H11545
H11546
H11545
H11545H11546
H11545H11546
H11545H11546
H11546H11545
H11546
H11545
H11546
H11545
+H11546
+H11546
Plasma
membrane
(a)
(b)
FIGURE 12–3 Transmembrane electrical potential. (a) The electro-
genic Na
H11001
K
H11001
ATPase produces a transmembrane electrical potential
of H1100260 mV (inside negative). (b) Blue arrows show the direction in
which ions tend to move spontaneously across the plasma membrane
in an animal cell, driven by the combination of chemical and elec-
trical gradients. The chemical gradient drives Na
H11001
and Ca
2H11001
inward
(producing depolarization) and K
H11001
outward (producing hyperpolar-
ization). The electrical gradient drives Cl
H11002
outward, against its con-
centration gradient (producing depolarization).
8885d_c12_425 2/20/04 1:16 PM Page 425 mac76 mac76:385_reb:
potential of that ion across the membrane. The force
(H9004G) that causes a cation (say, Na
H11001
) to pass sponta-
neously inward through an ion channel is a function of
the ratio of its concentrations on the two sides of the
membrane (C
in
/C
out
) and of the difference in electrical
potential (H9004H9274 or V
m
):
H9004G H11005 RT ln
H20898H20899
H11001 ZV
m
(12–1)
where R is the gas constant, T the absolute tempera-
ture, Z the charge on the ion, and the Faraday con-
stant. In a typical neuron or myocyte, the concentra-
tions of Na
H11001
, K
H11001
, Ca
2H11001
, and Cl
H11002
in the cytosol are very
different from those in the extracellular fluid (Table
12–2). Given these concentration differences, the rest-
ing V
m
of H1100260 mV, and the relationship shown in Equa-
tion 12–1, opening of a Na
H11001
or Ca
2H11001
channel will result
in a spontaneous inward flow of Na
H11001
or Ca
2H11001
(and
depolarization), whereas opening of a K
H11001
channel will
result in a spontaneous outward flux of K
H11001
(and
hyperpolarization) (Fig. 12–3b).
A given ionic species continues to flow through a
channel only as long as the combination of concentra-
tion gradient and electrical potential provides a driving
force, according to Equation 12–1. For example, as Na
H11001
flows down its concentration gradient it depolarizes
the membrane. When the membrane potential reaches
H1100170 mV, the effect of this membrane potential (to resist
further entry of Na
H11001
) exactly equals the effect of the
Na
H11001
concentration gradient (to cause more Na
H11001
to flow
inward). At this equilibrium potential (E), the driving
force (H9004G) tending to move an ion is zero. The equilib-
rium potential is different for each ionic species because
the concentration gradients differ for each ion.
The number of ions that must flow to change the
membrane potential significantly is negligible relative to
the concentrations of Na
H11001
, K
H11001
, and Cl
H11002
in cells and ex-
tracellular fluid, so the ion fluxes that occur during sig-
naling in excitable cells have essentially no effect on the
concentrations of those ions. However, because the in-
tracellular concentration of Ca
2H11001
is generally very low
(～10
H110027
M), inward flow of Ca
2H11001
can significantly alter
the cytosolic [Ca
2H11001
].
The membrane potential of a cell at a given time is
the result of the types and numbers of ion channels open
at that instant. In most cells at rest, more K
H11001
channels
C
in
H5007
C
out
than Na
H11001
, Cl
H11002
, or Ca
2H11001
channels are open and thus the
resting potential is closer to the E for K
H11001
(H1100298 mV)
than that for any other ion. When channels for Na
H11001
,
Ca
2H11001
, or Cl
H11002
open, the membrane potential moves to-
ward the E for that ion. The precisely timed opening
and closing of ion channels and the resulting transient
changes in membrane potential underlie the electrical
signaling by which the nervous system stimulates the
skeletal muscles to contract, the heart to beat, or se-
cretory cells to release their contents. Moreover, many
hormones exert their effects by altering the membrane
potentials of their target cells. These mechanisms are
not limited to complex animals; ion channels play im-
portant roles in the responses of bacteria, protists, and
plants to environmental signals.
To illustrate the action of ion channels in cell-to-cell
signaling, we describe the mechanisms by which a neu-
ron passes a signal along its length and across a synapse
to the next neuron (or to a myocyte) in a cellular cir-
cuit, using acetylcholine as the neurotransmitter.
The Nicotinic Acetylcholine Receptor Is a
Ligand-Gated Ion Channel
One of the best-understood examples of a ligand-gated
receptor channel is the nicotinic acetylcholine re-
ceptor (see Fig. 11–51). The receptor channel opens
in response to the neurotransmitter acetylcholine (and
to nicotine, hence the name). This receptor is found
in the postsynaptic membrane of neurons at certain
synapses and in muscle fibers (myocytes) at neuro-
muscular junctions.
Acetylcholine released by an excited neuron dif-
fuses a few micrometers across the synaptic cleft or neu-
romuscular junction to the postsynaptic neuron or my-
ocyte, where it interacts with the acetylcholine receptor
and triggers electrical excitation (depolarization) of the
receiving cell. The acetylcholine receptor is an allosteric
protein with two high-affinity binding sites for acetyl-
choline, about 3.0 nm from the ion gate, on the two H9251
Acetylcholine (Ach)
CH
3
CH
2
CH
2
ON
CH
3
CH
3
O
H11001
C CH
3
Chapter 12 Biosignaling426
TABLE 12–2 Ion Concentrations in Cells and Extracellular Fluids (mM)
K
H11001
Na
H11001
Ca
2H11001
Cl
H11002
Cell type In Out In Out In Out In Out
Squid axon 400 20 50 440 H113490.4 10 40–150 560
Frog muscle 124 2.3 10.4 109 H110210.1 2.1 1.5 78
8885d_c12_426 2/20/04 1:16 PM Page 426 mac76 mac76:385_reb:
subunits. The binding of acetylcholine causes a change
from the closed to the open conformation. The process
is positively cooperative: binding of acetylcholine to the
first site increases the acetylcholine-binding affinity of
the second site. When the presynaptic cell releases a
brief pulse of acetylcholine, both sites on the postsy-
naptic cell receptor are occupied briefly and the chan-
nel opens (Fig. 12–4). Either Na
H11001
or Ca
2H11001
can now pass,
and the inward flux of these ions depolarizes the plasma
membrane, initiating subsequent events that vary with
the type of tissue. In a postsynaptic neuron, depolar-
ization initiates an action potential (see below); at a neu-
romuscular junction, depolarization of the muscle fiber
triggers muscle contraction.
Normally, the acetylcholine concentration in the
synaptic cleft is quickly lowered by the enzyme acetyl-
cholinesterase, present in the cleft. When acetylcholine
levels remain high for more than a few milliseconds, the
receptor is desensitized (Fig. 12–1c). The receptor
channel is converted to a third conformation (Fig.
12–4c) in which the channel is closed and the acetyl-
choline is very tightly bound. The slow release (in tens
of milliseconds) of acetylcholine from its binding sites
eventually allows the receptor to return to its resting
state—closed and resensitized to acetylcholine levels.
Voltage-Gated Ion Channels Produce Neuronal
Action Potentials
Signaling in the nervous system is accomplished by net-
works of neurons, specialized cells that carry an elec-
trical impulse (action potential) from one end of the cell
(the cell body) through an elongated cytoplasmic ex-
tension (the axon). The electrical signal triggers release
of neurotransmitter molecules at the synapse, carrying
the signal to the next cell in the circuit. Three types of
voltage-gated ion channels are essential to this
signaling mechanism. Along the entire length of the
axon are voltage-gated Na
H11545
channels (Fig. 12–5; see
also Fig. 11–50), which are closed when the membrane
is at rest (V
m
H11005H1100260 mV) but open briefly when the
membrane is depolarized locally in response to acetyl-
choline (or some other neurotransmitter). The depo-
larization induced by the opening of Na
H11001
channels
causes voltage-gated K
H11545
channels to open, and the
resulting efflux of K
H11001
repolarizes the membrane locally.
A brief pulse of depolarization traverses the axon as lo-
cal depolarization triggers the brief opening of neigh-
boring Na
H11001
channels, then K
H11001
channels. After each
opening of a Na
H11001
channel, a short refractory period fol-
lows during which that channel cannot open again, and
thus a unidirectional wave of depolarization sweeps
from the nerve cell body toward the end of the axon.
The voltage sensitivity of ion channels is due to the pres-
ence at critical positions in the channel protein of
charged amino acid side chains that interact with the
electric field across the membrane. Changes in trans-
membrane potential produce subtle conformational
changes in the channel protein (see Fig. 11–50).
At the distal tip of the axon are voltage-gated
Ca
2H11545
channels. When the wave of depolarization
reaches these channels, they open, and Ca
2H11001
enters
from the extracellular space. The rise in cytoplasmic
[Ca
2H11001
] then triggers release of acetylcholine by exocy-
tosis into the synaptic cleft (step 3 in Fig. 12–5).
Acetylcholine diffuses to the postsynaptic cell (another
12.2 Gated Ion Channels 427
ACh
Continued
excitation
Na
H11545
, Ca
2H11545
Acetylcholine
binding sites
Outside
Inside
(a)
Resting
(gate closed)
(b)
Excited
(gate open)
(c)
Desensitized
(gate closed)
ACh
FIGURE 12–4 Three states of the acetylcholine receptor. Brief ex-
posure of (a) the resting (closed) ion channel to acetylcholine (ACh)
produces (b) the excited (open) state. Longer exposure leads to (c) de-
sensitization and channel closure.
8885d_c12_427 2/20/04 1:16 PM Page 427 mac76 mac76:385_reb:
neuron or a myocyte), where it binds to acetylcholine
receptors and triggers depolarization. Thus the message
is passed to the next cell in the circuit.
We see, then, that gated ion channels convey sig-
nals in either of two ways: by changing the cytosolic con-
centration of an ion (such as Ca
2H11001
), which then serves
as an intracellular second messenger (the hormone or
neurotransmitter is the first messenger), or by chang-
ing V
m
and affecting other membrane proteins that are
sensitive to V
m
. The passage of an electrical signal
through one neuron and on to the next illustrates both
types of mechanism.
Neurons Have Receptor Channels That Respond
to Different Neurotransmitters
Animal cells, especially those of the nervous system, con-
tain a variety of ion channels gated by ligands, voltage,
or both. The neurotransmitters 5-hydroxytryptamine
(serotonin), glutamate, and glycine can all act through
receptor channels that are structurally related to the
acetylcholine receptor. Serotonin and glutamate trigger
the opening of cation (K
H11001
, Na
H11001
, Ca
2H11001
) channels, whereas
glycine opens Cl
H11002
-specific channels. Cation and anion
channels are distinguished by subtle differences in the
amino acid residues that line the hydrophilic channel.
Cation channels have negatively charged Glu and Asp side
chains at crucial positions. When a few of these acidic
residues are experimentally replaced with basic residues,
the cation channel is converted to an anion channel.
Depending on which ion passes through a channel,
the ligand (neurotransmitter) for that channel either de-
polarizes or hyperpolarizes the target cell. A single neu-
ron normally receives input from several (or many)
other neurons, each releasing its own characteristic
neurotransmitter with its characteristic depolarizing or
hyperpolarizing effect. The target cell’s V
m
therefore
reflects the integrated input (Fig. 12–1d) from multi-
Chapter 12 Biosignaling428
Axon of
presynaptic
neuron
Voltage-
gated Na
H11001
channel
Voltage-
gated K
H11001
channel
Action
potential
Secretory
vesicles containing
acetylcholine
Synaptic
cleft
Acetylcholine
receptor ion
channels
Action
potential
Cell body of
postsynaptic
neuron
Volted-
gated Ca
2H11001
channel
Na
H11001
Na
H11001
Ca
2H11001
Na
H11001
,Ca
2H11001
Na
H11001
Na
H11001
K
H11001
K
H11001
K
H11001
H11001
H11001
H11001H11002
H11001H11002
H11001
H11001
H11001
H11002
H11001
H11001
H11001
H11001
H11001
H11001 H11002
H11002
H11002
H11002H11001
H11001
H11001 H11002
H11001
H11001
H11001
H11001
1
2
3
5
4
FIGURE 12–5 Role of voltage-gated and ligand-gated ion channels
in neural transmission. Initially, the plasma membrane of the pre-
synaptic neuron is polarized (inside negative) through the action of
the electrogenic Na
H11001
K
H11001
ATPase, which pumps 3 Na
H11001
out for every 2
K
H11001
pumped into the neuron (see Fig. 12–3). 1 A stimulus to this neu-
ron causes an action potential to move along the axon (white arrow),
away from the cell body. The opening of one voltage-gated Na
H11001
chan-
nel allows Na
H11001
entry, and the resulting local depolarization causes
the adjacent Na
H11001
channel to open, and so on. The directionality of
movement of the action potential is ensured by the brief refractory
period that follows the opening of each voltage-gated Na
H11001
channel.
2 When the wave of depolarization reaches the axon tip, voltage-
gated Ca
2H11001
channels open, allowing Ca
2H11001
entry into the presynaptic
neuron. 3 The resulting increase in internal [Ca
2H11001
] triggers exocytic
release of the neurotransmitter acetylcholine into the synaptic cleft.
4 Acetylcholine binds to a receptor on the postsynaptic neuron, caus-
ing its ligand-gated ion channel to open. 5 Extracellular Na
H11001
and
Ca
2H11001
enter through this channel, depolarizing the postsynaptic cell.
The electrical signal has thus passed to the cell body of the post-
synaptic neuron and will move along its axon to a third neuron by
this same sequence of events.
Glutamate
CH
2
COO
H11002
H
3
N CH
CH
2
COO
H11002
H11001
Serotonin
(5-hydroxytryptamine)
HO
N
H
CH
2
CH
2
NH
3
H11001
8885d_c12_428 2/20/04 1:16 PM Page 428 mac76 mac76:385_reb:
ple neurons. The cell responds with an action potential
only if the integrated input adds up to a net depolar-
ization of sufficient size.
The receptor channels for acetylcholine, glycine,
glutamate, and H9253-aminobutyric acid (GABA) are gated
by extracellular ligands. Intracellular second messen-
gers—such as cAMP, cGMP (3H11032,5H11032-cyclic GMP, a close
analog of cAMP), IP
3
(inositol 1,4,5-trisphosphate),
Ca
2H11001
, and ATP—regulate ion channels of another class,
which, as we shall see in Section 12.7, participate in the
sensory transductions of vision, olfaction, and gustation.
SUMMARY 12.2 Gated Ion Channels
■ Ion channels gated by ligands or membrane
potential are central to signaling in neurons
and other cells.
■ The acetylcholine receptor of neurons and
myocytes is a ligand-gated ion channel.
■ The voltage-gated Na
H11001
and K
H11001
channels of
neuronal membranes carry the action potential
along the axon as a wave of depolarization
(Na
H11001
influx) followed by repolarization (K
H11001
efflux).
■ The arrival of an action potential triggers
neurotransmitter release from the presynaptic
cell. The neurotransmitter (acetylcholine, for
example) diffuses to the postsynaptic cell,
binds to specific receptors in the plasma
membrane, and triggers a change in V
m
.
12.3 Receptor Enzymes
A fundamentally different mechanism of signal trans-
duction is carried out by the receptor enzymes. These
proteins have a ligand-binding domain on the extracel-
lular surface of the plasma membrane and an enzyme
active site on the cytosolic side, with the two domains
connected by a single transmembrane segment. Com-
monly, the receptor enzyme is a protein kinase that
phosphorylates Tyr residues in specific target proteins;
the insulin receptor is the prototype for this group. In
plants, the protein kinase of receptors is specific for Ser
or Thr residues. Other receptor enzymes synthesize the
intracellular second messenger cGMP in response to ex-
tracellular signals. The receptor for atrial natriuretic fac-
tor is typical of this type.
The Insulin Receptor Is a Tyrosine-Specific
Protein Kinase
Insulin regulates both metabolism and gene expression:
the insulin signal passes from the plasma membrane re-
ceptor to insulin-sensitive metabolic enzymes and to the
nucleus, where it stimulates the transcription of specific
genes. The active insulin receptor consists of two iden-
tical H9251 chains protruding from the outer face of the
plasma membrane and two transmembrane H9252 subunits
with their carboxyl termini protruding into the cytosol
(Fig. 12–6, step 1 ). The H9251 chains contain the insulin-
binding domain, and the intracellular domains of the H9252
chains contain the protein kinase activity that transfers
a phosphoryl group from ATP to the hydroxyl group of
Tyr residues in specific target proteins. Signaling
through the insulin receptor begins (step 1 ) when
binding of insulin to the H9251 chains activates the Tyr ki-
nase activity of the H9252 chains, and each H9251H9252 monomer
phosphorylates three critical Tyr residues near the car-
boxyl terminus of the H9252 chain of its partner in the dimer.
This autophosphorylation opens up the active site so
that the enzyme can phosphorylate Tyr residues of other
target proteins (Fig. 12–7).
One of these target proteins (Fig. 12–6, step 2 ) is
insulin receptor substrate-1 (IRS-1). Once phosphory-
lated on its Tyr residues, IRS-1 becomes the point of nu-
cleation for a complex of proteins (step 3 ) that carry
the message from the insulin receptor to end targets in
the cytosol and nucleus, through a long series of inter-
mediate proteins. First, a P –Tyr residue in IRS-1 is
bound by the SH2 domain of the protein Grb2. (SH2
is an abbreviation of Src homology 2; the sequences of
SH2 domains are similar to a domain in another protein
Tyr kinase, Src (pronounced sark).) A number of sig-
naling proteins contain SH2 domains, all of which bind
P –Tyr residues in a protein partner. Grb2 also contains
a second protein-binding domain, SH3, that binds to re-
gions rich in Pro residues. Grb2 binds to a proline-rich
region of Sos, recruiting Sos to the growing receptor
complex. When bound to Grb2, Sos catalyzes the re-
placement of bound GDP by GTP on Ras, one of a family
of guanosine nucleotide–binding proteins (G proteins)
that mediate a wide variety of signal transductions (Sec-
tion 12.4). When GTP is bound, Ras can activate a pro-
tein kinase, Raf-1 (step 4 ), the first of three protein
kinases—Raf-1, MEK, and ERK—that form a cascade in
which each kinase activates the next by phosphoryla-
tion (step 5 ). The protein kinase ERK is activated by
phosphorylation of both a Thr and a Tyr residue. When
activated, it mediates some of the biological effects of
insulin by entering the nucleus and phosphorylating pro-
teins such as Elk1, which modulates the transcription
of about 100 insulin-regulated genes (step 6 ).
The proteins Raf-1, MEK, and ERK are members of
three larger families, for which several nomenclatures
are employed. ERK is a member of the MAPK family
(mitogen-activated protein kinases; mitogens are sig-
nals that act from outside the cell to induce mitosis and
cell division). Soon after discovery of the first MAPK, that
enzyme was found to be activated by another protein
kinase, which came to be called MAP kinase kinase (MEK
12.3 Receptor Enzymes 429
8885d_c12_429 2/20/04 1:17 PM Page 429 mac76 mac76:385_reb:
belongs to this family); and when a third kinase that ac-
tivated MAP kinase kinase was discovered, it was given
the slightly ludicrous family name MAP kinase kinase
kinase (Raf-1 is a member of this family; Fig. 12–6).
Slightly less cumbersome are the acronyms for these
three families, MAPK, MAPKK, and MAPKKK. Kinases
in the MAPK and MAPKKK families are specific for Ser
or Thr residues, but MAPKKs (here, MEK) phosphory-
late both a Ser and a Tyr residue in their substrate, a
MAPK (here, ERK).
Biochemists now recognize the insulin pathway as
but one instance of a more general theme in which hor-
mone signals, via pathways similar to that shown in Fig-
ure 12–6, result in phosphorylation of target enzymes
by protein kinases. The target of phosphorylation is of-
ten another protein kinase, which then phosphorylates
a third protein kinase, and so on. The result is a cas-
cade of reactions that amplifies the initial signal by many
orders of magnitude (see Fig. 12–1b). Cascades such as
that shown in Figure 12–6 are called MAPK cascades.
Chapter 12 Biosignaling430
New proteins
DNA
Insulin
P
P
P
P
P
PP
P
P
P
P
P
P
Grb2
Sos
Ras
GTP
GDP
Raf-1
MEK
ERKERK
P
ERK
MEK
P
P
IRS-1
IRS-1
Elk1SRF
Elk1SRF
Insulin receptor
phosphorylates IRS-1
on its Tyr residues.
2
Activated Ras binds and
activates Raf-1.
4
ERK moves into
the nucleus and
phosphorylates
nuclear transcription
factors such as Elk1,
activating them.
6
Phosphorylated Elk1
joins SRF to stimulate
the transcription and
translation of a set of
genes needed for
cell division.
7
Cytosol
Nucleus
SH2 domain of Grb2 binds
to P –Tyr of IRS-1. Sos binds
to Grb2, then to Ras,
causing GDP release and
GTP binding to Ras.
3
Raf-1 phosphorylates
MEK on two Ser residues,
activating it. MEK
phosphorylates ERK
on a Thr and a Tyr residue,
activating it.
5
Insulin receptor binds
insulin and undergoes
autophosphorylation on its
carboxyl-terminal Tyr residues.
1
aa
bb
FIGURE 12–6 Regulation of gene expression by insulin. The insulin
receptor consists of two H9251 chains on the outer face of the plasma mem-
brane and two H9252 chains that traverse the membrane and protrude from
the cytoplasmic face. Binding of insulin to the H9251 chains triggers a con-
formational change that allows the autophosphorylation of Tyr residues
in the carboxyl-terminal domain of the H9252 subunits. Autophosphoryla-
tion further activates the Tyr kinase domain, which then catalyzes phos-
phorylation of other target proteins. The signaling pathway by which
insulin regulates the expression of specific genes consists of a cascade
of protein kinases, each of which activates the next. The insulin re-
ceptor is a Tyr-specific kinase; the other kinases (all shown in blue)
phosphorylate Ser or Thr residues. MEK is a dual-specificity kinase,
which phosphorylates both a Thr and a Tyr residue in ERK (extracel-
lular regulated kinase); MEK is mitogen-activated, ERK-activating ki-
nase; SRF is serum response factor. Abbreviations for other compo-
nents are explained in the text.
8885d_c12_430 2/20/04 1:17 PM Page 430 mac76 mac76:385_reb:
Inactive (unphosphorylated)
Tyr kinase domain
(a)
Activation loop
blocks substrate-
binding site
Tyr
1158
Tyr
1162
Tyr
1163
Asp
1132
Tyr
1158
Tyr
1162
Tyr
1163
Target protein
in substrate-
binding site
Active (triply phosphorylated)
Tyr kinase domain
(b)
Grb2 is not the only protein that associates with
phosphorylated IRS-1. The enzyme phosphoinositide 3-
kinase (PI-3K) binds IRS-1 through the former’s SH2
domain (Fig. 12–8). Thus activated, PI-3K converts the
membrane lipid phosphatidylinositol 4,5-bisphosphate
(see Fig. 10–15), also called PIP
2
, to phosphatidylinos-
itol 3,4,5-trisphosphate (PIP
3
). When bound to PIP
3
,
protein kinase B (PKB) is phosphorylated and activated
by yet another protein kinase, PDK1. The activated PKB
then phosphorylates Ser or Thr residues on its target
proteins, one of which is glycogen synthase kinase 3
(GSK3). In its active, nonphosphorylated form, GSK3
phosphorylates glycogen synthase, inactivating it and
thereby contributing to the slowing of glycogen synthe-
sis. (This mechanism is believed to be only part of the
explanation for the effects of insulin on glycogen me-
tabolism.) When phosphorylated by PKB, GSK3 is inac-
tivated. By thus preventing inactivation of glycogen
synthase in liver and muscle, the cascade of protein
phosphorylations initiated by insulin stimulates glyco-
gen synthesis (Fig. 12–8). In muscle, PKB triggers the
movement of glucose transporters (GLUT4) from inter-
nal vesicles to the plasma membrane, stimulating glu-
cose uptake from the blood (Fig. 12–8; see also Box
11–2). PKB also functions in several other signaling
pathways, including that triggered by H9004
9
-tetrahydro-
cannabinol (THC), the active ingredient of marijuana
12.3 Receptor Enzymes 431
FIGURE 12–7 Activation of the insulin-receptor Tyr kinase by au-
tophosphorylation. (a) In the inactive form of the Tyr kinase domain
(PDB ID 1IRK), the activation loop (blue) sits in the active site, and
none of the critical Tyr residues (black and red ball-and-stick struc-
tures) are phosphorylated. This conformation is stabilized by hydro-
gen bonding between Tyr
1162
and Asp
1132
. (b) When insulin binds to
the H9251 chains of insulin receptors, the Tyr kinase of each H9252 subunit of
the dimer phosphorylates three Tyr residues (Tyr
1158
, Tyr
1162
, and
Tyr
1163
) on the other H9252 subunit (shown here; PDB ID 1IR3). (Phos-
phoryl groups are depicted here as an orange space-filling phospho-
rus atom and red ball-and-stick oxygen atoms.) The effect of intro-
ducing three highly charged P –Tyr residues is to force a 30 ? change
in the position of the activation loop, away from the substrate-binding
site, which becomes available to bind to and phosphorylate a target
protein, shown here as a red arrow.
O
H
CH
3
(CH
2
)
3
H
CH
3
CH
3
HO
CH
3
H9004
9
-Tetrahydrocannabinol (THC)
8885d_c12_431 2/20/04 1:17 PM Page 431 mac76 mac76:385_reb:
P
P
PKB
PI-3K
PIP
2
PIP
3
P
GS
(inactive)
GSK3
(active)
GSK3
(inactive)
GS
(active)
IRS-1
IRS-1, phosphorylated
by the insulin receptor,
activates PI-3K by binding
to its SH2 domain. PI-3K
converts PIP
2
to PIP
3
.
Glucose
GLUT4
1
GSK3, inactivated by
phosphorylation, cannot
convert glycogen synthase
(GS) to its inactive form
by phosphorylation, so
GS remains active.
3
Synthesis of
glycogen
from glucose
is accelerated.
4
PKB stimulates movement
of glucose transporter GLUT4
from internal membrane vesicles
to the plasma membrane,
increasing the uptake of glucose.
5
Glycogen
2
P
P
P
P
PKB bound to PIP
3
is phosphorylated by
PDK1 (not shown).
Thus activated, PKB
phosphorylates GSK3
on a Ser residue,
inactivating it.
and hashish. THC activates the CB
1
receptor in the
plasma membrane of neurons in the brain, triggering a
signaling cascade that involves MAPKs. One conse-
quence of CB
1
activation is the stimulation of appetite,
one of the well-established effects of marijuana use. The
normal ligands for the CB
1
receptor are endocannabi-
noids such as anandamide, which serve to protect the
brain from the toxicity of excessive neuronal activity—
as in an epileptic seizure, for example. Hashish has for
centuries been used in the treatment of epilepsy.
As in all signaling pathways, there is a mecha-
nism for terminating signaling through the PI-
3K–PKB pathway. A PIP
3
-specific phosphatase (PTEN
in humans) removes the phosphate at the 3 position of
PIP
3
to produce PIP
2
, which no longer serves as a bind-
ing site for PKB, and the signaling chain is broken. In
various types of advanced cancer, tumor cells often have
a defect in the PTEN gene and thus have abnormally
high levels of PIP
3
and of PKB activity. The result seems
to be a continuing signal for cell division and thus tu-
mor growth. ■
O
N
H
OH
Anandamide (arachidonylethanolamide,
an endogenous cannabinoid)
What spurred the evolution of such complicated
regulatory machinery? This system allows one activated
receptor to activate several IRS-1 molecules, amplifying
the insulin signal, and it provides for the integration of
signals from several receptors, each of which can phos-
phorylate IRS-1. Furthermore, because IRS-1 can acti-
vate any of several proteins that contain SH2 domains,
a single receptor acting through IRS-1 can trigger two
or more signaling pathways; insulin affects gene ex-
pression through the Grb2-Sos-Ras-MAPK pathway and
glycogen metabolism through the PI-3K–PKB pathway.
The insulin receptor is the prototype for a number
of receptor enzymes with a similar structure and recep-
tor Tyr kinase activity. The receptors for epidermal
growth factor and platelet-derived growth factor, for ex-
ample, have structural and sequence similarities to the
insulin receptor, and both have a protein Tyr kinase ac-
tivity that phosphorylates IRS-1. Many of these recep-
tors dimerize after binding ligand; the insulin receptor
is already a dimer before insulin binds. The binding of
adaptor proteins such as Grb2 to P –Tyr residues is a
common mechanism for promoting protein-protein in-
teractions, a subject to which we return in Section 12.5.
In addition to the many receptors that act as protein
Tyr kinases, a number of receptorlike plasma membrane
proteins have protein Tyr phosphatase activity. Based on
the structures of these proteins, we can surmise that their
ligands are components of the extracellular matrix or the
Chapter 12 Biosignaling432
FIGURE 12–8 Activation of glycogen synthase by insulin. Transmission of the signal is
mediated by PI-3 kinase (PI-3K) and protein kinase B (PKB).
8885d_c12_432 2/20/04 1:17 PM Page 432 mac76 mac76:385_reb:
surfaces of other cells. Although their signaling roles are
not yet as well understood as those of the receptor Tyr
kinases, they clearly have the potential to reverse the ac-
tions of signals that stimulate these kinases.
A variation on the basic theme of receptor Tyr ki-
nases is seen in receptors that have no intrinsic protein
kinase activity but, when occupied by their ligand, bind
a soluble Tyr kinase. One example is the system that
regulates the formation of erythrocytes in mammals.
The cytokine (developmental signal) for this system is
erythropoietin (EPO), a 165 amino acid protein pro-
duced in the kidneys. When EPO binds to its plasma
membrane receptor (Fig. 12–9), the receptor dimerizes
and can now bind the soluble protein kinase JAK (Janus
kinase). This binding activates JAK, which phosphory-
lates several Tyr residues in the cytoplasmic domain of
the EPO receptor. A family of transcription factors, col-
lectively called STATs (signal transducers and activa-
tors of transcription), are also targets of the JAK kinase
activity. An SH2 domain in STAT5 binds P –Tyr residues
in the EPO receptor, positioning it for this phosphory-
lation by JAK. When STAT5 is phosphorylated in re-
sponse to EPO, it forms dimers, exposing a signal for its
transport into the nucleus. There, STAT5 causes the ex-
pression (transcription) of specific genes essential for
erythrocyte maturation. This JAK-STAT system oper-
ates in a number of other signaling pathways, including
that for the hormone leptin, described in detail in Chap-
ter 23 (see Fig. 23–34). Activated JAK can also trigger,
through Grb2, the MAPK cascade (Fig. 12–6), which
leads to altered expression of specific genes.
Src is another soluble protein Tyr kinase that asso-
ciates with certain receptors when they bind their lig-
ands. Src was the first protein found to have the char-
acteristic P –Tyr-binding domain that was subsequently
named the Src homology (SH2) domain. Yet another ex-
ample of a receptor’s association with a soluble protein
kinase is the Toll-like receptor (TLR4) system through
which mammals detect the bacterial lipopolysaccharide
(LPS), a potent toxin. We return to the Toll-like recep-
tor system in Section 12.6, in the context of the evolu-
tion of signaling proteins.
Receptor Guanylyl Cyclases Generate the Second
Messenger cGMP
Guanylyl cyclases (Fig. 12–10) are another type of re-
ceptor enzyme. When activated, a guanylyl cyclase pro-
duces guanosine 3H11541,5H11541-cyclic monophosphate (cyclic
GMP, cGMP) from GTP:
12.3 Receptor Enzymes 433
SH2 domain
dimerization
NLS
Affects gene
expression in
nucleus
MAPK
cascade
MAPK
Erythropoietin
EPO receptor
P
P
P
P
P
P
P
P
JAKJAK
P
STAT
P
STAT
P
STAT
STAT
Grb2
FIGURE 12–9 The JAK-STAT transduction mechanism for the ery-
thropoietin receptor. Binding of erythropoietin (EPO) causes dimer-
ization of the EPO receptor, which allows the soluble Tyr kinase JAK
to bind to the internal domain of the receptor and phosphorylate it on
several Tyr residues. The STAT protein STAT5 contains an SH2 domain
and binds to the P –Tyr residues on the receptor, bringing the recep-
tor into proximity with JAK. Phosphorylation of STAT5 by JAK allows
two STAT molecules to dimerize, each binding the other’s P –Tyr
residue. Dimerization of STAT5 exposes a nuclear localization se-
quence (NLS) that targets STAT5 for transport into the nucleus. In the
nucleus, STAT causes the expression of genes controlled by EPO. A
second signaling pathway is also triggered by autophosphorylation of
JAK that is associated with EPO binding to its receptor. The adaptor
protein Grb2 binds P –Tyr in JAK and triggers the MAPK cascade, as
in the insulin system (see Fig. 12–6).
H11002
O
H
P
O
H11002
O
P
O
P
H
H
H
N
O
O
PP
i
O
O
O
O
H11002
O
H11002
OO
H
N
N
HN
CH
2
5H11032
Guanosine 3H11032,5H11032-cyclic monophosphate
(cGMP)
H
P
O
H11002
O
OO
H
H
H
N
O
OH
O
O
H
N
N
HN
CH
NH
2
NH
2
2
GTP
3H11032
8885d_c12_433 2/20/04 1:17 PM Page 433 mac76 mac76:385_reb:
Cyclic GMP is a second messenger that carries different
messages in different tissues. In the kidney and intes-
tine it triggers changes in ion transport and water re-
tention; in cardiac muscle (a type of smooth muscle) it
signals relaxation; in the brain it may be involved both
in development and in adult brain function. Guanylyl cy-
clase in the kidney is activated by the hormone atrial
natriuretic factor (ANF), which is released by cells
in the atrium of the heart when the heart is stretched
by increased blood volume. Carried in the blood to the
kidney, ANF activates guanylyl cyclase in cells of the
collecting ducts (Fig. 12–10a). The resulting rise in
[cGMP] triggers increased renal excretion of Na
H11001
and,
consequently, of water, driven by the change in osmotic
pressure. Water loss reduces the blood volume, coun-
tering the stimulus that initially led to ANF secretion.
Vascular smooth muscle also has an ANF receptor—
guanylyl cyclase; on binding to this receptor, ANF causes
relaxation (vasodilation) of the blood vessel, which in-
creases blood flow while decreasing blood pressure.
A similar receptor guanylyl cyclase in the plasma
membrane of intestinal epithelial cells is activated by an
intestinal peptide, guanylin, which regulates Cl
H11002
secre-
tion in the intestine. This receptor is also the target of a
heat-stable peptide endotoxin produced by Escherichia
coli and other gram-negative bacteria. The elevation in
[cGMP] caused by the endotoxin increases Cl
H11002
secretion
and consequently decreases reabsorption of water by
the intestinal epithelium, producing diarrhea.
A distinctly different type of guanylyl cyclase is a
cytosolic protein with a tightly associated heme group
(Fig. 12–10b), an enzyme activated by nitric oxide (NO).
Nitric oxide is produced from arginine by Ca
2H11001
-
dependent NO synthase, present in many mammalian
tissues, and diffuses from its cell of origin into nearby
cells. NO is sufficiently nonpolar to cross plasma mem-
branes without a carrier. In the target cell, it binds to
the heme group of guanylyl cyclase and activates cGMP
production. In the heart, cGMP reduces the forcefulness
of contractions by stimulating the ion pump(s) that ex-
pel Ca
2H11001
from the cytosol.
This NO-induced relaxation of cardiac muscle is
the same response brought about by nitroglyc-
erin tablets and other nitrovasodilators taken to relieve
angina, the pain caused by contraction of a heart de-
prived of O
2
because of blocked coronary arteries. Ni-
tric oxide is unstable and its action is brief; within sec-
onds of its formation, it undergoes oxidation to nitrite
or nitrate. Nitrovasodilators produce long-lasting relax-
ation of cardiac muscle because they break down over
several hours, yielding a steady stream of NO. The value
of nitroglycerin as a treatment for angina was discov-
ered serendipitously in factories producing nitroglycerin
as an explosive in the 1860s. Workers with angina re-
ported that their condition was much improved during
the work week but returned on weekends. The physi-
cians treating these workers heard this story so often
that they made the connection, and a drug was born.
The effects of increased cGMP synthesis diminish
after the stimulus ceases, because a specific phospho-
diesterase (cGMP PDE) converts cGMP to the inactive
5H11032-GMP. Humans have several isoforms of cGMP PDE,
with different tissue distributions. The isoform in the
blood vessels of the penis is inhibited by the drug
sildenafil (Viagra), which therefore causes cGMP levels
to remain elevated once raised by an appropriate stim-
ulus, accounting for the usefulness of this drug in the
treatment of erectile dysfunction. ■
Most of the actions of cGMP in animals are believed
to be mediated by cGMP-dependent protein kinase,
also called protein kinase G or PKG, which, when ac-
Citrulline
NH
2
NH
3
O
NH
(CH
2
)
3
CH COO
H11002
H11001
NADP
H11001
NADPH
O
NO synthase
2
Ca
2H11001
Arginine
NH
2
NH
3
C
NH
(CH
2
)
3
CH COO
H11002
H11001
NH
2
H11001
C
H11001 NO
Chapter 12 Biosignaling434
(a) (b)
Extracellular
ligand-
binding
(receptor)
domains
Guanylin
and
endotoxin
receptors
Intracellular
catalytic
(cGMP-
forming)
domains
Membrane-spanning
guanylyl cyclases
Soluble NO-
activated
guanylyl cyclase
NH
3
H11001
H
3
N
H11001
ANF
receptor
COO
H5008
COO
H5008
Heme Fe
FIGURE 12–10 Two types (isozymes) of guanylyl cyclase that par-
ticipate in signal transduction. (a) One isozyme exists in two similar
membrane-spanning forms that are activated by their extracellular lig-
ands: atrial natriuretic factor, ANF (receptors in cells of the renal col-
lecting ducts and the smooth muscle of blood vessels), and guanylin
(receptors in intestinal epithelial cells). The guanylin receptor is also
the target of a type of bacterial endotoxin that triggers severe diarrhea.
(b) The other isozyme is a soluble enzyme that is activated by intra-
cellular nitric oxide (NO); this form is found in many tissues, includ-
ing smooth muscle of the heart and blood vessels.
8885d_c12_434 2/20/04 1:19 PM Page 434 mac76 mac76:385_reb:
tivated by cGMP, phosphorylates Ser and Thr residues
in target proteins. The catalytic and regulatory domains
of this enzyme are in a single polypeptide (M
r
～80,000).
Part of the regulatory domain fits snugly in the substrate-
binding site. Binding of cGMP forces this part of the
regulatory domain out of the binding site, activating the
catalytic domain.
Cyclic GMP has a second mode of action in the ver-
tebrate eye: it causes ion-specific channels to open in
the retinal rod and cone cells. We return to this role of
cGMP in the discussion of vision in Section 12.7.
SUMMARY 12.3 Receptor Enzymes
■ The insulin receptor is the prototype of
receptor enzymes with Tyr kinase activity.
When insulin binds to its receptor, each H9251H9252
monomer of the receptor phosphorylates the H9252
chain of its partner, activating the receptor’s
Tyr kinase activity. The kinase catalyzes the
phosphorylation of Tyr residues on other
proteins such as IRS-1.
■ P –Tyr residues in IRS-1 serve as binding sites
for proteins with SH2 domains. Some of these
proteins, such as Grb2, have two or more
protein-binding domains and can serve as
adaptors that bring two proteins into proximity.
■ Further protein-protein interactions result in
GTP binding to and activation of the Ras
protein, which in turn activates a protein
kinase cascade that ends with the
phosphorylation of target proteins in the
cytosol and nucleus. The result is specific
metabolic changes and altered gene expression.
■ Several signals, including atrial natriuretic
factor and the intestinal peptide guanylin, act
through receptor enzymes with guanylyl
cyclase activity. The cGMP produced acts as a
second messenger, activating cGMP-dependent
protein kinase (PKG). This enzyme alters
metabolism by phosphorylating specific enzyme
targets.
■ Nitric oxide (NO) is a short-lived messenger
that acts by stimulating a soluble guanylyl
cyclase, raising [cGMP] and stimulating PKG.
12.4 G Protein–Coupled Receptors and
Second Messengers
A third mechanism of signal transduction, distinct from
gated ion channels and receptor enzymes, is defined
by three essential components: a plasma membrane
receptor with seven transmembrane helical segments,
an enzyme in the plasma membrane that generates
an intracellular second messenger, and a guanosine
nucleotide–binding protein (G protein). The G pro-
tein, stimulated by the activated receptor, exchanges
bound GDP for GTP; the GTP-protein dissociates from
the occupied receptor and binds to a nearby enzyme,
altering its activity. The human genome encodes more
than 1,000 members of this family of receptors, spe-
cialized for transducing messages as diverse as light,
smells, tastes, and hormones. The H9252-adrenergic recep-
tor, which mediates the effects of epinephrine on many
tissues, is the prototype for this type of transducing
system.
The H9252-Adrenergic Receptor System Acts through the
Second Messenger cAMP
Epinephrine action begins when the hormone binds to a
protein receptor in the plasma membrane of a hormone-
sensitive cell. Adrenergic receptors (“adrenergic” re-
flects the alternative name for epinephrine, adrenaline)
are of four general types, H9251
1
, H9251
2
, H9252
1
, and H9252
2
, defined by
subtle differences in their affinities and responses to a
group of agonists and antagonists. Agonists are struc-
tural analogs that bind to a receptor and mimic the ef-
fects of its natural ligand; antagonists are analogs that
bind without triggering the normal effect and thereby
block the effects of agonists. In some cases, the affinity
of the synthetic agonist or antagonist for the receptor
is greater than that of the natural agonist (Fig. 12–11).
The four types of adrenergic receptors are found in dif-
ferent target tissues and mediate different responses to
epinephrine. Here we focus on the H9252-adrenergic re-
ceptors of muscle, liver, and adipose tissue. These
receptors mediate changes in fuel metabolism, as de-
scribed in Chapter 23, including the increased break-
down of glycogen and fat. Adrenergic receptors of the
H9252
1
and H9252
2
subtypes act through the same mechanism,
so in our discussion, “H9252-adrenergic” applies to both
types.
The H9252-adrenergic receptor is an integral protein
with seven hydrophobic regions of 20 to 28 amino
acid residues that “snake” back and forth across the
plasma membrane seven times. This protein is a mem-
ber of a very large family of receptors, all with seven
transmembrane helices, that are commonly called ser-
pentine receptors, G protein–coupled receptors
(GPCR), or 7 transmembrane segment (7tm) re-
ceptors. The binding of epinephrine to a site on the
12.4 G Protein–Coupled Receptors and Second Messengers 435
S
O
O
O
N
N
Sildenafil (Viagra)
N
N
N
HN
O
8885d_c12_435 2/20/04 1:19 PM Page 435 mac76 mac76:385_reb:
receptor deep within the membrane (Fig. 12–12, step
1 ) promotes a conformational change in the receptor’s
intracellular domain that affects its interaction with the
second protein in the signal-transduction pathway, a
heterotrimeric GTP-binding stimulatory G protein, or
G
S
, on the cytosolic side of the plasma membrane.
Alfred G. Gilman and Martin Rodbell discovered that
when GTP is bound to G
s
, G
s
stimulates the production
of cAMP by adenylyl cyclase (see below) in the plasma
membrane. The function of G
s
as a molecular switch re-
sembles that of another class of G proteins typified by
Ras, discussed in Section 12.3 in the context of the in-
sulin receptor. Structurally, G
s
and Ras are quite distinct;
G proteins of the Ras type are monomers (M
r
～20,000),
whereas the G proteins that interact with serpentine
Chapter 12 Biosignaling436
FIGURE 12–11 Epinephrine and its synthetic analogs. Epinephrine,
also called adrenaline, is released from the adrenal gland and regulates
energy-yielding metabolism in muscle, liver, and adipose tissue. It also
serves as a neurotransmitter in adrenergic neurons. Its affinity for its re-
ceptor is expressed as a dissociation constant for the receptor-ligand
complex. Isoproterenol and propranolol are synthetic analogs, one an
agonist with an affinity for the receptor that is higher than that of epi-
nephrine, and the other an antagonist with extremely high affinity.
HO
OH
CH CH
2
CH
3
NH
HO
HO
OH
CH CH
2
CH
3
CH
3
NH CH
OH
CHCH
2
CH
3
CH
3
NH CHCH
2
HO
Epinephrine
Isoproterenol
(agonist)
Propranolol
(antagonist)
k
d
(H9262M)
5
0.4
0.0046O
2
The occupied receptor
causes replacement of
the GDP bound to G
s
by GTP, activating G
s
.
1
Epinephrine binds to
its specific receptor.
ATP
cAMP
5H11032-AMP
cyclic nucleotide
phosphodiesterase
GTP GDP
5
cAMP
activates
PKA.
6
Phosphorylation of
cellular proteins by
PKA causes the
cellular response to
epinephrine.
7
cAMP is degraded,
reversing the
activation of PKA.
E
NH
3
H11002
OOC
H11001
Rec
H9253
H9251
H9252
G
s
G
s
GDP
GTP
AC
H9251
4
Adenylyl cyclase
catalyzes the
formation of cAMP.
3
G
s
( subunit) moves
to adenylyl cyclase
and activates it.
H9251
FIGURE 12–12 Transduction of the epinephrine signal: the H9252-
adrenergic pathway. The seven steps of the mechanism that couples
binding of epinephrine (E) to its receptor (Rec) with activation of adenyl-
yl cyclase (AC) are discussed further in the text. The same adenylyl
cyclase molecule in the plasma membrane may be regulated by a
stimulatory G protein (G
s
), as shown, or an inhibitory G protein (G
i
,
not shown). G
s
and G
i
are under the influence of different hormones.
Hormones that induce GTP binding to G
i
cause inhibition of adenyl-
yl cyclase, resulting in lower cellular [cAMP].
Alfred G. Gilman Martin Rodbell, 1925–1998
8885d_c12_436 2/20/04 1:19 PM Page 436 mac76 mac76:385_reb:
G
s
GTP
AC
H9251
receptors are trimers of three different subunits, H9251 (M
r
43,000), H9252 (M
r
37,000), and H9253 (M
r
7,500 to 10,000).
When the nucleotide-binding site of G
s
(on the H9251
subunit) is occupied by GTP, G
s
is active and can acti-
vate adenylyl cyclase (AC in Fig. 12–12); with GDP
bound to the site, G
s
is inactive. Binding of epinephrine
enables the receptor to catalyze displacement of bound
GDP by GTP, converting G
s
to its active form (step
2 ). As this occurs, the H9252 and H9253 subunits of G
s
dissoci-
ate from the H9251 subunit, and G
sH9251
, with its bound GTP,
moves in the plane of the membrane from the receptor
to a nearby molecule of adenylyl cyclase (step 3 ). The
G
sH9251
is held to the membrane by a covalently attached
palmitoyl group (see Fig. 11–14).
Adenylyl cyclase (Fig. 12–13) is an integral protein
of the plasma membrane, with its active site on the cyto-
solic face. It catalyzes the synthesis of cAMP from ATP:
The association of active G
sH9251
with adenylyl cyclase stim-
ulates the cyclase to catalyze cAMP synthesis (Fig.
12–12, step 4 ), raising the cytosolic [cAMP]. This stim-
ulation by G
sH9251
is self-limiting; G
sH9251
is a GTPase that turns
itself off by converting its bound GTP to GDP (Fig.
12–14). The now inactive G
sH9251
dissociates from adenylyl
cyclase, rendering the cyclase inactive. After G
sH9251
reas-
sociates with the H9252 and H9253 subunits (G
sH9252H9253
), G
s
is again
available to interact with a hormone-bound receptor.
Trimeric G Proteins: Molecular On/Off Switches
H11002
O
H
P
O
H11002
O
P
O
P
H
H
H
N
O
O
PP
i
O
O
O
H11002
O
H11002
OO
H
N
N
N
CH
2
5H11032
Adenosine 3H11032,5H11032-cyclic
monophosphate
(cAMP)
H
P
O
H11002
O
OO
H
H
H
N
O
OH
O
H
N
N
N
CH
NH
2
NH
2
2
ATP
3H11032
12.4 G Protein–Coupled Receptors and Second Messengers 437
FIGURE 12–13 Interaction of G
sH9251
with adenylyl cyclase. (PDB ID
1AZS) The soluble catalytic core of the adenylyl cyclase (AC, blue),
severed from its membrane anchor, was cocrystallized with G
sH9251
(green)
to give this crystal structure. The plant terpene forskolin (yellow) is a
drug that strongly stimulates the enzyme, and GTP (red) bound to G
sH9251
triggers interaction of G
sH9251
with adenylyl cyclase.
FIGURE 12–14 Self-inactivation of G
s
. The steps are further described
in the text. The protein’s intrinsic GTPase activity, in many cases stim-
ulated by RGS proteins (regulators of G protein signaling), determines
how quickly bound GTP is hydrolyzed to GDP and thus how long the
G protein remains active.
H9253H9252
H9251
H9253 H9252
GDP
GTP
G
s
G
s
GTP
GDP
P
i
GDP
G
s
1
G
s
with GDP
bound is turned
off; it cannot
activate adenylyl
cyclase.
2
Contact of G
s
with
hormone-receptor
complex causes dis-
placement of bound
GDP by GTP.
3
G
s
with GTP bound
dissociates into a
and bg subunits.
G
sa
-GTP is turned
on; it can activate
adenylyl cyclase.
4
GTP bound to G
sa
is hydrolyzed by the protein’s
intrinsic GTPase; G
sa
thereby turns itself off. The
inactive a subunit reassociates with the bg subunit.
H9251
H9251
8885d_c12_437 2/20/04 1:19 PM Page 437 mac76 mac76:385_reb:
One downstream effect of epinephrine is to activate
glycogen phosphorylase b. This conversion is promoted
by the enzyme phosphorylase b kinase, which catalyzes
the phosphorylation of two specific Ser residues in phos-
phorylase b, converting it to phosphorylase a (see Fig.
6–31). Cyclic AMP does not affect phosphorylase b ki-
nase directly. Rather, cAMP-dependent protein ki-
nase, also called protein kinase A or PKA, which is
allosterically activated by cAMP (Fig. 12–12, step 5 ),
catalyzes the phosphorylation of inactive phosphorylase
b kinase to yield the active form.
The inactive form of PKA contains two catalytic sub-
units (C) and two regulatory subunits (R) (Fig. 12–15a),
which are similar in sequence to the catalytic and reg-
ulatory domains of PKG (cGMP-dependent protein ki-
nase). The tetrameric R
2
C
2
complex is catalytically in-
active, because an autoinhibitory domain of each R
subunit occupies the substrate-binding site of each C
subunit. When cAMP binds to two sites on each R sub-
unit, the R subunits undergo a conformational change
and the R
2
C
2
complex dissociates to yield two free,
catalytically active C subunits. This same basic mecha-
nism—displacement of an autoinhibitory domain—
mediates the allosteric activation of many types of pro-
tein kinases by their second messengers (as in Figs 12–7
and 12–23, for example).
As indicated in Figure 12–12 (step 6 ), PKA regu-
lates a number of enzymes (Table 12–3). Although the
proteins regulated by cAMP-dependent phosphorylation
have diverse functions, they share a region of sequence
similarity around the Ser or Thr residue that undergoes
phosphorylation, a sequence that marks them for regu-
lation by PKA. The catalytic site of PKA (Fig. 12–15b)
interacts with several residues near the Thr or Ser
residue in the target protein, and these interactions de-
fine the substrate specificity. Comparison of the se-
quences of a number of protein substrates for PKA has
yielded the consensus sequence—the specific neigh-
boring residues needed to mark a Ser or Thr residue for
phosphorylation (see Table 12–3).
Signal transduction by adenylyl cyclase entails sev-
eral steps that amplify the original hormone signal (Fig.
Chapter 12 Biosignaling438
(b)
R
C
R
RR
C
C C
4 cAMP4 cAMP
(a)
Inactive PKA
Regulatory subunits:
empty cAMP sites
Regulatory subunits:
autoinhibitory
domains buried
Catalytic subunits:
substrate-binding
sites blocked by
autoinhibitory
domains of R subunits
Active PKA
Catalytic subunits:
open substrate-
binding sites
+
FIGURE 12–15 Activation of cAMP-dependent protein kinase, PKA.
(a) A schematic representation of the inactive R
2
C
2
tetramer, in which
the autoinhibitory domain of a regulatory (R) subunit occupies the
substrate-binding site, inhibiting the activity of the catalytic (C) sub-
unit. Cyclic AMP activates PKA by causing dissociation of the C sub-
units from the inhibitory R subunits. Activated PKA can phosphorylate
a variety of protein substrates (Table 12–3) that contain the PKA con-
sensus sequence (X–Arg–(Arg/Lys)–X–(Ser/Thr)–B, where X is any
residue and B is any hydrophobic residue), including phosphorylase
b kinase. (b) The substrate-binding region of a catalytic subunit re-
vealed by x-ray crystallography (derived from PDB ID 1JBP). Enzyme
side chains known to be critical in substrate binding and specificity
are in blue. The peptide substrate (red) lies in a groove in the enzyme
surface, with its Ser residue (yellow) positioned in the catalytic site.
In the inactive R
2
C
2
tetramer, the autoinhibitory domain of R lies in
this groove, blocking access to the substrate.
8885d_c12_438 2/20/04 1:20 PM Page 438 mac76 mac76:385_reb:
CH
2
N
O
OH
P
O
H11002
H11002
O
H
N
N
N
H
HH
NH
2
3H11032
5H11032
O
O
OH
Cyclic AMP
Adenosine 5H11032-monophosphate (AMP)
H
2
O
CH
2
N
O
OHP
O
H11002
H
N
N
N
H
HH
3H11032
5H11032
O
OO
NH
2
12–16). First, the binding of one hormone molecule to
one receptor catalytically activates several G
s
molecules.
Next, by activating a molecule of adenylyl cyclase, each
active G
sH9251
molecule stimulates the catalytic synthesis of
many molecules of cAMP. The second messenger cAMP
now activates PKA, each molecule of which catalyzes
the phosphorylation of many molecules of the target
protein—phosphorylase b kinase in Figure 12–16. This
The intracellular signal therefore persists only as long
as the hormone receptor remains occupied by epineph-
rine. Methyl xanthines such as caffeine and theophylline
(a component of tea) inhibit the phosphodiesterase, in-
creasing the half-life of cAMP and thereby potentiating
agents that act by stimulating adenylyl cyclase.
The H9252-Adrenergic Receptor Is Desensitized
by Phosphorylation
As noted earlier, signal-transducing systems undergo
desensitization when the signal persists. Desensitization
of the H9252-adrenergic receptor is mediated by a protein
kinase that phosphorylates the receptor on the intra-
cellular domain that normally interacts with G
s
(Fig.
12–17). When the receptor is occupied by epinephrine,
12.4 G Protein–Coupled Receptors and Second Messengers 439
x molecules
Epinephrine-receptor
complex
x molecules
Active PKAInactive PKA
10x molecules
Active
phosphorylase b
kinase
100x molecules
Inactive
phosphorylase b
kinase
Active glycogen
phosphorylase a
1,000x molecules
Inactive glycogen
phosphorylase b
Glycogen Glucose 1-phosphate
Hepatocyte
ATP Cyclic AMP
adenylyl
cyclase
20x molecules
Blood glucose
10,000x molecules
Glucose
10,000x molecules
Epinephrine
G
Sa
many
steps
FIGURE 12–16 Epinephrine cascade. Epinephrine triggers a series of
reactions in hepatocytes in which catalysts activate catalysts, resulting
in great amplification of the signal. Binding of a small number of mol-
ecules of epinephrine to specific H9252-adrenergic receptors on the cell
surface activates adenylyl cyclase. To illustrate amplification, we show
20 molecules of cAMP produced by each molecule of adenylyl cyclase,
the 20 cAMP molecules activating 10 molecules of PKA, each PKA
molecule activating 10 molecules of the next enzyme (a total of 100),
and so forth. These amplifications are probably gross underestimates.
kinase activates glycogen phosphorylase b, which leads
to the rapid mobilization of glucose from glycogen. The
net effect of the cascade is amplification of the hormonal
signal by several orders of magnitude, which accounts
for the very low concentration of epinephrine (or any
other hormone) required for hormone activity.
Cyclic AMP, the intracellular second messenger in
this system, is short-lived. It is quickly degraded by
cyclic nucleotide phosphodiesterase to 5H11032-AMP
(Fig. 12–12, step
7
), which is not active as a second
messenger:
8885d_c12_439 2/20/04 1:20 PM Page 439 mac76 mac76:385_reb:
Chapter 12 Biosignaling440
TABLE 12–3 Some Enzymes and Other Proteins Regulated by cAMP-Dependent Phosphorylation (by PKA)
Enzyme/protein Sequence phosphorylated
*
Pathway/process regulated
Glycogen synthase RASCTSSS Glycogen synthesis
Phosphorylase b kinase
H9251 subunit VEFRRLSI
Glycogen breakdown
H9252 subunit RTKRSGSV
Pyruvate kinase (rat liver) GVLRRASVAZL Glycolysis
Pyruvate dehydrogenase complex (type L) GYLRRASV Pyruvate to acetyl-CoA
Hormone-sensitive lipase PMRRSV Triacylglycerol mobilization and fatty
acid oxidation
Phosphofructokinase-2/fructose 2,6-bisphosphatase LQRRRGSSIPQ Glycolysis/gluconeogenesis
Tyrosine hydroxylase FIGRRQSL Synthesis of L-DOPA, dopamine,
norepinephrine, and epinephrine
Histone H1 AKRKASGPPVS DNA condensation
Histone H2B KKAKASRKESYSVYVYK DNA condensation
Cardiac phospholamban (cardiac pump regulator) AIRRAST Intracellular [Ca
2H11001
]
Protein phosphatase-1 inhibitor-1 IRRRRPTP Protein dephosphorylation
PKA consensus sequence
?
XR(R/K)X(S/T)B Many
*The phosphorylated S or T residue is shown in red. All residues are given as their one-letter abbreviations (see Table 3–1).
?
X is any amino acid; B is any hydrophobic amino acid.
}
P
P
Binding of epinephrine (E)
to b-adrenergic receptor
triggers dissociation of
G
sbg
from G
sa
(not shown).
1
G
sbg
recruits bARK to the membrane,
where it phosphorylates Ser
residues at the carboxyl
terminus of the receptor.
2
barr binds to the
phosphorylated
carboxyl-terminal
domain of the receptor.
3
EE
E
bARK
b
arr
G
sbg
G
sbg
P
P
P
P
P
P
In endocytic vesicle,
arrestin dissociates;
receptor is dephosphorylated
and returned to cell surface.
5
Receptor-arrestin
complex enters the cell
by endocytosis.
4
FIGURE 12–17 Desensitization of the H9252-adrenergic receptor in the
continued presence of epinephrine. This process is mediated by two
proteins: H9252-adrenergic protein kinase (H9252ARK) and H9252-arrestin (H9252arr;
arrestin 2).
8885d_c12_440 2/20/04 1:20 PM Page 440 mac76 mac76:385_reb:
H9252-adrenergic receptor kinase (H9252ARK) phosphory-
lates Ser residues near the carboxyl terminus of the re-
ceptor. Normally located in the cytosol, H9252ARK is drawn
to the plasma membrane by its association with th G
sH9252H9253
subunits and is thus positioned to phosphorylate the re-
ceptor. The phosphorylation creates a binding site for
the protein H9252-arrestin (H9252arr), also called arrestin 2,
and binding of H9252-arrestin effectively prevents interac-
tion between the receptor and the G protein. The bind-
ing of H9252-arrestin also facilitates receptor sequestration,
the removal of receptors from the plasma membrane by
endocytosis into small intracellular vesicles. Receptors
in the endocytic vesicles are dephosphorylated, then re-
turned to the plasma membrane, completing the circuit
and resensitizing the system to epinephrine. H9252-Adrenergic
receptor kinase is a member of a family of G protein–
coupled receptor kinases (GRKs), all of which phos-
phorylate serpentine receptors on their carboxyl-terminal
cytosolic domains and play roles similar to that of H9252ARK
in desensitization and resensitization of their receptors. At
least five different GRKs and four different arrestins are
encoded in the human genome; each GRK is capable of
desensitizing a subset of the serpentine receptors, and
each arrestin can interact with many different types of
phosphorylated receptors.
While preventing the signal from a serpentine re-
ceptor from reaching its associated G protein, arrestins
can also initiate a second signaling cascade, by acting
as scaffold proteins that bring together several pro-
tein kinases that function in a cascade. For example, the
H9252-arrestin associated with the serpentine receptor for
angiotensin, a potent regulator of blood pressure, binds
the three protein kinases Raf-1, MEK1, and ERK (Fig.
12–18), serving as a scaffold that facilitates any signal-
ing process, such as insulin signaling (Fig. 12–6), that
requires these three protein kinases to interact. This is
one of many known examples of cross-talk between sys-
tems triggered by different ligands (angiotensin and in-
sulin, in this case).
Cyclic AMP Acts as a Second Messenger for a
Number of Regulatory Molecules
Epinephrine is only one of many hormones, growth fac-
tors, and other regulatory molecules that act by chang-
ing the intracellular [cAMP] and thus the activity of PKA
(Table 12–4). For example, glucagon binds to its re-
ceptors in the plasma membrane of adipocytes, activat-
ing (via a G
s
protein) adenylyl cyclase. PKA, stimulated
by the resulting rise in [cAMP], phosphorylates and ac-
tivates two proteins critical to the conversion of stored
fat to fatty acids (perilipin and hormone-sensitive tri-
acylglycerol lipase; see Fig. 17–3), leading to the mobi-
lization of fatty acids. Similarly, the peptide hormone
ACTH (adrenocorticotropic hormone, also called corti-
cotropin), produced by the anterior pituitary, binds to
specific receptors in the adrenal cortex, activating
adenylyl cyclase and raising the intracellular [cAMP].
PKA then phosphorylates and activates several of the
enzymes required for the synthesis of cortisol and other
steroid hormones. The catalytic subunit of PKA can also
move into the nucleus, where it phosphorylates a pro-
tein that alters the expression of specific genes.
Some hormones act by inhibiting adenylyl cyclase,
lowering cAMP levels, and suppressing protein phos-
phorylation. For example, the binding of somatostatin to
its receptor leads to activation of an inhibitory G pro-
tein, or G
i
, structurally homologous to G
s
, that inhibits
adenylyl cyclase and lowers [cAMP]. Somatostatin there-
fore counterbalances the effects of glucagon. In adipose
tissue, prostaglandin E
1
(PGE
1
; see Fig. 10–18b) inhibits
adenylyl cyclase, thus lowering [cAMP] and slowing the
12.4 G Protein–Coupled Receptors and Second Messengers 441
FIGURE 12–18 H9252-Arrestin uncouples the serpentine receptor from its
G protein and brings together the three enzymes of the MAPK cas-
cade. The effect is that one stimulus triggers two distinct response path-
ways: the path activated by the G protein and the MAPK cascade.
MEK1
MAPKK
ERK1/2
MAPK
E
barr
P P
Raf-1
MAPKKK
Corticotropin (ACTH)
Corticotropin-releasing hormone (CRH)
Dopamine [D
1
,D
2
]
*
Epinephrine (H9252-adrenergic)
Follicle-stimulating hormone (FSH)
Glucagon
Histamine [H
2
]
*
Luteinizing hormone (LH)
Melanocyte-stimulating hormone (MSH)
Odorants (many)
Parathyroid hormone
Prostaglandins E
1
,E
2
(PGE
1
, PGE
2
)
Serotonin [5-HT-1a, 5-HT-2]
*
Somatostatin
Tastants (sweet, bitter)
Thyroid-stimulating hormone (TSH)
Some Signals That Use cAMP as
Second Messenger
TABLE 12–4
*
Receptor subtypes in square brackets. Subtypes may have different transduction mechanisms. For
example, serotonin is detected in some tissues by receptor subtypes 5-HT-1a and 5-HT-1b, which
act through adenylyl cyclase and cAMP, and in other tissues by receptor subtype 5-HT-1c, acting
through the phospholipase C–IP
3
mechanism (see Table 12–5).
8885d_c12_441 2/20/04 1:20 PM Page 441 mac76 mac76:385_reb:
mobilization of lipid reserves triggered by epinephrine
and glucagon. In certain other tissues PGE
1
stimulates
cAMP synthesis, because its receptors are coupled to
adenylyl cyclase through a stimulatory G protein, G
s
. In
tissues with H9251
2
-adrenergic receptors, epinephrine low-
ers [cAMP], because the H9251
2
receptors are coupled to
adenylyl cyclase through an inhibitory G protein, G
i
. In
short, an extracellular signal such as epinephrine or
PGE
1
can have quite different effects on different tis-
sues or cell types, depending on three factors: the type
of receptor in each tissue, the type of G protein (G
s
or
G
i
) with which the receptor is coupled, and the set of
PKA target enzymes in the cells.
A fourth factor that explains how so many signals
can be mediated by a single second messenger (cAMP)
is the confinement of the signaling process to a specific
region of the cell by scaffold proteins. AKAPs (Akinase
anchoring proteins) are bivalent; one part binds to the
R subunit of PKA, and another to a specific structure
within the cell, confining the PKA to the vicinity of that
structure. For example, specific AKAPs bind PKA to
microtubules, actin filaments, Ca
2H11001
channels, mito-
chondria, and the nucleus. Different types of cells have
different AKAPs, so cAMP might stimulate phosphory-
lation of mitochondrial proteins in one cell and phos-
phorylation of actin filaments in another. In studies of
the intracellular localization of biochemical changes,
biochemistry meets cell biology, and techniques that
cross this boundary become invaluable (Box 12–2).
Two Second Messengers Are Derived
from Phosphatidylinositols
A second class of serpentine receptors are coupled
through a G protein to a plasma membrane phospholi-
pase C (PLC) that is specific for the plasma membrane
lipid phosphatidylinositol 4,5-bisphosphate (see Fig.
10–15). This hormone-sensitive enzyme catalyzes the
formation of two potent second messengers: diacyl-
glycerol and inositol 1,4,5-trisphosphate, or IP
3
(not to be confused with PIP
3
, p. 431).
When a hormone of this class (Table 12–5) binds its
specific receptor in the plasma membrane (Fig. 12–19,
step 1 ), the receptor-hormone complex catalyzes
GTP-GDP exchange on an associated G protein, G
q
H
P
O
O
O
H
H
HH
H
HH
4
32
1
6
Inositol 1,4,5-trisphosphate (IP
3
)
O
O
H11002
H11002
O
H11002
O
P
O
H11002
OPO
O
H11002
O
OH
O
5
O
H11002
(step 2 ), activating it exactly as the H9252-adrenergic re-
ceptor activates G
s
(Fig. 12–12). The activated G
q
in turn
activates a specific membrane-bound PLC (step 3 ),
which catalyzes the production of the two second mes-
sengers diacylglycerol and IP
3
by hydrolysis of phos-
phatidylinositol 4,5-bisphosphate in the plasma mem-
brane (step 4 ).
Inositol trisphosphate, a water-soluble compound,
diffuses from the plasma membrane to the endoplasmic
reticulum, where it binds to specific IP
3
receptors and
causes Ca
2H11001
channels within the ER to open. Seques-
tered Ca
2H11001
is thus released into the cytosol (step 5 ),
and the cytosolic [Ca
2H11001
] rises sharply to about 10
H110026
M.
One effect of elevated [Ca
2H11001
] is the activation of pro-
tein kinase C (PKC). Diacylglycerol cooperates with
Ca
2H11001
in activating PKC, thus also acting as a second
messenger (step 6 ). PKC phosphorylates Ser or Thr
residues of specific target proteins, changing their cat-
alytic activities (step
7
). There are a number of
isozymes of PKC, each with a characteristic tissue dis-
tribution, target protein specificity, and role.
The action of a group of compounds known as
tumor promoters is attributable to their effects
on PKC. The best understood of these are the phorbol
esters, synthetic compounds that are potent activators
of PKC. They apparently mimic cellular diacylglycerol
as second messengers, but unlike naturally occurring di-
acylglycerols they are not rapidly metabolized. By con-
tinuously activating PKC, these synthetic tumor pro-
moters interfere with the normal regulation of cell
growth and division (discussed in Section 12.10). ■
Calcium Is a Second Messenger in Many
Signal Transductions
In many cells that respond to extracellular signals, Ca
2H11001
serves as a second messenger that triggers intracellu-
lar responses, such as exocytosis in neurons and en-
docrine cells, contraction in muscle, and cytoskeletal
rearrangement during amoeboid movement. Normally,
cytosolic [Ca
2H11001
] is kept very low (H1102110
H110027
M) by the ac-
tion of Ca
2H11001
pumps in the ER, mitochondria, and plasma
membrane. Hormonal, neural, or other stimuli cause
either an influx of Ca
2H11001
into the cell through specific
CH
2
CO
HO
OH
CH
3
CH
3
O
CH
3
(CH
2
)
12
CO
O
HO
Myristoylphorbol acetate
(a phorbol ester)
CH
3
O
CH
3
CH
3
Chapter 12 Biosignaling442
8885d_c12_442 2/20/04 1:21 PM Page 442 mac76 mac76:385_reb:
12.4 G Protein–Coupled Receptors and Second Messengers 443
Phospholipase C
(PLC)
Hormone (H) binds to a
specific receptor.
The occupied
receptor causes
GDP-GTP exchange
on G
q
.
G
q
, with bound GTP,
moves to PLC and
activates it.
Active PLC cleaves phosphatidyl-
inositol 4,5-bisphosphate to inositol
trisphosphate (IP
3
) and diacylglycerol.
Endoplasmic
reticulum
IP
3
binds to a specific
receptor on the endoplasmic
reticulum, releasing
sequestered Ca
2H11001
.
Diacylglycerol and Ca
2H11001
activate
protein kinase C at the surface
of the plasma membrane.
Protein
kinase C
IP
3
Ca
2H11001
Ca
2H11001
channel
Diacylglycerol
Plasma
membrane
Extracellular
space
Cytosol
Phosphorylation of cellular
proteins by protein kinase C
produces some of the cellular
responses to the hormone.
Receptor
GTP
G
q
G
q
GTP
H
GDP
GDP
1
2
3
4
5
6
7
FIGURE 12–19 Hormone-activated phospholipase C and IP
3
. Two in-
tracellular second messengers are produced in the hormone-sensitive
phosphatidylinositol system: inositol 1,4,5-trisphosphate (IP
3
) and
diacylglycerol. Both contribute to the activation of protein kinase C.
By raising cytosolic [Ca
2H11001
], IP
3
also activates other Ca
2H11001
-dependent
enzymes; thus Ca
2H11001
also acts as a second messenger.
Acetylcholine [muscarinic M
1
]
H9251
1
-Adrenergic agonists
Angiogenin
Angiotensin II
ATP [P
2x
and P
2y
]
*
Auxin
Gastrin-releasing peptide
Glutamate
Gonadotropin-releasing hormone (GRH)
Histamine [H
1
]
*
Light (Drosophila)
Oxytocin
Platelet-derived growth factor (PDGF)
Serotonin [5-HT-1c]
*
Thyrotropin-releasing hormone (TRH)
Vasopressin
TABLE 12–5 Some Signals That Act through Phospholipase C and IP
3
*
Receptor subtypes are in square brackets; see footnote to Table 12–4.
8885d_c12_443 2/20/04 1:21 PM Page 443 mac76 mac76:385_reb:
Ca
2H11001
channels in the plasma membrane or the release
of sequestered Ca
2H11001
from the ER or mitochondria, in
either case raising the cytosolic [Ca
2H11001
] and triggering a
cellular response.
Very commonly, [Ca
2H11001
] does not simply rise and
then decrease, but rather oscillates with a period of a
few seconds (Fig. 12–20), even when the extracellular
concentration of hormone remains constant. The mech-
anism underlying [Ca
2H11001
] oscillations presumably entails
feedback regulation by Ca
2H11001
of either the phospholipase
Chapter 12 Biosignaling444
Cytosolic [Ca
2
H11001
] (n
M
)
0
(b)
100
100
200 300 400
600
200
300
400
500
0 0.5 1.0
[Ca
2H11001
] (mM)
(a)
FIGURE 12–20 Triggering of oscillations in intracellular [Ca
2H11545
] by
extracellular signals. (a) A dye (fura) that undergoes fluorescence
changes when it binds Ca
2H11001
is allowed to diffuse into cells, and its
instantaneous light output is measured by fluorescence microscopy.
Fluorescence intensity is represented by color; the color scale relates
intensity of color to [Ca
2H11001
], allowing determination of the absolute
[Ca
2H11001
]. In this case, thymocytes (cells of the thymus) have been stim-
ulated with extracellular ATP, which raises their internal [Ca
2H11001
]. The
cells are heterogeneous in their responses; some have high intracel-
lular [Ca
2H11001
] (red), others much lower (blue). (b) When such a probe
is used to measure [Ca
2H11001
] in a single hepatocyte, we observe that the
agonist norepinephrine (added at the arrow) causes oscillations of
[Ca
2H11001
] from 200 to 500 nM. Similar oscillations are induced in other
cell types by other extracellular signals.
Adenylyl cyclase (brain)
Ca
2H11001
/calmodulin-dependent protein kinases (CaM
kinases I to IV)
Ca
2H11001
-dependent Na
H11001
channel (Paramecium)
Ca
2H11001
-release channel of sarcoplasmic reticulum
Calcineurin (phosphoprotein phosphatase 2B)
cAMP phosphodiesterase
cAMP-gated olfactory channel
cGMP-gated Na
H11001
,Ca
2H11001
channels (rod and cone cells)
Glutamate decarboxylase
Myosin light chain kinases
NAD
H11001
kinase
Nitric oxide synthase
Phosphoinositide 3-kinase
Plasma membrane Ca
2H11001
ATPase (Ca
2H11001
pump)
RNA helicase (p68)
Some Proteins Regulated
by Ca
2H11545
and Calmodulin
TABLE 12–6
that generates IP
3
or the ion channel that regulates Ca
2H11001
release from the ER, or both. Whatever the mechanism,
the effect is that one kind of signal (hormone concen-
tration, for example) is converted into another (fre-
quency and amplitude of intracellular [Ca
2H11001
] “spikes”).
Changes in intracellular [Ca
2H11001
] are detected by
Ca
2+
-binding proteins that regulate a variety of Ca
2H11001
-
dependent enzymes. Calmodulin (CaM) (M
r
17,000)
is an acidic protein with four high-affinity Ca
2H11001
-binding
sites. When intracellular [Ca
2H11001
] rises to about 10
H110026
M
(1 H9262M), the binding of Ca
2H11001
to calmodulin drives a con-
formational change in the protein (Fig. 12–21). Calmod-
ulin associates with a variety of proteins and, in its Ca
2H11001
-
bound state, modulates their activities. Calmodulin is a
member of a family of Ca
2H11001
-binding proteins that also
includes troponin (p. 185), which triggers skeletal mus-
cle contraction in response to increased [Ca
2H11001
]. This
family shares a characteristic Ca
2H11001
-binding structure,
the EF hand (Fig. 12–21c).
Calmodulin is also an integral subunit of a family of
enzymes, the Ca
2H11545
/calmodulin-dependent protein
kinases (CaM kinases I–IV). When intracellular
[Ca
2H11001
] increases in response to some stimulus, calmod-
ulin binds Ca
2H11001
, undergoes a change in conformation,
and activates the CaM kinase. The kinase then phos-
phorylates a number of target enzymes, regulating their
activities. Calmodulin is also a regulatory subunit of
phosphorylase b kinase of muscle, which is activated by
Ca
2H11001
. Thus Ca
2H11001
triggers ATP-requiring muscle con-
tractions while also activating glycogen breakdown, pro-
viding fuel for ATP synthesis. Many other enzymes are
also known to be modulated by Ca
2H11001
through calmod-
ulin (Table 12–6).
8885d_c12_444 2/20/04 1:21 PM Page 444 mac76 mac76:385_reb:
SUMMARY 12.4 G Protein–Coupled Receptors and
Second Messengers
■ A large family of plasma membrane receptors
with seven transmembrane segments act
through heterotrimeric G proteins. On ligand
binding, these receptors catalyze the exchange
of GTP for GDP bound to an associated G
protein, forcing dissociation of the H9251 subunit of
the G protein. This subunit stimulates or
inhibits the activity of a nearby membrane-bound
enzyme, changing the level of its second
messenger product.
■ The H9252-adrenergic receptor binds epinephrine,
then through a stimulatory G protein, G
s
,
activates adenylyl cyclase in the plasma
membrane. The cAMP produced by adenylyl
cyclase is an intracellular second messenger
that stimulates cAMP-dependent protein kinase,
which mediates the effects of epinephrine by
phosphorylating key proteins, changing their
enzymatic activities or structural features.
■ The cascade of events in which a single
molecule of hormone activates a catalyst that
in turn activates another catalyst, and so on,
results in large signal amplification; this is
characteristic of most hormone-activated
systems.
■ Some receptors stimulate adenylyl cyclase
through G
s
; others inhibit it through G
i
. Thus
cellular [cAMP] reflects the integrated input of
two (or more) signals.
■ Cyclic AMP is eventually eliminated by cAMP
phosphodiesterase, and G
s
turns itself off by
hydrolysis of its bound GTP to GDP. When the
epinephrine signal persists, H9252-adrenergic
receptor–specific protein kinase and arrestin 2
temporarily desensitize the receptor and cause
it to move into intracellular vesicles. In some
cases, arrestin also acts as a scaffold protein,
bringing together protein components of a
signaling pathway such as the MAPK cascade.
■ Some serpentine receptors are coupled to a
plasma membrane phospholipase C that cleaves
PIP
2
to diacylglycerol and IP
3
. By opening Ca
2H11001
channels in the endoplasmic reticulum, IP
3
raises cytosolic [Ca
2H11001
]. Diacylglycerol and Ca
2H11001
act together to activate protein kinase C, which
phosphorylates and changes the activity of
specific cellular proteins. Cellular [Ca
2H11001
] also
regulates a number of other enzymes, often
through calmodulin.
12.4 G Protein–Coupled Receptors and Second Messengers 445
(b)
(c)
EF hand
(a)
Ca
2+
E helix
F helix
FIGURE 12–21 Calmodulin. This is the protein mediator of many Ca
2H11001
-stimu-
lated enzymatic reactions. Calmodulin has four high-affinity Ca
2H11001
-binding sites
(K
d
H11015 0.1 to 1 H9262M). (a) A ribbon model of the crystal structure of calmodulin
(PDB ID 1CLL). The four Ca
2H11001
-binding sites are occupied by Ca
2H11001
(purple).
The amino-terminal domain is on the left; the carboxyl-terminal domain on the
right. (b) Calmodulin associated with a helical domain (red) of one of the
many enzymes it regulates, calmodulin-dependent protein kinase II (PDB ID
1CDL). Notice that the long central H9251 helix visible in (a) has bent back on
itself in binding to the helical substrate domain. The central helix is clearly
more flexible in solution than in the crystal. (c) Each of the four Ca
2H11001
-binding
sites occurs in a helix-loop-helix motif called the EF hand, also found in many
other Ca
2H11001
-binding proteins.
8885d_c12_445 2/20/04 3:16 PM Page 445 mac76 mac76:385_reb:
Chapter 12 Biosignaling446
BOX 12–2 WORKING IN BIOCHEMISTRY
FRET: Biochemistry Visualized in a Living Cell
Fluorescent probes are commonly used to detect rapid
biochemical changes in single living cells. They can be
designed to give an essentially instantaneous report
(within nanoseconds) on the changes in intracellular
concentration of a second messenger or in the activ-
ity of a protein kinase. Furthermore, fluorescence mi-
croscopy has sufficient resolution to reveal where in
the cell such changes are occurring. In one widely
used procedure, the fluorescent probes are derived
from a naturally occurring fluorescent protein, the
green fluorescent protein (GFP) of the jellyfish Ae-
quorea victoria (Fig. 1).
When excited by absorption of a photon of light,
GFP emits a photon (that is, it fluoresces) in the green
region of the spectrum. GFP is an 11-stranded H9252 bar-
rel, and the light-absorbing/emitting center of the pro-
tein (its chromophore) comprises the tripeptide
Ser
65
–Tyr
66
–Gly
67
, located within the barrel (Fig. 2).
Variants of this protein, with different fluorescence
spectra, can be produced by genetic engineering of
the GFP gene. For example, in the yellow fluorescent
protein (YFP), Ala
206
in GFP is replaced by a Lys
residue, changing the wavelength of light absorption
and fluorescence. Other variants of GFP fluoresce blue
(BFP) or cyan (CFP) light, and a related protein
(mRFP1) fluoresces red light (Fig. 3). GFP and its
variants are compact structures that retain their abil-
ity to fold into their native H9252-barrel conformation even
when fused with another protein. Investigators are us-
ing these fluorescent hybrid proteins as spectroscopic
rulers to measure distances between interacting com-
ponents within a cell.
FIGURE 1 Aequorea victoria, a jellyfish abundant in Puget Sound,
Washington State.
FIGURE 2 Green fluorescent protein (GFP), with the fluorescent chro-
mophore shown in ball-and-stick form (derived from PDB ID 1GFL).
Chromophore
(Ser
65
Tyr
66
Gly
67
)
100
BFP
CFP GFP
mRFP1
80
60
Relative fluorescence
40
20
0
400 500 600
Wavelength (nm)
700
YFP
FIGURE 3 Emission spectra of GFP variants.
8885d_c12_446 2/20/04 1:21 PM Page 446 mac76 mac76:385_reb:
12.4 G Protein–Coupled Receptors and Second Messengers 447
An excited fluorescent molecule such as GFP or
YFP can dispose of the energy from the absorbed pho-
ton in either of two ways: (1) by fluorescence, emitting
a photon of slightly longer wavelength (lower energy)
than the exciting light, or (2) by nonradiative fluores-
cence resonance energy transfer (FRET), in which
the energy of the excited molecule (the donor) passes
directly to a nearby molecule (the acceptor) without
emission of a photon, exciting the acceptor (Fig. 4).
The acceptor can now decay to its ground state by flu-
orescence; the emitted photon has a longer wavelength
(lower energy) than both the original exciting light and
the fluorescence emission of the donor. This second
mode of decay (FRET) is possible only when donor and
acceptor are close to each other (within 1 to 50 ?); the
efficiency of FRET is inversely proportional to the sixth
power of the distance between donor and acceptor.
Thus very small changes in the distance between donor
and acceptor register as very large changes in FRET,
measured as the fluorescence of the acceptor molecule
when the donor is excited. With sufficiently sensitive
light detectors, this fluorescence signal can be located
to specific regions of a single, living cell.
FRET has been used to measure [cAMP] in living
cells. The gene for GFP is fused with that for the reg-
ulatory subunit (R) of cAMP-dependent protein ki-
nase, and the gene for BFP is fused with that for the
catalytic subunit (C) (Fig. 5). When these two hybrid
proteins are expressed in a cell, BFP (donor; excita-
tion at 380 nm, emission at 460 nm) and GFP (ac-
ceptor; excitation at 475 nm, emission at 545 nm) in
the inactive PKA (R
2
C
2
tetramer) are close enough to
undergo FRET. Wherever in the cell [cAMP] increases,
the R
2
C
2
complex dissociates into R
2
and 2C and the
FRET signal is lost, because donor and acceptor are
now too far apart for efficient FRET. Viewed in the
fluorescence microscope, the region of higher [cAMP]
has a minimal GFP signal and higher BFP signal. Mea-
suring the ratio of emission at 460 nm and 545 nm
gives a sensitive measure of the change in [cAMP]. By
determining this ratio for all regions of the cell, the
investigator can generate a false color image of the
433
nm
433
nm
476
nm
527
nm
CFP YFP
protein–
protein
interaction
Genetically
engineered hybrid
proteins
FRET
FIGURE 4 When the donor protein (CFP) is excited with mono-
chromatic light of wavelength 433 nm, it emits fluorescent light at
476 nm (left). When the (red) protein fused with CFP interacts with
the (purple) protein fused with YFP, that interaction brings CFP and
YFP close enough to allow fluorescence resonance energy transfer
(FRET) between them. Now, when CFP absorbs light of 433 nm, in-
stead of fluorescing at 476 nm, it transfers energy directly to YFP,
which then fluoresces at its characteristic emission wavelength, 527
nm. The ratio of light emission at 527 and 476 nm is therefore a
measure of the interaction of the red and purple protein.
460 nm
380 nm
545 nm
BFP
380 nm
(inactive)
(active)
cAMP-dependent
protein kinase
(PKA)
GFP
R
no emission
at 545 nm
R
C
R
RR
C
C C
cAMPcAMP
+
FRET
433 nm
FIGURE 5 Measuring [cAMP] with FRET. Gene fusion creates hy-
brid proteins that exhibit FRET when the PKA regulatory and cat-
alytic subunits are associated (low [cAMP]). When [cAMP] rises, the
subunits dissociate, and FRET ceases. The ratio of emission at 460
nm (dissociated) and 545 nm (complexed) thus offers a sensitive
measure of [cAMP].
(continued on next page)
8885d_c12_447 2/20/04 1:22 PM Page 447 mac76 mac76:385_reb:
12.5 Multivalent Scaffold Proteins and
Membrane Rafts
About 10% of the 30,000 to 35,000 genes in the human
genome encode signaling proteins—receptors, G pro-
teins, enzymes that generate second messengers, pro-
tein kinases (H11022500), proteins involved in desensitiza-
tion, and ion channels. Not every signaling protein is
expressed in a given cell type, but most cells doubtless
contain many such proteins. How does one protein find
another in a signaling pathway, and how are their inter-
actions regulated? As is becoming clear, the reversible
phosphorylation of Tyr, Ser, and Thr residues in signal-
ing proteins creates docking sites for other proteins,
and many signaling proteins are multivalent in that
they can interact with several different proteins simul-
taneously to form multiprotein signaling complexes. In
this section we present a few examples to illustrate the
general principles of protein interactions in signaling.
Protein Modules Bind Phosphorylated Tyr, Ser, or Thr
Residues in Partner Proteins
We have seen that the protein Grb2 in the insulin sig-
naling pathway (Fig. 12–6) binds through its SH2 do-
main to other proteins that contain exposed P –Tyr
residues. The human genome encodes at least 87 SH2-
containing proteins, many already known to participate
in signaling. The P –Tyr residue is bound in a deep
pocket in an SH2 domain, with each of its phosphate oxy-
gens participating in hydrogen-bonding or electrostatic
interactions; the positive charges on two Arg residues
figure prominently in the binding. Subtle differences
in the structure of SH2 domains in different proteins
account for the specificities of their interactions with
various P –Tyr-containing proteins. The three to five
residues on the carboxyl-terminal side of the P –Tyr
residue are critical in determining the specificity of in-
teractions with SH2 domains (Fig. 12–22).
PTB domains (phosphotyrosine-binding domains)
also bind P –Tyr in partner proteins, but their critical
sequences and three-dimensional structures distinguish
them from SH2 domains. The human genome encodes
24 proteins that contain PTB domains, including IRS-1,
which we have already met in its role as a scaffold pro-
tein in insulin-signal transduction (Fig. 12–6).
Many of the signaling protein kinases, including
PKA, PKC, PKG, and members of the MAPK cascade,
phosphorylate Ser or Thr residues in their target pro-
teins, which in some cases acquire the ability to inter-
act with partner proteins through the phosphorylated
residue, triggering a downstream process. An alphabet
soup of domains that bind P –Ser or P –Thr residues
has been identified, and more are sure to be found. Each
domain favors a certain sequence around the phosphor-
ylated residue, so the domains represent families of
highly specific recognition sites, able to bind to a spe-
cific subset of phosphorylated proteins.
Chapter 12 Biosignaling448
BOX 12–2 WORKING IN BIOCHEMISTRY (continued from previous page)
cell in which the ratio, or relative [cAMP], is repre-
sented by the intensity of the color. Images recorded
at timed intervals reveal changes in [cAMP] over time.
A variation of this technology has been used to
measure the activity of PKA in a living cell (Fig. 6).
Researchers create a phosphorylation target for PKA
by producing a hybrid protein containing four ele-
ments: YFP (acceptor); a short peptide with a Ser
residue surrounded by the consensus sequence for
PKA; a P –Ser-binding domain (called 14-3-3); and
CFP (donor). When the Ser residue is not phosphor-
ylated, 14-3-3 has no affinity for the Ser residue and
the hybrid protein exists in an extended form, with
the donor and acceptor too far apart to generate a
FRET signal. Wherever PKA is active in the cell, it
phosphorylates the Ser residue of the hybrid protein,
and 14-3-3 binds to the P –Ser. In doing so, it draws
YFP and CFP together and a FRET signal is detected
with the fluorescence microscope, revealing the pres-
ence of active PKA.
ATP
PKA consensus
sequence
14-3-3
(Phosphoserine-
binding domain)
ADP
PKA
YFP
433
nm
476
nm
CFP
Ser
433
nm
FRET
527
nm
P
FIGURE 6 Measuring the activity of PKA with FRET. An engineered
protein links YFP and CFP via a peptide that contains a Ser residue
surrounded by the consensus sequence for phosphorylation by PKA,
and the 14-3-3 phosphoserine binding domain. Active PKA phos-
phorylates the Ser residue, which docks with the 14-3-3 binding do-
main, bringing the fluorescence proteins close enough to allow FRET
to occur, revealing the presence of active PKA.
8885d_c12_448 2/20/04 1:22 PM Page 448 mac76 mac76:385_reb:
FIGURE 12–22 Structure of an SH2 domain and its interaction with a P –Tyr residue
in a partner protein. (PDB ID 1SHC) The SH2 domain is shown as a gray surface contour
representation. The phosphorus of the phosphate group in the interacting P –Tyr is
visible as an orange sphere; most of the residue is obscured in this view. The next few
residues toward the carboxyl end of the partner protein are shown in red. The SH2
domain interacts with P –Tyr (which, as the phosphorylated residue, is assigned the
index position 0) and also with the next three residues toward the carboxyl terminus
(designated H110011, H110012, H110013). The residues important in the P –Tyr residue are conserved in
all SH2 domains. Some SH2 domains (Src, Fyn, Hck, Nck) favor negatively charged
residues in the H110011 and H110012 positions; others (PLC-H92531, SHP-2) have a long hydrophobic
groove that selects for aliphatic residues in positions H110011 to H110015. These differences define
subclasses of SH2 domains that have different partner specificities.
In some cases, the domain-binding partner is inter-
nal. Phosphorylation of some protein kinases inhibits
their activity by favoring the interaction of an SH2 do-
main with a P –Tyr in another domain of the same en-
zyme. For example, the soluble protein Tyr kinase Src,
when phosphorylated on a critical Tyr residue, is ren-
dered inactive as an SH2 domain needed to bind to the
substrate protein instead binds to an internal P –Tyr
(Fig. 12–23). Glycogen synthase kinase 3 (GSK3) is in-
active when phosphorylated on a Ser residue in its auto-
inhibitory domain (Fig. 12–23b). Dephosphorylation of
that domain frees the enzyme to bind and phosphory-
late its target proteins. Similarly, the polar head group
of the phospholipid PIP
3
, protruding from the inner leaf-
let of the plasma membrane, provides points of attach-
ment for proteins that contain SH3 and other domains.
12.5 Multivalent Scaffold Proteins and Membrane Rafts 449
(a)
Autoinhibited
Src
kinase
SH3
SH3
SH2
SH2
P
Tyr
Tyr
TyrHO
Pro
Pro
Active
site
Active; substrate
positioned for
phosphorylation
P
Tyr
Tyr
Tyr
Autoinhibited
(b)
Glycogen
synthase
Active
site
GSK3
Ser
Ser
HO Ser
Ser
Active; substrate
positioned for
phosphorylation
P
SerP
GSK3
FIGURE 12–23 Mechanism of autoinhibition
of Src Tyr kinase and GSK3. (a) In the active
form of Src kinase, an SH2 domain binds a
P –Tyr in the substrate, and an SH3 domain
binds a proline-rich region of the substrate,
lining up the active site of the kinase with
several target Tyr residues in the substrate.
When Src is phosphorylated on a specific Tyr
residue, the SH2 domain binds to the internal
P –Tyr instead of to the P –Tyr of the
substrate, preventing productive binding of
the kinase to its protein substrate; the enzyme
is thus autoinhibited. (b) In the autoinhibited
glycogen synthase kinase 3 (GSK3), an
internal P –Ser residue is bound to an
internal P –Ser-binding domain (top).
Dephosphorylation of this internal Ser residue
leaves the P –Ser-binding site of GSK3 avail-
able to bind P –Ser in a protein substrate,
and thus to position the kinase to phosphory-
late neighboring Ser residues (bottom).
8885d_c12_449 2/20/04 1:22 PM Page 449 mac76 mac76:385_reb:
Most of the proteins involved in signaling at the
plasma membrane have one or more protein- or phos-
pholipid-binding domains; many have three or more, and
thus are multivalent in their interactions with other sig-
naling proteins. Figure 12–24 shows a few of the many
multivalent proteins known to participate in signaling.
A remarkable picture of signaling pathways has
emerged from studies of many signaling proteins and
the multiple binding domains they contain (Fig. 12–25).
An initial signal results in phosphorylation of the re-
ceptor or a target protein, triggering the assembly of
large multiprotein complexes, held together on scaffolds
made from adaptor proteins with multivalent binding ca-
pacities. Some of these complexes have several protein
kinases that activate each other in turn, producing a cas-
cade of phosphorylation and a great amplification of the
initial signal. Animal cells also have phosphotyrosine
phosphatases (PTPases), which remove the phosphate
from P –Tyr residues, reversing the effect of phosphor-
ylation. Some of these phosphatases are receptorlike
membrane proteins, presumably controlled by extracel-
lular factors not yet identified; other PTPases are solu-
ble and contain SH2 domains. In addition, animal cells
have protein phosphoserine and phosphothreonine
phosphatases, which reverse the effects of Ser- and Thr-
specific protein kinases. We can see, then, that signal-
ing occurs in protein circuits, effectively hard-wired
from signal receptor to response effector and able to be
switched off instantly by the hydrolysis of a single phos-
phate ester bond.
The multivalency of signaling proteins allows for the
assembly of many different combinations of signaling
modules, each combination presumably suited to partic-
ular signals, cell types, and metabolic circumstances. The
large variety of protein kinases and of phosphoprotein-
binding domains, each with its own specificity (the con-
sensus sequence required in its substrate), provides for
many permutations and combinations and many differ-
ent signaling circuits of extraordinary complexity. And
given the variety of specific phosphatases that reverse
Chapter 12 Biosignaling450
SH2
Adaptor Grb2
Shc
SH3
DNA STAT
PTB
Tyr kinase Src
Scaffold
Kinase
Ras signaling
Phosphatase
Signal regulation
Transcription
Phospholipid second-
messenger signaling
SH2 SH3
SH2
SH2SH3
Tyr phosphatase Shp2SH2SH2
SH3 SH2SH2 C2PH RasGAPGTPase-activating
SH2 SH3SH2 C2PH PH PH PLC PLCgPLC
TA
SH2 SOCSSOCS
Binding domains
proline-rich protein
or membrane lipid PIP
3
Tyr– P
Tyr–
PIP
3
phospholipids (Ca
2+
-dependent)
DNA
transcriptional activation
carboxyl-terminal domain
marking protein for attachment
of ubiquitin
P
FIGURE 12–24 Some binding modules of signaling proteins. Each
protein is represented by a line (with the amino terminus to the left);
symbols indicate the location of conserved binding domains (with
specificities as listed in the key; PH denotes plextrin homology; other
abbreviations explained in the text); green boxes indicate catalytic ac-
tivities. The name of each protein is given at its carboxyl-terminal end.
These signaling proteins interact with phosphorylated proteins or
phospholipids in many permutations and combinations to form inte-
grated signaling complexes.
8885d_c12_450 2/20/04 1:22 PM Page 450 mac76 mac76:385_reb:
the action of protein kinases, some under specific types
of external control, a cell can quickly “disconnect” the
entire protein circuitry of a signaling pathway. Together,
these mechanisms confer a huge capacity for cellular reg-
ulation in response to signals of many types.
Membrane Rafts and Caveolae May Segregate
Signaling Proteins
Membrane rafts are regions of the membrane bilayer en-
riched in sphingolipids, sterols, and certain proteins, in-
cluding many attached to the bilayer by GPI anchors
(Chapter 11). Some receptor Tyr kinases, such as the
receptors for epidermal growth factor (EGF) and
platelet-derived growth factor (PDGF), appear to be lo-
calized in rafts; other signaling proteins, such as the small
G protein Ras (which is prenylated) and the hetero-
trimeric G protein G
s
(also prenylated, on the H9251 and H9253
subunits), are not. Growing evidence suggests that this
sequestration of signaling proteins is functionally signif-
icant. When cholesterol is removed from rafts by treat-
ment with cyclodextrin (which binds cholesterol and
removes it from membranes), the rafts are disrupted
and a number of signaling pathways become defective.
How might localization in rafts influence signaling
through a receptor? There are several possibilities. If a
receptor Tyr kinase in a raft is phosphorylated, and the
phosphotyrosine phosphatase that reverses this phos-
phorylation is in another raft, then dephosphorylation
of the Tyr kinase will be slowed or prevented. If two sig-
naling proteins must interact during transduction of a
signal, the probability of encounters between these pro-
teins is greatly enhanced if both are in the same raft.
Interactions between scaffold proteins might be strong
enough to pull into a raft a signaling protein not nor-
mally located there, or strong enough to pull receptors
out of a raft. For example, the EGF receptor in isolated
fibroblasts is normally concentrated in the specialized
rafts called caveolae (see Fig. 11–21), but treatment
with EGF causes the receptor to leave the raft. This mi-
gration depends on the receptor’s protein kinase activ-
ity; mutant receptors lacking this activity remain in the
rafts during treatment with EGF. Caveolin, an integral
membrane protein localized in caveolae, is phosphory-
lated on Tyr residues in response to insulin, and phos-
phorylation may allow the now-activated EGF receptor
to draw its binding partners into the raft. Finally, an-
other example of the clustering of signaling proteins in
rafts is the H9252-adrenergic receptor. This receptor is seg-
regated in membrane rafts that also contain the G pro-
teins, adenylyl cyclase, PKA, and a specific protein phos-
phatase, PP2, providing a highly integrated signaling
unit. Spatial segregation of signaling proteins in rafts
adds yet another dimension to the already complex
processes initiated by extracellular signals.
SUMMARY 12.5 Multivalent Scaffold Proteins and
Membrane Rafts
■ Many signaling proteins have domains that bind
phosphorylated Tyr, Ser, or Thr residues in
other proteins; the binding specificity for each
domain is determined by sequences that adjoin
the phosphorylated residue.
■ SH2 and PTB domains bind to proteins
containing P –Tyr residues; other domains
bind P –Ser and P –Thr residues in various
contexts.
■ Plextrin homology domains bind the membrane
phospholipid PIP
3
.
■ Many signaling proteins are multivalent, with
several different binding modules. By combining
the substrate specificities of various protein
kinases with the specificities of domains that
bind phosphorylated Ser, Thr, or Tyr residues,
and with phosphatases that can rapidly
inactivate a pathway, cells create a large
number of multiprotein signaling complexes.
■ Membrane rafts and caveolae sequester groups
of signaling proteins in small regions of the
plasma membrane, enhancing their interactions
and making signaling more efficient.
12.5 Multivalent Scaffold Proteins and Membrane Rafts 451
14-3-3
MEK
ERK
MP1
IRS-1
Insulin
receptor
P
P
P
P P
P PKC
PKB
Raf-1
Grb2
Sos
PI-3K
Ras
PIP
3
PIP
3
FIGURE 12–25 Insulin-induced formation of supramolecular signal-
ing complexes. The binding of insulin to its receptor sets off a series
of events that lead eventually to the formation of membrane-associated
complexes involving the 12 signaling proteins shown here, as well as
others. Phosphorylation of Tyr residues in the insulin receptor initiates
complex formation, and dephosphorylation of any of the phospho-
proteins breaks the circuit. Four general types of interaction hold the
complex together: the binding of a protein to a second phosphopro-
tein through SH2 or PTB domains in the first (red); the binding of SH3
domains in the first with proline-rich domains in the second (orange);
the binding of PH domains in one protein to the phospholipid PIP
3
in
the plasma membrane (blue); or the association of a protein (RAS) with
the plasma membrane through a lipid covalently bound to the pro-
tein (yellow). Two proteins shown here are not described in the text:
14-3-3, which binds a P –Ser in Raf and mediates its interaction with
MEK; and MP1, a scaffold protein that cements the links between Raf,
MEK, and ERK.
8885d_c12_451 2/20/04 1:22 PM Page 451 mac76 mac76:385_reb:
12.6 Signaling in Microorganisms
and Plants
Much of what we have said here about signaling relates
to mammalian tissues or cultured cells from such tis-
sues. Bacteria, eukaryotic microorganisms, and vascu-
lar plants must also respond to a variety of external sig-
nals, such as O
2
, nutrients, light, noxious chemicals, and
so on. We turn here to a brief consideration of the kinds
of signaling machinery used by microorganisms and
plants.
Bacterial Signaling Entails Phosphorylation in a
Two-Component System
E. coli responds to a number of nutrients in its envi-
ronment, including sugars and amino acids, by swim-
ming toward them, propelled by one or a few flagella. A
family of membrane proteins have binding domains on
the outside of the plasma membrane to which specific
attractants (sugars or amino acids) bind (Fig. 12–26).
Ligand binding causes another domain on the inside of
the plasma membrane to phosphorylate itself on a His
residue. This first component of the two-component
system, the receptor His kinase, then catalyzes the
transfer of the phosphoryl group from the His residue
to an Asp residue on a second, soluble protein, the re-
sponse regulator; this phosphoprotein moves to the
base of the flagellum, carrying the signal from the mem-
brane receptor. The flagellum is driven by a rotary mo-
tor that can propel the cell through its medium or cause
it to stall, depending on the direction of the motor’s ro-
tation. Information from the receptor allows the cell to
determine whether it is moving toward or away from the
source of the attractant. If its motion is toward the at-
tractant, the response regulator signals the cell to con-
tinue in a straight line; if away from it, the cell tumbles
momentarily, acquiring a new direction. Repetition of
this behavior results in a random path, biased toward
movement in the direction of increasing attractant
concentration.
E. coli detects not only sugars and amino acids but
also O
2
, extremes of temperature, and other environ-
mental factors, using this basic two-component system.
Two-component systems have been detected in many
other bacteria, including gram-positive and gram-
negative eubacteria and archaebacteria, as well as in
protists and fungi. Clearly this signaling mechanism de-
veloped early in the course of cellular evolution and has
been conserved.
Various signaling systems used by animal cells also
have analogs in the prokaryotes. As the full genomic se-
quences of more, and more diverse, bacteria become
known, researchers have discovered genes that encode
proteins similar to protein Ser or Thr kinases, Ras-like
proteins regulated by GTP binding, and proteins with
SH3 domains. Receptor Tyr kinases have not been
detected in bacteria, but P –Tyr residues do occur in
some bacterial proteins, so there must be an enzyme
that phosphorylates Tyr residues.
Signaling Systems of Plants Have Some of the Same
Components Used by Microbes and Mammals
Like animals, vascular plants must have a means of com-
munication between tissues to coordinate and direct
growth and development; to adapt to conditions of O
2
,
nutrients, light, and temperature; and to warn of the
presence of noxious chemicals and damaging pathogens
(Fig. 12–27). At least a billion years of evolution have
passed since the plant and animal branches of the eu-
karyotes diverged, which is reflected in the differences
in signaling mechanisms: some plant mechanisms are
conserved—that is, are similar to those in animals (pro-
tein kinases, scaffold proteins, cyclic nucleotides, elec-
trogenic ion pumps, and gated ion channels); some are
similar to bacterial two-component systems; and some
are unique to plants (light-sensing mechanisms, for ex-
Chapter 12 Biosignaling452
AAttractant
Receptor His kinase
(component 1)
A
His
A
His
His
ATP
ADP
E. coli
Response regulator
(component 2)
Plasma
membrane
Rotary motor
(controls flagellum)
Phosphorylated
form of component 2
reverses direction
of motor
Asp
Asp
PP
FIGURE 12–26 The two-component
signaling mechanism in bacterial
chemotaxis. When an attractant ligand
(A) binds to the receptor domain of the
membrane-bound receptor, a protein
His kinase in the cytosolic domain
(component 1) is activated and
autophosphorylates on a His residue.
This phosphoryl group is then trans-
ferred to an Asp residue on component
2 (in some cases a separate protein; in
others, another domain of the receptor
protein). After phosphorylation on Asp,
component 2 moves to the base of the
flagellum, where it determines the
direction of rotation of the flagellar
motor.
8885d_c12_452 2/20/04 1:23 PM Page 452 mac76 mac76:385_reb:
ample) (Table 12–7). The genome of the widely stud-
ied plant Arabidopsis thaliana, for example, encodes
about 1,000 protein Ser/Thr kinases, including about 60
MAPKs and nearly 400 membrane-associated receptor
kinases that phosphorylate Ser or Thr residues; a vari-
ety of protein phosphatases; scaffold proteins that bring
other proteins together in signaling complexes; enzymes
for the synthesis and degradation of cyclic nucleotides;
and 100 or more ion channels, including about 20 gated
by cyclic nucleotides. Inositol phospholipids are pres-
ent, as are kinases that interconvert them by phospho-
rylation of inositol head groups.
However, some types of signaling proteins common
in animal tissues are not present in plants, or are rep-
resented by only a few genes. Cyclic nucleotide–
dependent protein kinases (PKA and PKG) appear to
be absent, for example. Heterotrimeric G proteins and
protein Tyr kinase genes are much less prominent in
the plant genome, and serpentine (G protein–coupled)
receptors, the largest gene family in the human
genome (H110221,000 genes), are very sparsely represented
in the plant genome. DNA-binding nuclear steroid re-
ceptors are certainly not prominent, and may be ab-
sent from plants. Although plants lack the most widely
conserved light-sensing mechanism present in animals
(rhodopsin, with retinal as pigment), they have a rich
collection of other light-detecting mechanisms not
found in animal tissues—phytochromes and cryp-
tochromes, for example (Chapter 19).
The kinds of compounds that elicit signals in plants
are similar to certain signaling molecules in mammals
(Fig. 12–28). Instead of prostaglandins, plants have jas-
monate; instead of steroid hormones, brassinosteroids.
12.6 Signaling in Microorganisms and Plants 453
Gravity
Light
Humidity
Temperature
Wind
Herbivores
Pathogens
Pathogens
Parasites
Microorganisms
O
2
Minerals
Toxic molecules
Water status
CO
2
C
2
H
4
FIGURE 12–27 Some stimuli that produce responses in plants.
TABLE 12–7 Signaling Components Present in Mammals, Plants, or Bacteria
Signaling protein Mammals Plants Bacteria
Ion channels H11001H11001H11001
Electrogenic ion pumps H11001H11001H11001
Two-component His kinases H11001H11001H11001
Adenylyl cyclase H11001H11001H11001
Guanylyl cyclase H11001H11001 ?
Receptor protein kinases (Ser/Thr) H11001H11001 ?
Ca
2H11001
as second messenger H11001H11001 ?
Ca
2H11001
channels H11001H11001 ?
Calmodulin, CaM-binding protein H11001H11001H11002
MAPK cascade H11001H11001H11002
Cyclic nucleotide–gated channels H11001H11001H11002
IP
3
-gated Ca
2H11001
channels H11001H11001H11002
Phosphatidylinositol kinases H11001H11001H11002
Serpentine receptors H11001H11001/H11002H11001
Trimeric G proteins H11001H11001/H11002H11002
PI-specific phospholipase C H11001 ? H11002
Tyrosine kinase receptors H11001 ? H11002
SH2 domains H11001 ??
Nuclear steroid receptors H11001H11002H11002
Protein kinase A H11001H11002H11002
Protein kinase G H11001H11002H11002
8885d_c12_453 2/20/04 1:23 PM Page 453 mac76 mac76:385_reb:
About 100 different small peptides serve as plant signals,
and both plants and animals use compounds derived
from aromatic amino acids as signals.
Plants Detect Ethylene through a Two-Component
System and a MAPK Cascade
The receptors for the plant hormone ethylene
(CH
2
UCH
2
) are related in primary sequence to the
receptor His kinases of the bacterial two-component
systems and probably evolved from them; the cyanobac-
terial origin of chloroplasts (see Fig. 1–36) may have
brought the bacterial signaling genes into the plant cell
nucleus. In Arabidopsis, the two-component signaling
system is contained within a single protein. The first
downstream component affected by ethylene signaling
is a protein Ser/Thr kinase (CTR-1; Fig. 12–29) with se-
quence homology to Raf, the protein kinase that begins
the MAPK cascade in the mammalian response to in-
sulin (see the comparison in Fig. 12–30). In plants, in
the absence of ethylene, the CTR-1 kinase is active and
inhibits the MAPK cascade, preventing transcription of
ethylene-responsive genes. Exposure to ethylene inac-
tivates the CTR-1 kinase, thereby activating the MAPK
cascade that leads to activation of the transcription fac-
tor EIN3. Active EIN3 stimulates the synthesis of a sec-
ond transcription factor (ERF1), which in turn activates
transcription of a number of ethylene-responsive genes;
the gene products affect processes ranging from
seedling development to fruit ripening.
Although apparently derived from the bacterial two-
component signaling system, the ethylene system in
Arabidopsis is different in that the His kinase activity
that defines component 1 in bacteria is not essential to
the transduction in Arabidopsis. The genome of the
cyanobacterium Anabaena encodes proteins with both
an ethylene-binding domain and an active His kinase do-
main. It seems likely that in the course of evolution, the
ethylene receptor of vascular plants was derived from
that of a cyanobacterial endosymbiont, and that the bac-
terial His kinase became a Ser/Thr kinase in the plant.
Chapter 12 Biosignaling454
O
COO
H11002
Jasmonate
Plants Animals
O
COO
H11002
COO
H11002
Prostaglandin E
1
OH
HO
8
12
OH
Estradiol
H
3
HO
C
Brassinolide
(a brassinosteroid)
HO
H
O
O
HO
OH
OH
Serotonin
(5-hydroxytryptamine)
HO
N
H
Indole-3-acetate
(an auxin)
N
H
H11001
NH
3
Cytosol
Nucleus
MAPK
cascade
DNA
mRNA
DNA
mRNA
Ethylene-
response
proteins
Ethylene
Ethylene receptor
Two-component
system Plasma
membrane
CTR-1
MAPKKK
EIN2
EIN3
11
22
ERF1
FIGURE 12–28 Structural similarities between plant and animal sig-
nals. The plant signals jasmonate, indole-3-acetate, and brassinolide
resemble the mammalian signals prostaglandin E
1
, serotonin, and
estradiol.
FIGURE 12–29 Transduction mechanism for detection of ethylene
by plants. The ethylene receptor in the plasma membrane (red) is a
two-component system contained within a single protein, which has
both a receptor domain (component 1) and a response regulator do-
main (component 2). The receptor controls (in ways we do not yet un-
derstand) the activity of CTR1, a protein kinase similar to MAPKKKs
and therefore presumed to be part of a MAPK cascade. CTR1 is a neg-
ative regulator of the ethylene response; when CTR1 is inactive, the
ethylene signal passes through the gene product EIN2 (thought to be
a nuclear envelope protein), which somehow causes increased syn-
thesis of ERF1, a transcription factor; ERF1 in turn stimulates expres-
sion of proteins specific to the ethylene response.
8885d_c12_454 2/20/04 1:25 PM Page 454 mac76 mac76:385_reb:
Receptorlike Protein Kinases Transduce Signals from
Peptides and Brassinosteroids
One common motif in plant signaling involves recep-
torlike kinases (RLKs) with a single helical segment
in the plasma membrane that connects a receptor do-
main on the outside of the membrane with a protein
Ser/Thr kinase on the cytoplasmic side. This type of re-
ceptor participates in the defense mechanism triggered
by infection with a bacterial pathogen (Fig. 12–30a). The
signal to turn on the genes needed for defense against
infection is a peptide (flg22) released by breakdown of
flagellin, the major protein of the bacterial flagellum.
Binding of flg22 to the FLS2 receptor of Arabidopsis
induces receptor dimerization and autophosphorylation
on Ser and Thr residues, and the downstream effect is
activation of a MAPK cascade like that described above
for insulin action (Fig. 12–6). The final kinase in this
cascade activates a specific transcription factor, trig-
gering synthesis of the proteins that defend against the
bacterial infection. The steps between receptor phos-
phorylation and the MAPK cascade are not yet known.
A phosphoprotein phosphatase (KAPP) associates with
the active receptor protein and inactivates it by de-
phosphorylation to end the response.
The MAPK cascade in the plant’s defense against
bacterial pathogens is remarkably similar to the innate
immune response triggered by bacterial lipopolysac-
charide and mediated by the Toll-like receptors in mam-
mals (Fig. 12–30b). Other membrane receptors use sim-
ilar mechanisms to activate a MAPK cascade, ultimately
activating transcription factors and turning on the genes
essential to the defense response.
Most of the several hundred RLKs in plants are
presumed to act in similar ways: ligand binding in-
duces dimerization and autophosphorylation, and the
12.6 Signaling in Microorganisms and Plants 455
(a) (b)Plant (Arabidopsis) Mammal
Dimeric
receptor
flg22
Protein
kinase
domain
Transcription
factors WRKY22/29
Immune-
response proteins
Transcription
factors Jun, Fos
Transcription
factors NFkB
Immune-
response proteins
MAPK
cascade
Protein
kinase
IRAK
LPS
Flagellin
Receptors
Plasma
membrane
Ser, Thr
MAPK
cascade
MAPK
cascade
P
FIGURE 12–30 Similarities between the signaling pathways that trig-
ger immune responses in plants and animals. (a) In the plant Ara-
bidopsis thaliana, the peptide flg22, derived from the flagella of a bac-
terial pathogen, binds to its receptor in the plasma membrane, causing
the receptors to form dimers and triggering autophosphorylation of the
cytosolic protein kinase domain on a Ser or Thr residue (not a Tyr).
Autophosphorylation activates the receptor protein kinase, which then
phosphorylates downstream proteins. The activated receptor also ac-
tivates (by means unknown) a MAPKKK. The resulting kinase cascade
leads to phosphorylation of a nuclear protein that normally inhibits
the transcription factors WRKY22 and 29, triggering proteolytic degra-
dation of the inhibitor and freeing the transcription factors to stimu-
late gene expression related to the immune response. (b) In mammals,
the toxic bacterial lipopolysaccharide (LPS; see Fig. 7–32) is detected
by plasma membrane receptors that associate with and activate a sol-
uble protein kinase (IRAK). The major flagellar protein of pathogenic
bacteria acts through a similar receptor to activate IRAK. Then IRAK
initiates two distinct MAPK cascades that end in the nucleus, causing
the synthesis of proteins needed in the immune response. Jun, Fos,
and NFH9260B are transcription factors.
8885d_c12_455 2/20/04 1:25 PM Page 455 mac76 mac76:385_reb:
activated receptor kinase triggers downstream responses
by phosphorylating key proteins at Ser or Thr residues.
The ligands for these kinases have been identified in
only a few cases: brassinosteroids, the peptide trigger
for the self-incompatibility response that prevents self-
pollination, and CLV1 peptide, a factor involved in reg-
ulating the fate of stem cells (undifferentiated cells) in
plant development.
SUMMARY 12.6 Signaling in Microorganisms
and Plants
■ Bacteria and unicellular eukaryotes have a
variety of sensory systems that allow them to
sample and respond to their environment. In
the two-component system, a receptor His
kinase senses the signal and autophosphory-
lates a His residue, then phosphorylates the
response regulator on an Asp residue.
■ Plants respond to many environmental stimuli,
and employ hormones and growth factors to
coordinate the development and metabolic
activities of their tissues. Plant genomes
encode hundreds of signaling proteins,
including some very similar to those used in
signal transductions in mammalian cells.
■ Two-component signaling mechanisms common
in bacteria have been acquired in altered forms
by plants. Cyanobacteria use typical
two-component systems in the detection of
chemical signals and light; plants use related
proteins—which autophosphorylate on Ser/Thr,
not His, residues—to detect ethylene.
■ Plant receptorlike kinases (RLKs), with an
extracellular ligand-binding domain, a single
transmembrane segment, and a cytosolic
protein kinase domain, participate in detecting
a wide variety of stimuli, including peptides
that originate from pathogens, brassinosteroid
hormones, self-incompatible pollen, and
developmental signals. RLKs autophosphorylate
Ser/Thr residues, then activate downstream
proteins that in some cases are MAPK
cascades. The end result of many such signals
is increased transcription of specific genes.
12.7 Sensory Transduction in Vision,
Olfaction, and Gustation
The detection of light, smells, and tastes (vision, olfac-
tion, and gustation, respectively) in animals is accom-
plished by specialized sensory neurons that use signal-
transduction mechanisms fundamentally similar to those
that detect hormones, neurotransmitters, and growth
factors. An initial sensory signal is amplified greatly by
mechanisms that include gated ion channels and intra-
cellular second messengers; the system adapts to con-
tinued stimulation by changing its sensitivity to the
stimulus (desensitization); and sensory input from sev-
eral receptors is integrated before the final signal goes
to the brain.
Light Hyperpolarizes Rod and Cone Cells of the
Vertebrate Eye
In the vertebrate eye, light entering through the pupil
is focused on a highly organized collection of light-
sensitive neurons (Fig. 12–31). The light-sensing cells
are of two types: rods (about 10
9
per retina), which
sense low levels of light but cannot discriminate colors,
and cones (about 3 H11003 10
6
per retina), which are less
sensitive to light but can discriminate colors. Both cell
types are long, narrow, specialized sensory neurons with
two distinct cellular compartments: the outer segment
contains dozens of membranous disks loaded with the
membrane protein rhodopsin, and the inner segment
contains the nucleus and many mitochondria, which
produce the ATP essential to phototransduction.
Chapter 12 Biosignaling456
Light
To optic
nerve
Ganglion
neurons
Interconnecting
neurons
Rod
Cone
Light
Lens
Eye
Retina Optic
nerve
FIGURE 12–31 Light reception in the vertebrate eye. The lens of the
eye focuses light on the retina, which is composed of layers of neu-
rons. The primary photosensory neurons are rod cells (yellow), which
are responsible for high-resolution and night vision, and cone cells of
three subtypes (pink), which initiate color vision. The rods and cones
form synapses with several ranks of interconnecting neurons that con-
vey and integrate the electrical signals. The signals eventually pass
from ganglion neurons through the optic nerve to the brain.
8885d_c12_456 2/20/04 1:25 PM Page 456 mac76 mac76:385_reb:
Like other neurons, rods and cones have a trans-
membrane electrical potential (V
m
), produced by the
electrogenic pumping of the Na
H11001
K
H11001
ATPase in the plasma
membrane of the inner segment (Fig. 12–32). Also con-
tributing to the membrane potential is an ion channel
in the outer segment that permits passage of either Na
H11001
or Ca
2H11001
and is gated (opened) by cGMP. In the dark,
rod cells contain enough cGMP to keep this channel open.
The membrane potential is therefore determined by the
net difference between the Na
H11001
and K
H11001
pumped by the
inner segment (which polarizes the membrane) and the
influx of Na
H11001
through the ion channels of the outer seg-
ment (which tends to depolarize the membrane).
The essence of signaling in the retinal rod or cone
cell is a light-induced decrease in the concentration of
cGMP, which causes the cGMP-gated ion channel to
close. The plasma membrane then becomes hyperpolar-
ized by the Na
H11001
K
H11001
ATPase. Rod and cone cells synapse
with interconnecting neurons (Fig. 12–31) that carry
information about the electrical activity to the ganglion
neurons near the inner surface of the retina. The gan-
glion neurons integrate the output from many rod or cone
cells and send the resulting signal through the optic
nerve to the visual cortex of the brain.
Light Triggers Conformational Changes in the
Receptor Rhodopsin
Visual transduction begins when light falls on rhodopsin,
many thousands of molecules of which are present in
each disk of the outer segments of rod and cone cells.
Rhodopsin (M
r
40,000) is an integral protein with seven
membrane-spanning H9251 helices (Fig. 12–33), the charac-
teristic serpentine architecture. The amino-terminal do-
main projects into the disk, and the carboxyl-terminal
domain faces the cytosol of the outer segment. The
light-absorbing pigment (chromophore) 11-cis-retinal
is covalently attached to opsin, the protein component
of rhodopsin, through a Schiff base to a Lys residue. The
retinal lies near the middle of the bilayer (Fig. 12–33),
oriented with its long axis approximately in the plane of
the membrane. When a photon is absorbed by the reti-
nal component of rhodopsin, the energy causes a pho-
tochemical change; 11-cis-retinal is converted to all-
trans-retinal (see Figs 1–18b, 10–21). This change in
the structure of the chromophore causes conformational
changes in the rhodopsin molecule—the first stage in
visual transduction.
Excited Rhodopsin Acts through the G Protein
Transducin to Reduce the cGMP Concentration
In its excited conformation, rhodopsin interacts with a
second protein, transducin, which hovers nearby on
the cytoplasmic face of the disk membrane (Fig. 12–33).
Transducin (T) belongs to the same family of hetero-
trimeric GTP-binding proteins as G
s
and G
i
. Although
12.7 Sensory Transduction in Vision, Olfaction, and Gustation 457
Inner segment
Outer segment
Na
H11545
K
H11545
ATPase
Ion channel
open
Ion channel
closed
Light
Na
H11545
Na
H11545
Na
H11545
Na
H11545
Ca
2H11545
V
m
H11005H1100245 mV
V
m
H11005H1100275 mV
cGMP
FIGURE 12–32 Light-induced hyperpolarization of rod cells. The rod
cell consists of an outer segment that is filled with stacks of membra-
nous disks (not shown) containing the photoreceptor rhodopsin and
an inner segment that contains the nucleus and other organelles. Cones
have a similar structure. ATP in the inner segment powers the Na
H11001
K
H11001
ATPase, which creates a transmembrane electrical potential by pump-
ing 3 Na
H11001
out for every 2 K
H11001
pumped in. The membrane potential is
reduced by the flow of Na
H11001
and Ca
2H11001
into the cell through cGMP-
gated cation channels in the plasma membrane of the outer segment.
When rhodopsin absorbs light, it triggers degradation of cGMP (green
dots) in the outer segment, causing closure of the cation channel.
Without cation influx through this channel, the cell becomes hyper-
polarized. This electrical signal is passed to the brain through the ranks
of neurons shown in Figure 12–31.
8885d_c12_457 2/20/04 1:25 PM Page 457 mac76 mac76:385_reb:
specialized for visual transduction, transducin shares
many functional features with G
s
and G
i
. It can bind ei-
ther GDP or GTP. In the dark, GDP is bound, all three
subunits of the protein (T
H9251
, T
H9252
, and T
H9253
) remain together,
and no signal is sent. When rhodopsin is excited by light,
it interacts with transducin, catalyzing the replacement
of bound GDP by GTP from the cytosol (Fig. 12–34,
steps 1 and 2 ). Transducin then dissociates into T
H9251
and T
H9252H9253
, and the T
H9251
-GTP carries the signal from the ex-
cited receptor to the next element in the transduction
pathway, cGMP phosphodiesterase (PDE); this enzyme
converts cGMP to 5H11032-GMP (steps 3 and 4 ). Note that
this is not the same cyclic nucleotide phosphodiesterase
that hydrolyzes cAMP to terminate the H9252-adrenergic re-
sponse. The cGMP-specific PDE is unique to the visual
cells of the retina.
PDE is an integral protein with its active site on the
cytoplasmic side of the disk membrane. In the dark, a
tightly bound inhibitory subunit very effectively sup-
presses PDE activity. When T
H9251
-GTP encounters PDE,
the inhibitory subunit is released, and the enzyme’s ac-
tivity immediately increases by several orders of mag-
nitude. Each molecule of active PDE degrades many
molecules of cGMP to the biologically inactive 5H11032-GMP,
lowering [cGMP] in the outer segment within a fraction
of a second. At the new, lower [cGMP], the cGMP-gated
ion channels close, blocking reentry of Na
H11001
and Ca
2H11001
into the outer segment and hyperpolarizing the mem-
brane of the rod or cone cell (step 5 ). Through this
process, the initial stimulus—a photon—changes the V
m
of the cell.
Amplification of the Visual Signal Occurs in the Rod
and Cone Cells
Several steps in the visual-transduction process result in
great amplification of the signal. Each excited rhodopsin
molecule activates at least 500 molecules of transducin,
each of which can activate a molecule of PDE. This phos-
phodiesterase has a remarkably high turnover number,
each activated molecule hydrolyzing 4,200 molecules of
cGMP per second. The binding of cGMP to cGMP-gated
ion channels is cooperative (at least three cGMP mole-
cules must be bound to open one channel), and a rela-
tively small change in [cGMP] therefore registers as a
large change in ion conductance. The result of these
amplifications is exquisite sensitivity to light. Absorption
of a single photon closes 1,000 or more ion channels and
changes the cell’s membrane potential by about 1 mV.
The Visual Signal Is Quickly Terminated
As your eyes move across this line, the images of the
first words disappear rapidly—before you see the next
series of words. In that short interval, a great deal of
biochemistry has taken place. Very shortly after illumi-
nation of the rod or cone cells stops, the photosensory
system shuts off. The H9251 subunit of transducin (with
bound GTP) has intrinsic GTPase activity. Within mil-
liseconds after the decrease in light intensity, GTP is hy-
drolyzed and T
H9251
reassociates with T
H9252H9253
. The inhibitory
subunit of PDE, which had been bound to T
H9251
-GTP, is re-
leased and reassociates with PDE, strongly inhibiting
that enzyme. To return [cGMP] to its “dark” level, the
enzyme guanylyl cyclase converts GTP to cGMP (step
7
in Fig. 12–34) in a reaction that is inhibited by high
[Ca
2H11001
] (H11022100 nM). Calcium levels drop during illumina-
tion, because the steady-state [Ca
2H11001
] in the outer seg-
ment is the result of outward pumping of Ca
2H11001
through
the Na
H11001
-Ca
2H11001
exchanger of the plasma membrane and
inward movement of Ca
2H11001
through open cGMP-gated
channels. In the dark, this produces a [Ca
2H11001
] of about
500 nM—enough to inhibit cGMP synthesis. After brief
illumination, Ca
2H11001
entry slows and [Ca
2H11001
] declines (step
Chapter 12 Biosignaling458
H9251
H9252 H9253
Cytosol
Transducin
Disk
compartment Rhodopsin
FIGURE 12–33 Likely structure of rhodopsin complexed with the G
protein transducin. (PDB ID 1BAC) Rhodopsin (red) has seven trans-
membrane helices embedded in the disk membranes of rod outer seg-
ments and is oriented with its carboxyl terminus on the cytosolic side
and its amino terminus inside the disk. The chromophore 11-cis reti-
nal (blue), attached through a Schiff base linkage to Lys
256
of the sev-
enth helix, lies near the center of the bilayer. (This location is similar
to that of the epinephrine-binding site in the H9252-adrenergic receptor.)
Several Ser and Thr residues near the carboxyl terminus are substrates
for phosphorylations that are part of the desensitization mechanism
for rhodopsin. Cytosolic loops that interact with the G protein trans-
ducin are shown in orange; their exact positions are not yet known.
The three subunits of transducin (green) are shown in their likely
arrangement. Rhodopsin is palmitoylated at its carboxyl terminus, and
both the H9251 and H9253 subunits of transducin have attached lipids (yellow)
that assist in anchoring them to the membrane.
8885d_c12_458 2/20/04 1:26 PM Page 458 mac76 mac76:385_reb:
6 ). The inhibition of guanylyl cyclase by Ca
2H11001
is re-
lieved, and the cyclase converts GTP to cGMP to return
the system to its prestimulus state (step
7
).
Rhodopsin Is Desensitized by Phosphorylation
Rhodopsin itself also undergoes changes in response to
prolonged illumination. The conformational change in-
duced by light absorption exposes several Thr and Ser
residues in the carboxyl-terminal domain. These
residues are quickly phosphorylated by rhodopsin ki-
nase (step 8 in Fig. 12–34), which is functionally and
structurally homologous to the H9252-adrenergic kinase
(H9252ARK) that desensitizes the H9252-adrenergic receptor
(Fig. 12–17). The Ca
2H11001
-binding protein recoverin in-
hibits rhodopsin kinase at high [Ca
2H11001
], but the inhibi-
tion is relieved when [Ca
2H11001
] drops after illumination, as
described above. The phosphorylated carboxyl-terminal
domain of rhodopsin is bound by the protein arrestin
1, preventing further interaction between activated
rhodopsin and transducin. Arrestin 1 is a close homolog
of arrestin 2 (H9252arr; Fig. 12–17). On a relatively long time
scale (seconds to minutes), the all-trans-retinal of an
excited rhodopsin molecule is removed and replaced by
11-cis-retinal, to produce rhodopsin that is ready for an-
other round of excitation (step 9 in Fig. 12–34).
Humans cannot synthesize retinal from simpler
precursors and must obtain it in the diet in the
form of vitamin A (see Fig. 10–21). Given the role of
retinal in the process of vision, it is not surprising that
dietary deficiency of vitamin A causes night blindness
(poor vision at night or in dim light). ■
12.7 Sensory Transduction in Vision, Olfaction, and Gustation 459
Ca
2+
Na
+
,Ca
2+
4 Na
+
Ca
2+
Light absorption
converts 11-cis-
retinal to
all-trans-retinal,
activating rhodopsin.
1
Activated rhodopsin
catalyzes replacement
of GDP by GTP
on transducin (T),
which then dissociates
into T
a
-GTP and T
bg
.
2
T
α
-GTP activates
cGMP phosphodiesterase
(PDE) by binding and
removing its inhibitory
subunit (I).
3
Active PDE reduces
[cGMP] to below the
level needed to keep
cation channels open.
4
Reduction of [Ca
2+
]
activates guanylyl
cyclase (GC) and
inhibits PDE; [cGMP]
rises toward “dark”
level, reopening cation
channels and returning
V
m
to prestimulus level.
7
Slowly, arrestin dissociates,
rhodopsin is dephosphorylated,
and all-trans-retinal is
replaced with 11-cis-retinal.
Rhodopsin is ready for
another phototransduction
cycle.
9
Rhodopsin kinase (RK)
phosphorylates “bleached”
rhodopsin; low [Ca
2+
]
and recoverin (Recov)
stimulate this reaction.
Arrestin (Arr) binds
phosphorylated carboxyl
terminus, inactivating
rhodopsin.
8
Cation channels
close, preventing
influx of Na
+
and
Ca
2+
; membrane is
hyperpolarized.
This signal passes
to the brain.
Plasma
membrane
Disk membrane
5
Continued efflux of
Ca
2+
through the
Na
+
-Ca
2+
exchanger
reduces cytosolic
[Ca
2+
].
6
P
P
P
P
P
RK
Recov
P
GDP
GTP
cGMP
↓[Ca
2+
]
Recovery/Adaptation
Excitation
Rod
I
b
g
T
Rh
Rh
Rh
Arr
Rh
GC
PDE
PDE
cGMP
5H11032-GMP
cGMP
cGMP
cGMP
cGMP
I
T
a
?
GTP
T
a
?
GTP
T
a
?
GDP
GTP
FIGURE 12–34 Molecular consequences of photon absorption by rhodopsin in the rod outer
segment. The top half of the figure (steps 1 to 5 ) describes excitation; the bottom (steps 6
to 9 ), recovery and adaptation after illumination.
8885d_c12_459 2/20/04 1:26 PM Page 459 mac76 mac76:385_reb:
Cone Cells Specialize in Color Vision
Color vision in cone cells involves a path of sensory
transduction essentially identical to that described
above, but triggered by slightly different light receptors.
Three types of cone cells are specialized to detect light
from different regions of the spectrum, using three re-
lated photoreceptor proteins (opsins). Each cone cell
expresses only one kind of opsin, but each type is closely
related to rhodopsin in size, amino acid sequence, and
presumably three-dimensional structure. The differ-
ences among the opsins, however, are great enough to
place the chromophore, 11-cis-retinal, in three slightly
different environments, with the result that the three
photoreceptors have different absorption spectra (Fig.
12–35). We discriminate colors and hues by integrating
the output from the three types of cone cells, each con-
taining one of the three photoreceptors.
Color blindness, such as the inability to distin-
guish red from green, is a fairly common, genet-
ically inherited trait in humans. The various types of
color blindness result from different opsin mutations.
One form is due to loss of the red photoreceptor; af-
fected individuals are red
H11546
dichromats (they see only
two primary colors). Others lack the green pigment and
are green
H11002
dichromats. In some cases, the red and
green photoreceptors are present but have a changed
amino acid sequence that causes a change in their
absorption spectra, resulting in abnormal color vision.
Depending on which pigment is altered, such indivi-
duals are red-anomalous trichromats or green-
anomalous trichromats. Examination of the genes for
the visual receptors has allowed the diagnosis of color
blindness in a famous “patient” more than a century af-
ter his death (Box 12–3)! ■
Vertebrate Olfaction and Gustation Use Mechanisms
Similar to the Visual System
The sensory cells used to detect odors and tastes have
much in common with the rod and cone cells that de-
tect light. Olfactory neurons have a number of long thin
cilia extending from one end of the cell into a mucous
layer that overlays the cell. These cilia present a large
surface area for interaction with olfactory signals. The
receptors for olfactory stimuli are ciliary membrane pro-
teins with the familiar serpentine structure of seven
transmembrane H9251 helices. The olfactory signal can be
any one of the many volatile compounds for which there
are specific receptor proteins. Our ability to discrimi-
nate odors stems from hundreds of different olfactory
receptors in the tongue and nasal passages and from the
brain’s ability to integrate input from different types of
olfactory receptors to recognize a “hybrid” pattern, ex-
tending our range of discrimination far beyond the num-
ber of receptors.
The olfactory stimulus arrives at the sensory cells
by diffusion through the air. In the mucous layer over-
laying the olfactory neurons, the odorant molecule binds
directly to an olfactory receptor or to a specific binding
protein that carries the odorant to a receptor (Fig.
12–36). Interaction between odorant and receptor
triggers a change in receptor conformation that results
in the replacement of bound GDP by GTP on a G pro-
tein, G
olf
, analogous to transducin and to G
s
of the H9252-
adrenergic system. The activated G
olf
then activates
adenylyl cyclase of the ciliary membrane, which syn-
thesizes cAMP from ATP, raising the local [cAMP]. The
cAMP-gated Na
H11001
and Ca
2H11001
channels of the ciliary mem-
brane open, and the influx of Na
H11001
and Ca
2H11001
produces a
small depolarization called the receptor potential. If
a sufficient number of odorant molecules encounter re-
ceptors, the receptor potential is strong enough to cause
the neuron to fire an action potential. This is relayed to
the brain in several stages and registers as a specific
smell. All these events occur within 100 to 200 ms.
Some olfactory neurons may use a second trans-
duction mechanism. They have receptors coupled
through G proteins to PLC rather than to adenylyl cy-
clase. Signal reception in these cells triggers production
of IP
3
(Fig. 12–19), which opens IP
3
-gated Ca
2H11001
chan-
nels in the ciliary membrane. Influx of Ca
2H11001
then depo-
larizes the ciliary membrane and generates a receptor
potential or regulates Ca
2H11001
-dependent enzymes in the
olfactory pathway.
In either type of olfactory neuron, when the stimu-
lus is no longer present, the transducing machinery shuts
Chapter 12 Biosignaling460
Blue
pigment
Rhodopsin
Red
pigment
100
90
80
70
60
50
40
30
20
10
0
Relative absorbance
400 450 500 550 600 650
Green
pigment
Wavelength (nm)
FIGURE 12–35 Absorption spectra of purified rhodopsin and the red,
green, and blue receptors of cone cells. The spectra, obtained from
individual cone cells isolated from cadavers, peak at about 420, 530,
and 560 nm, and the maximum absorption for rhodopsin is at about
500 nm. For reference, the visible spectrum for humans is about 380
to 750 nm.
8885d_c12_460 2/20/04 1:26 PM Page 460 mac76 mac76:385_reb:
12.7 Sensory Transduction in Vision, Olfaction, and Gustation 461
Ciliary
membrane
Mucous
layer
Air
cAMPATP
GTP
GDP
Olfactory
neuron
Dendrite
Axon
Cilia
AC
Ca
2+
Cl
–
G
olf
b g
O
O
BP
O
Odorant (O) arrives
at the mucous layer
and binds directly to
an olfactory receptor
(OR) or to a binding
protein (BP) that
carries it to the OR.
1
Activated OR catalyzes
GDP-GTP exchange
on a G protein (G
olf
),
causing its dissociation
into a and bg.
2
Ca
2+
reduces the
affinity of the cation
channel for cAMP,
lowering the sensitivity
of the system to odorant.
6
G
olf
hydrolyzes GTP to
GDP, shutting itself off.
PDE hydrolyzes cAMP.
Receptor kinase phosphorylates
OR, inactivating it. Odorant
is removed by metabolism.
7
Ca
2+
-gated chloride
channels open. Efflux
of Cl
–
depolarizes the
cell, triggering an
electrical signal
to the brain.
5
G
a
-GTP activates
adenylyl cyclase,
which catalyzes
cAMP synthesis,
raising [cAMP].
3
cAMP-gated cation
channels open. Ca
2+
enters, raising
internal [Ca
2+
].
4
OR
O
a
GDP
a
GTP
FIGURE 12–36 Molecular events of olfaction. These interactions oc-
cur in the cilia of olfactory receptor cells.
FIGURE 7 (a)H11021?/AuH11022 (b)H11021?/AuH11022
BOX 12–3 BIOCHEMISTRY IN MEDICINE
Color Blindness: John Dalton’s Experiment
from the Grave
The chemist John Dalton (of atomic theory fame) was
color-blind. He thought it probable that the vitreous
humor of his eyes (the fluid that fills the eyeball be-
hind the lens) was tinted blue, unlike the colorless
fluid of normal eyes. He proposed that after his death,
his eyes should be dissected and the color of the vit-
reous humor determined. His wish was honored. The
day after Dalton’s death in July 1844, Joseph Ransome
dissected his eyes and found the vitreous humor to be
perfectly colorless. Ransome, like many scientists, was
reluctant to throw samples away. He placed Dalton’s
eyes in a jar of preservative (Fig. 1), where they stayed
for a century and a half.
Then, in the mid-1990s, molecular biologists in
England took small samples of Dalton’s retinas and ex-
tracted DNA. Using the known gene sequences for the
opsins of the red and green photopigments, they am-
plified the relevant sequences (using techniques de-
scribed in Chapter 9) and determined that Dalton had
the opsin gene for the red photopigment but lacked
the opsin gene for the green photopigment. Dalton was
a green
H11002
dichromat. So, 150 years after his death, the
experiment Dalton started—by hypothesizing about
the cause of his color blindness—was finally finished.
FIGURE 1 Dalton’s eyes.
8885d_c12_461 2/20/04 1:26 PM Page 461 mac76 mac76:385_reb:
itself off in several ways. A cAMP phosphodiesterase
returns [cAMP] to the prestimulus level. G
olf
hydrolyzes
its bound GTP to GDP, thereby inactivating itself. Phos-
phorylation of the receptor by a specific kinase prevents
its interaction with G
olf
, by a mechanism analogous to
that used to desensitize the H9252-adrenergic receptor and
rhodopsin. And lastly, some odorants are enzymatically
destroyed by oxidases.
The sense of taste in vertebrates reflects the activ-
ity of gustatory neurons clustered in taste buds on the
surface of the tongue. In these sensory neurons, ser-
pentine receptors are coupled to the heterotrimeric G
protein gustducin (very similar to the transducin of rod
and cone cells). Sweet-tasting molecules are those that
bind receptors in “sweet” taste buds. When the mole-
cule (tastant) binds, gustducin is activated by replace-
ment of bound GDP with GTP and then stimulates cAMP
production by adenylyl cyclase. The resulting elevation
of [cAMP] activates PKA, which phosphorylates K
H11001
channels in the plasma membrane, causing them to
close. Reduced efflux of K
H11001
depolarizes the cell (Fig.
12–37). Other taste buds specialize in detecting bitter,
sour, or salty tastants, using various combinations of sec-
ond messengers and ion channels in the transduction
mechanisms.
G Protein–Coupled Serpentine Receptor Systems
Share Several Features
We have now looked at four systems (hormone signal-
ing, vision, olfaction, and gustation) in which membrane
receptors are coupled to second messenger–generating
enzymes through G proteins. It is clear that signaling
mechanisms arose early in evolution; serpentine recep-
tors, heterotrimeric G proteins, and adenylyl cyclase are
found in virtually all eukaryotic organisms. Even the
common brewer’s yeast Saccharomyces uses serpentine
receptors and G proteins to detect the opposite mating
type. Overall patterns have been conserved, and the in-
troduction of variety has given modern organisms the
ability to respond to a wide range of stimuli (Table 12–8).
Of the 35,000 or so genes in the human genome, as many
as 1,000 encode serpentine receptors, including hun-
dreds for olfactory stimuli and a number of “orphan re-
ceptors” for which the natural ligand is not yet known.
All well-studied transducing systems that act
through heterotrimeric G proteins share certain com-
mon features (Fig. 12–38). The receptors have seven
transmembrane segments, a domain (generally the loop
between transmembrane helices 6 and 7) that interacts
with a G protein, and a carboxyl-terminal cytoplasmic
domain that undergoes reversible phosphorylation on
several Ser or Thr residues. The ligand-binding site (or,
in the case of light reception, the light receptor) is
buried deep in the membrane and includes residues
from several of the transmembrane segments. Ligand
binding (or light) induces a conformational change in
the receptor, exposing a domain that can interact with
a G protein. Heterotrimeric G proteins activate or in-
hibit effector enzymes (adenylyl cyclase, PDE, or PLC),
which change the concentration of a second messenger
(cAMP, cGMP, IP
3
, or Ca
2H11001
). In the hormone-detecting
Chapter 12 Biosignaling462
Acetylcholine (muscarinic)
Adenosine
Angiotensin
ATP (extracellular)
Bradykinin
Calcitonin
Cannabinoids
Catecholamines
Cholecystokinin
Corticotropin-releasing factor (CRF)
Cyclic AMP (Dictyostelium discoideum)
Dopamine
Follicle-stimulating hormone (FSH)
H9253-Aminobutyric acid (GABA)
Glucagon
Glutamate
Growth hormone–releasing hormone (GHRH)
Histamine
Leukotrienes
Light
Luteinizing hormone (LH)
Melatonin
Odorants
Opioids
Oxytocin
Platelet-activating factor
Prostaglandins
Secretin
Serotonin
Somatostatin
Tastants
Thyrotropin
Thyrotropin-releasing hormone (TRH)
Vasoactive intestinal peptide
Vasopressin
Yeast mating factors
Some Signals Transduced by G Protein–Coupled
Serpentine Receptors
TABLE 12–8
8885d_c12_462 2/20/04 1:26 PM Page 462 mac76 mac76:385_reb:
systems, the final output is an activated protein kinase
that regulates some cellular process by phosphorylating
a protein critical to that process. In sensory neurons,
the output is a change in membrane potential and a con-
sequent electrical signal that passes to another neuron
in the pathway connecting the sensory cell to the brain.
All these systems self-inactivate. Bound GTP is con-
verted to GDP by the intrinsic GTPase activity of G pro-
teins, often augmented by GTPase-activating proteins
(GAPs) or RGS proteins (regulators of G-protein sig-
naling). In some cases, the effector enzymes that are
the targets of G protein modulation also serve as GAPs.
12.7 Sensory Transduction in Vision, Olfaction, and Gustation 463
Basolateral
membrane
Apical
membrane
cAMPATP
GTP
Olfactory
neuron
AC
K
H11545
G
gust
H9252 H9253
SR
Sweet-tasting
molecule (S) binds to
sweet-taste receptor (SR),
activating the G protein
gustducin (G
gust
).
1
Gustducin subunit
activates adenylyl
cyclase (AC) of the
apical membrane,
raising [cAMP].
H9251
Taste cell
2
PKA, activated by cAMP,
phosphorylates a K
H11545
channel
in the basolateral membrane,
causing it to close. The
reduced efflux of K
H11545
depolarizes the cell.
3
P
S
GTPGDP
GDP
PKA
H9251 H9251
Vasopressin Epinephrine Light Odorants
Sweet
tastant
↓[cAMP]
↓
↑[cAMP]
↑
P
↓[cGMP] ↑[IP
3
] ↑[cAMP] ↑[cAMP]
↓P
Ca
2H11545
,Na
H11545
↑P
Ca
2H11545
↑P
Ca
2H11545
,Na
H11545
↓P
K
H11545
VR
G
i
AC
PKA
-AR
G
s
AC
PKA
↑
PKA
Rh
T
PDE
OR
1
G
olf
PLC
OR
2
G
olf
AC
SR
G
gust
AC
H9252
FIGURE 12–38 Common features of signaling systems that detect
hormones, light, smells, and tastes. Serpentine receptors provide sig-
nal specificity, and their interaction with G proteins provides signal
amplification. Heterotrimeric G proteins activate effector enzymes:
adenylyl cyclase (AC), phospholipase C (PLC), and phosphodiesterases
(PDE) that degrade cAMP or cGMP. Changes in concentration of the
second messengers (cAMP, cGMP, IP
3
) result in alterations of enzy-
matic activities by phosphorylation or alterations in the permeability
(P) of surface membranes to Ca
2H11001
, Na
H11001
, and K
H11001
. The resulting depo-
larization or hyperpolarization of the sensory cell (the signal) is passed
through relay neurons to sensory centers in the brain. In the best-
studied cases, desensitization includes phosphorylation of the recep-
tor and binding of a protein (arrestin) that interrupts receptor–G pro-
tein interactions. VR is the vasopressin receptor; other receptor and G
protein abbreviations are as used in earlier illustrations.
FIGURE 12–37 Transduction mechanism for sweet tastants.
8885d_c12_463 2/20/04 1:27 PM Page 463 mac76 mac76:385_reb:
Disruption of G-Protein Signaling Causes Disease
Biochemical studies of signal transductions have
led to an improved understanding of the patho-
logical effects of toxins produced by the bacteria that
cause cholera and pertussis (whooping cough). Both
toxins are enzymes that interfere with normal signal
transductions in the host animal. Cholera toxin, se-
creted by Vibrio cholerae found in contaminated drink-
ing water, catalyzes the transfer of ADP-ribose from
NAD
H11001
to the H9251 subunit of G
s
(Fig. 12–39), blocking its
GTPase activity and thereby rendering G
s
permanently
activated. This results in continuous activation of the
adenylyl cyclase of intestinal epithelial cells and chron-
ically high [cAMP], which triggers constant secretion of
Cl
H11002
, HCO
3
H11002
, and water into the intestinal lumen. The re-
sulting dehydration and electrolyte loss are the major
pathologies in cholera. The pertussis toxin, produced
by Bordetella pertussis, catalyzes ADP-ribosylation of
G
i
, preventing displacement of GDP by GTP and block-
ing inhibition of adenylyl cyclase by G
i
. ■
SUMMARY 12.7 Sensory Transduction in Vision,
Olfaction, and Gustation
■ Vision, olfaction, and gustation in vertebrates
employ serpentine receptors, which act
through heterotrimeric G proteins to change
the V
m
of the sensory neuron.
■ In rod and cone cells of the retina, light
activates rhodopsin, which stimulates
replacement of GDP by GTP on the G protein
transducin. The freed H9251 subunit of transducin
activates cGMP phosphodiesterase, which lowers
[cGMP] and thus closes cGMP-dependent ion
channels in the outer segment of the neuron.
The resulting hyperpolarization of the rod or
cone cell carries the signal to the next neuron
in the pathway, and eventually to the brain.
■ In olfactory neurons, olfactory stimuli, acting
through serpentine receptors and G proteins,
trigger either an increase in [cAMP] (by
activating adenylyl cyclase) or an increase in
[Ca
2H11001
] (by activating PLC). These second
messengers affect ion channels and thus
the V
m
.
■ Gustatory neurons have serpentine receptors
that respond to tastants by altering [cAMP],
which in turn changes V
m
by gating ion
channels.
■ There is a high degree of conservation of
signaling proteins and transduction
mechanisms across species.
Chapter 12 Biosignaling464
H9252
H9253
H9251
G
s
H9252
H9253
H9251
G
s CH
2
O
OH
P
O
H11002
H
N
H
HH
NH
2
O
O
H
OO
C
O
2
P
O
H11002
O
O
Rib Adenine
H11001
O
NH
2
C
NAD
H11001
H11001
cholera
toxin
CH
2
OH
P
O
H11002
H
H
HH
O
O
H
OO
O
P
O
H11002
O
O
Rib Adenine
N
Arg NH
ADP-ribose
Normal G
s
: GTPase activity
terminates the signal
from receptor to adenylyl
cyclase.
ADP-ribosylated G
s
:
GTPase activity is inactivated;
G
s
constantly activates
adenylyl cyclase.
NHArg
FIGURE 12–39 Toxins produced by bacteria that cause cholera and
whooping cough (pertussis). These toxins are enzymes that catalyze
transfer of the ADP-ribose moiety of NAD
H11001
to an Arg residue (cholera
toxin) or a Cys residue (pertussis toxin) of G proteins: G
s
in the case
of cholera (as shown here) and G
I
in whooping cough. The G proteins
thus modified fail to respond to normal hormonal stimuli. The pathol-
ogy of both diseases results from defective regulation of adenylyl cy-
clase and overproduction of cAMP.
8885d_c12_464 2/20/04 1:27 PM Page 464 mac76 mac76:385_reb:
12.8 Regulation of Transcription
by Steroid Hormones
The large group of steroid, retinoic acid (retinoid), and
thyroid hormones exert at least part of their effects by
a mechanism fundamentally different from that of other
hormones: they act in the nucleus to alter gene expres-
sion. We therefore discuss their mode of action in detail
in Chapter 28, along with other mechanisms for regu-
lating gene expression. Here we give a brief overview.
Steroid hormones (estrogen, progesterone, and cor-
tisol, for example), too hydrophobic to dissolve readily
in the blood, are carried on specific carrier proteins from
their point of release to their target tissues. In target
cells, these hormones pass through the plasma mem-
branes by simple diffusion and bind to specific receptor
proteins in the nucleus (Fig. 12–40). Hormone binding
triggers changes in the conformation of the receptor
proteins so that they become capable of interacting with
specific regulatory sequences in DNA called hormone
response elements (HREs), thus altering gene ex-
pression (see Fig. 28–31). The bound receptor-hormone
complex can either enhance or suppress the expression
of specific genes adjacent to HREs. Hours or days are
required for these regulators to have their full effect—
the time required for the changes in RNA synthesis and
subsequent protein synthesis to become evident in al-
tered metabolism.
The specificity of the steroid-receptor interac-
tion is exploited in the use of the drug tamox-
ifen to treat breast cancer. In some types of breast can-
cer, division of the cancerous cells depends on the
continued presence of the hormone estrogen. Tamox-
ifen competes with estrogen for binding to the estrogen
receptor, but the tamoxifen-receptor complex has little
or no effect on gene expression; tamoxifen is an antag-
onist of estrogen. Consequently, tamoxifen administered
after surgery or during chemotherapy for hormone-
dependent breast cancer slows or stops the growth of
remaining cancerous cells.
12.8 Regulation of Transcription by Steroid Hormones 465
1
1
Hormone (H), carried to the target
tissue on serum binding proteins,
diffuses across the plasma membrane
and binds to its specific receptor
protein (Rec) in the nucleus.
2
3
3
Binding regulates transcription of the
adjacent gene(s), increasing or decreasing
the rate of mRNA formation.
4
4
Altered levels of the hormone-
regulated gene product produce the
cellular response to the hormone.
Plasma
membrane
Nucleus
Rec
RNA polymerase
HRE
Gene
transcription
mRNA
translation
on ribosomes
New
protein
Altered cell
function
H
Serum binding protein
with bound hormone
2
Hormone binding changes the
conformation of Rec; it forms homo-
or heterodimers with other hormone-
receptor complexes and binds to
specific regulatory regions called
hormone response elements (HREs)
in the DNA adjacent to specific genes.
FIGURE 12–40 General mechanism by which steroid and thyroid
hormones, retinoids, and vitamin D regulate gene expression. The de-
tails of transcription and protein synthesis are discussed in Chapters
26 and 27. At least some steroids also act through plasma membrane
receptors by a completely different mechanism.
O
CH
3
N
CH
3
CH
3
Tamoxifen
8885d_c12_465 2/20/04 1:27 PM Page 465 mac76 mac76:385_reb:
Another steroid analog, the drug RU486, is used to ter-
minate early (preimplantation) pregnancies. An antag-
onist of the hormone progesterone, RU486 binds to the
progesterone receptor and blocks hormone actions es-
sential to implantation of the fertilized ovum in the
uterus. ■
The classic mechanism for steroid hormone action
through nuclear receptors does not explain certain ef-
fects of steroids that are too fast to be the result of al-
tered protein synthesis. For example, the estrogen-
mediated dilation of blood vessels is known to be
independent of gene transcription or protein synthesis,
as is the steroid-induced decrease in cellular [cAMP].
Another transduction mechanism is probably responsi-
ble for some of these effects. A plasma membrane pro-
tein predicted to have seven transmembrane helical seg-
ments binds progesterone with very high affinity and
mediates the inhibition of adenylyl cyclase by that hor-
mone, accounting for the decrease in [cAMP]. A second
nonclassical mechanism involves the rapid activation of
the MAPK cascade by progesterone, acting through the
soluble progesterone receptor. This is the same recep-
tor that, in the nucleus, causes the much slower changes
in gene expression that constitute the classic mecha-
nism of progesterone action. How the MAPK cascade is
activated is not yet clear.
SUMMARY 12.8 Regulation of Transcription
by Steroid Hormones
■ Steroid hormones enter cells and bind to
specific receptor proteins.
■ The hormone-receptor complex binds specific
regions of DNA, the hormone response
elements, and regulates the expression of
nearby genes by interacting with transcription
factors.
■ Two other, faster-acting mechanisms produce
some of the effects of steroids. Progesterone
triggers a rapid drop in [cAMP], mediated by a
plasma membrane receptor, and binding of
progesterone to the classic soluble steroid
receptor activates a MAPK cascade.
12.9 Regulation of the Cell Cycle
by Protein Kinases
One of the most dramatic roles for protein phosphory-
lation is the regulation of the eukaryotic cell cycle. Dur-
ing embryonic growth and later development, cell divi-
sion occurs in virtually every tissue. In the adult
organism most tissues become quiescent. A cell’s “deci-
sion” to divide or not is of crucial importance to the or-
ganism. When the regulatory mechanisms that limit cell
division are defective and cells undergo unregulated
division, the result is catastrophic—cancer. Proper cell
division requires a precisely ordered sequence of bio-
chemical events that assures every daughter cell a full
complement of the molecules required for life. Investi-
gations into the control of cell division in diverse eu-
karyotic cells have revealed universal regulatory mech-
anisms. Protein kinases and protein phosphorylation are
central to the timing mechanism that determines entry
into cell division and ensures orderly passage through
these events.
The Cell Cycle Has Four Stages
Cell division in eukaryotes occurs in four well-defined
stages (Fig. 12–41). In the S (synthesis) phase, the
DNA is replicated to produce copies for both daughter
Chapter 12 Biosignaling466
O
N
CH
3
CH
3
RU486
(mifepristone)
OH
CC
CH
3
G1
6–12 h
S
6–8 h
G2
3–4 h
M
1 h
G1 Phase
RNA and protein
synthesis. No DNA
synthesis.
Restriction point
A cell that passes this
point is committed
to pass into S phase.
Reentry point
A cell returning
from G0
enters at early
G1 phase.
G0 Phase
Terminally
differentiated
cells withdraw
from cell cycle
indefinitely.
M Phase
Mitosis (nuclear
division) and
cytokinesis
(cell division)
yield two
daughter cells.
G2 Phase
No DNA
synthesis.
RNA and
protein
synthesis
continue.
S Phase
DNA synthesis
doubles the
amount of DNA
in the cell. RNA
and protein also
synthesized.
G0
FIGURE 12–41 Eukaryotic cell cycle. The durations (in hours) of the
four stages vary, but those shown are typical.
8885d_c12_466 2/20/04 1:27 PM Page 466 mac76 mac76:385_reb:
cells. In the G2 phase (G indicates the gap between
divisions), new proteins are synthesized and the cell
approximately doubles in size. In the M phase (mitosis),
the maternal nuclear envelope breaks down, matching
chromosomes are pulled to opposite poles of the cell,
each set of daughter chromosomes is surrounded by a
newly formed nuclear envelope, and cytokinesis pinches
the cell in half, producing two daughter cells. In em-
bryonic or rapidly proliferating tissue, each daughter
cell divides again, but only after a waiting period (G1).
In cultured animal cells the entire process takes about
24 hours.
After passing through mitosis and into G1, a cell ei-
ther continues through another division or ceases to di-
vide, entering a quiescent phase (G0) that may last
hours, days, or the lifetime of the cell. When a cell in
G0 begins to divide again, it reenters the division cycle
through the G1 phase. Differentiated cells such as he-
patocytes or adipocytes have acquired their specialized
function and form; they remain in the G0 phase.
Levels of Cyclin-Dependent Protein Kinases Oscillate
The timing of the cell cycle is controlled by a family of
protein kinases with activities that change in response
to cellular signals. By phosphorylating specific proteins
at precisely timed intervals, these protein kinases or-
chestrate the metabolic activities of the cell to produce
orderly cell division. The kinases are heterodimers with
a regulatory subunit, cyclin, and a catalytic subunit,
cyclin-dependent protein kinase (CDK). In the ab-
sence of cyclin, the catalytic subunit is virtually inac-
tive. When cyclin binds, the catalytic site opens up, a
residue essential to catalysis becomes accessible (Fig.
12–42), and the activity of the catalytic subunit in-
creases 10,000-fold. Animal cells have at least ten dif-
ferent cyclins (designated A, B, and so forth) and at
least eight cyclin-dependent kinases (CDK1 through
CDK8), which act in various combinations at specific
points in the cell cycle. Plants also use a family of CDKs
to regulate their cell division.
12.9 Regulation of the Cell Cycle by Protein Kinases 467
(b)
(a)
(c)
FIGURE 12–42 Activation of cyclin-dependent protein kinases (CDKs) by cyclin
and phosphorylation. CDKs, a family of related enzymes, are active only when
associated with cyclins, another protein family. The crystal structure of CDK2 with
and without cyclin reveals the basis for this activation. (a) Without cyclin (PDB
ID 1HCK), CDK2 folds so that one segment, the T loop (red), obstructs the
binding site for protein substrates and thus inhibits protein kinase activity. The
binding site for ATP (blue) is also near the T loop. (b) When cyclin binds (PDB ID
1FIN), it forces conformational changes that move the T loop away from the
active site and reorient an amino-terminal helix (green), bringing a residue critical
to catalysis (Glu
51
) into the active site. (c) Phosphorylation of a Thr residue (dark
orange space-filling structure) in the T loop produces a negatively charged residue
that is stabilized by interaction with three Arg residues (red ball-and-stick struc-
tures), holding CDK in its active conformation (PDB ID 1JST).
8885d_c12_467 2/23/04 9:12 AM Page 467 mac76 mac76:
In a population of animal cells undergoing synchro-
nous division, some CDK activities show striking oscil-
lations (Fig. 12–43). These oscillations are the result of
four mechanisms for regulating CDK activity: phosphor-
ylation or dephosphorylation of the CDK, controlled
degradation of the cyclin subunit, periodic synthesis of
CDKs and cyclins, and the action of specific CDK-
inhibiting proteins.
Regulation of CDKs by Phosphorylation The activity of a
CDK is strikingly affected by phosphorylation and de-
phosphorylation of two critical residues in the protein
(Fig. 12–44a). Phosphorylation of Tyr
15
near the amino
terminus renders CDK2 inactive; the P –Tyr residue is
in the ATP-binding site of the kinase, and the negatively
charged phosphate group blocks the entry of ATP. A
specific phosphatase dephosphorylates this P –Tyr
residue, permitting the binding of ATP. Phosphorylation
Chapter 12 Biosignaling468
Kinase activity
Time
G1 S G2 M
Cyclin E–CDK2
Cyclin A–CDK2
Cyclin B–CDK1
G1
FIGURE 12–43 Variations in the activities of specific CDKs during
the cell cycle in animals. Cyclin E–CDK2 activity peaks near the G1
phase–S phase boundary, when the active enzyme triggers synthesis
of enzymes required for DNA synthesis (see Fig. 12–46). Cyclin
A–CDK2 activity rises during the S and G2 phases, then drops sharply
in the M phase, as cyclin B–CDK1 peaks.
U
Cyclin
U U U U
Cyclin
Tyr
No cyclin
present;
CDK is
inactive.
2
Cyclin
synthesis
leads to its
accumulation.
Cyclin-CDK complex forms, but phosphorylation
on Tyr
15
blocks ATP-binding site; still inactive.
3
Phosphorylation of Thr
160
in
T loop and removal of Tyr
15
phosphoryl group activates
cyclin-CDK manyfold.
4
1
Cyclin is
degraded
by proteasome,
leaving CDK
inactive.
8
CDK phosphorylates
phosphatase, which
activates more CDK.
5
CDK phosphorylates
DBRP, activating it.
6
DBRP triggers
addition of ubiquitin
molecules to cyclin
by ubiquitin ligase.
7
P
i
CDKCDK
CDK
Tyr Thr
Thr
Cyclin
CDK
PhosphatasePhosphatase
DBRPDBRP
P
P
P
P
P
P
(a)
(b)
FIGURE 12–44 Regulation of CDK by phosphorylation and prote-
olysis. (a) The cyclin-dependent protein kinase activated at the time
of mitosis (the M phase CDK) has a “T loop” that can fold into the
substrate-binding site. When Thr
160
in the T loop is phosphorylated,
the loop moves out of the substrate-binding site, activating the CDK
manyfold. (b) The active cyclin-CDK complex triggers its own inac-
tivation by phosphorylation of DBRP (destruction box recognizing
protein). DBRP and ubiquitin ligase then attach several molecules of
ubiquitin (U) to cyclin, targeting it for destruction by proteasomes,
proteolytic enzyme complexes.
8885d_c12_468 2/20/04 1:28 PM Page 468 mac76 mac76:385_reb:
of Thr
160
in the “T loop” of CDK, catalyzed by the CDK-
activating kinase, forces the T loop out of the substrate-
binding cleft, permitting substrate binding and catalytic
activity.
One circumstance that triggers this control mecha-
nism is the presence of single-strand breaks in DNA,
which leads to arrest of the cell cycle in G2. A specific
protein kinase (called Rad3 in yeast), which is activated
by single-strand breaks, triggers a cascade leading to the
inactivation of the phosphatase that dephosphorylates
Tyr
15
of CDK. The CDK remains inactive and the cell is
arrested in G2. The cell will not divide until the DNA is
repaired and the effects of the cascade are reversed.
Controlled Degradation of Cyclin Highly specific and pre-
cisely timed proteolytic breakdown of mitotic cyclins
regulates CDK activity throughout the cell cycle.
Progress through mitosis requires first the activation
then the destruction of cyclins A and B, which activate
the catalytic subunit of the M-phase CDK. These cyclins
contain near their amino terminus the sequence
Arg–Thr–Ala–Leu–Gly–Asp–Ile–Gly–Asn, the “destruc-
tion box,” which targets them for degradation. (This us-
age of “box” derives from the common practice, in dia-
gramming the sequence of a nucleic acid or protein, of
enclosing within a box a short sequence of nucleotide
or amino acid residues with some specific function. It
does not imply any three-dimensional structure.) The
protein DBRP (destruction box recognizing protein)
recognizes this sequence and initiates the process of cy-
clin degradation by bringing together the cyclin and an-
other protein, ubiquitin. Cyclin and activated ubiqui-
tin are covalently joined by the enzyme ubiquitin ligase
(Fig. 12–44b). Several more ubiquitin molecules are
then appended, providing the signal for a proteolytic en-
zyme complex, or proteasome, to degrade cyclin.
What controls the timing of cyclin breakdown? A
feedback loop occurs in the overall process shown in
Figure 12–44. Increased CDK activity activates cyclin
proteolysis. Newly synthesized cyclin associates with
and activates CDK, which phosphorylates and activates
DBRP. Active DBRP then causes proteolysis of cyclin.
Lowered [cyclin] causes a decline in CDK activity, and
the activity of DBRP also drops through slow, constant
dephosphorylation and inactivation by a DBRP phos-
phatase. The cyclin level is ultimately restored by syn-
thesis of new cyclin molecules.
The role of ubiquitin and proteasomes is not limited
to the regulation of cyclin; as we shall see in Chapter 27,
both also take part in the turnover of cellular proteins,
a process fundamental to cellular housekeeping.
Regulated Synthesis of CDKs and Cyclins The third mech-
anism for changing CDK activity is regulation of the rate
of synthesis of cyclin or CDK or both. For example, cy-
clin D, cyclin E, CDK2, and CDK4 are synthesized only
when a specific transcription factor, E2F, is present in
the nucleus to activate transcription of their genes. Syn-
thesis of E2F is in turn regulated by extracellular sig-
nals such as growth factors and cytokines (inducers
of cell division), compounds found to be essential for
the division of mammalian cells in culture. These growth
factors induce the synthesis of specific nuclear tran-
scription factors essential to the production of the
enzymes of DNA synthesis. Growth factors trigger phos-
phorylation of the nuclear proteins Jun and Fos, tran-
scription factors that promote the synthesis of a variety
of gene products, including cyclins, CDKs, and E2F. In
turn, E2F controls production of several enzymes es-
sential for the synthesis of deoxynucleotides and DNA,
enabling cells to enter the S phase (Fig. 12–45).
Inhibition of CDKs Finally, specific protein inhibitors
bind to and inactivate specific CDKs. One such protein
is p21, which we discuss below.
These four control mechanisms modulate the ac-
tivity of specific CDKs that, in turn, control whether a
cell will divide, differentiate, become permanently qui-
escent, or begin a new cycle of division after a period
of quiescence. The details of cell cycle regulation, such
as the number of different cyclins and kinases and the
12.9 Regulation of the Cell Cycle by Protein Kinases 469
Growth factors,
cytokines
Phosphorylation of
Jun and Fos in nucleus
transcriptional
regulation
Passage from
G1 to S phase
MAPK
cascade
Transcription
factor E2F
transcriptional
regulation
Cyclins,
CDKs
Enzymes for
DNA synthesis
FIGURE 12–45 Regulation of cell division by growth factors. The
path from growth factors to cell division leads through the enzyme
cascade that activates MAPK; phosphorylation of the nuclear tran-
scription factors Jun and Fos; and the activity of the transcription fac-
tor E2F, which promotes synthesis of several enzymes essential for
DNA synthesis.
8885d_c12_469 2/20/04 1:28 PM Page 469 mac76 mac76:385_reb:
combinations in which they act, differ from species to
species, but the basic mechanism has been conserved
in the evolution of all eukaryotic cells.
CDKs Regulate Cell Division by Phosphorylating
Critical Proteins
We have examined how cells maintain close control of
CDK activity, but how does the activity of CDK control
the cell cycle? The list of target proteins that CDKs are
known to act upon continues to grow, and much remains
to be learned. But we can see a general pattern behind
CDK regulation by inspecting the effect of CDKs on the
structures of laminin and myosin and on the activity of
retinoblastoma protein.
The structure of the nuclear envelope is maintained
in part by highly organized meshworks of intermediate
filaments composed of the protein laminin. Breakdown
of the nuclear envelope before segregation of the sister
chromatids in mitosis is partly due to the phosphoryla-
tion of laminin by a CDK, which causes laminin filaments
to depolymerize.
A second kinase target is the ATP-driven actin-
myosin contractile machinery that pinches a dividing
cell into two equal parts during cytokinesis. After the
division, CDK phosphorylates a small regulatory subunit
of myosin, causing dissociation of myosin from actin fil-
aments and inactivating the contractile machinery. Sub-
sequent dephosphorylation allows reassembly of the
contractile apparatus for the next round of cytokinesis.
A third and very important CDK substrate is the
retinoblastoma protein, pRb; when DNA damage is
detected, this protein participates in a mechanism that
arrests cell division in G1 (Fig. 12–46). Named for the
retinal tumor cell line in which it was discovered, pRb
functions in most, perhaps all, cell types to regulate cell
division in response to a variety of stimuli. Unphosphor-
ylated pRb binds the transcription factor E2F; while
bound to pRb, E2F cannot promote transcription of a
group of genes necessary for DNA synthesis (the genes
for DNA polymerase H9251, ribonucleotide reductase, and
other proteins; Chapter 25). In this state, the cell cycle
cannot proceed from the G1 to the S phase, the step
that commits a cell to mitosis and cell division. The pRb-
E2F blocking mechanism is relieved when pRb is phos-
phorylated by cyclin E–CDK2, which occurs in response
to a signal for cell division to proceed.
When the protein kinases ATM and ATR detect dam-
age to DNA, such as a single-strand break, they activate
p53 to serve as a transcription factor that stimulates the
synthesis of the protein p21 (Fig. 12–46). This protein
inhibits the protein kinase activity of cyclin E–CDK2. In
the presence of p21, pRb remains unphosphorylated and
bound to E2F, blocking the activity of this transcription
factor, and the cell cycle is arrested in G1. This gives
the cell time to repair its DNA before entering the S
phase, thereby avoiding the potentially disastrous trans-
fer of a defective genome to one or both daughter cells.
SUMMARY 12.9 Regulation of the Cell Cycle
by Protein Kinases
■ Progression through the cell cycle is regulated
by the cyclin-dependent protein kinases
(CDKs), which act at specific points in the
cycle, phosphorylating key proteins and
modulating their activities. The catalytic
subunit of CDKs is inactive unless associated
with the regulatory cyclin subunit.
■ The activity of a cyclin-CDK complex changes
during the cell cycle through differential
synthesis of CDKs, specific degradation of
cyclin, phosphorylation and dephosphorylation
of critical residues in CDKs, and binding of
inhibitory proteins to specific cyclin-CDKs.
Chapter 12 Biosignaling470
Cell division
blocked by p53
Cell division
occurs normally
CDK2
Inactive
CDK2
Active
Cyclin E
Active p53
DNA
damage
↑[p21]
p21
p21
pRb
P
pRb
pRb
E2F
Inactive
E2F
Enzymes
for DNA
synthesis
transcriptional
regulation
transcriptional
regulation
Passage
from
G1 to S
Active
Cyclin E
FIGURE 12–46 Regulation of passage from G1 to S by phosphory-
lation of pRb. When the retinoblastoma protein, pRb, is phosphory-
lated, it cannot bind and inactivate EF2, a transcription factor that pro-
motes synthesis of enzymes essential to DNA synthesis. If the
regulatory protein p53 is activated by ATM and ATR, protein kinases
that detect damaged DNA, it stimulates the synthesis of p21, which
can bind to and inhibit cyclin E–CDK2 and thus prevent phosphory-
lation of pRb. Unphosphorylated pRb binds and inactivates E2F, block-
ing passage from G1 to S until the DNA has been repaired.
8885d_c12_470 2/20/04 1:28 PM Page 470 mac76 mac76:385_reb:
12.10 Oncogenes, Tumor Suppressor Genes,
and Programmed Cell Death
Tumors and cancer are the result of uncontrolled cell
division. Normally, cell division is regulated by a family
of extracellular growth factors, proteins that cause rest-
ing cells to divide and, in some cases, differentiate.
Defects in the synthesis, regulation, or recognition of
growth factors can lead to cancer.
Oncogenes Are Mutant Forms of the Genes
for Proteins That Regulate the Cell Cycle
Oncogenes were originally discovered in tumor-causing
viruses, then later found to be closely similar to or de-
rived from genes in the animal host cells, proto-
oncogenes, which encode growth-regulating proteins.
During viral infections, the DNA sequence of a proto-
oncogene is sometimes copied by the virus and incor-
porated into its genome (Fig. 12–47). At some point
during the viral infection cycle, the gene can become
defective by truncation or mutation. When this viral
oncogene is expressed in its host cell during a subse-
quent infection, the abnormal protein product interferes
with normal regulation of cell growth, sometimes re-
sulting in a tumor.
Proto-oncogenes can become oncogenes without a
viral intermediary. Chromosomal rearrangements, chem-
ical agents, and radiation are among the factors that can
cause oncogenic mutations. The mutations that produce
oncogenes are genetically dominant; if either of a pair
of chromosomes contains a defective gene, that gene
product sends the signal “divide” and a tumor will re-
sult. The oncogenic defect can be in any of the proteins
involved in communicating the “divide” signal. We know
of oncogenes that encode secreted proteins, growth fac-
tors, transmembrane proteins (receptors), cytoplasmic
proteins (G proteins and protein kinases), and the nu-
clear transcription factors that control the expression
of genes essential for cell division (Jun, Fos).
12.10 Oncogenes, Tumor Suppressor Genes, and Programmed Cell Death 471
Normal cell
is infected with
retrovirus.
Retrovirus
Gene for regulatory
growth protein
(proto-oncogene)
Host cell now has
retroviral genome
incorporated near
proto-oncogene.
Forming virus
encapsulates
proto-oncogene
and viral genome.
Retrovirus with
proto-oncogene
infection cycles
Mutation creates
oncogene.
Retrovirus with
oncogene invades
normal cell.
Transformed cell,
producing defective
regulatory protein
3
4
5
2
1
FIGURE 12–47 Conversion of a regulatory gene to a viral oncogene.
1 A normal cell is infected by a retrovirus (Chapter 26), which
2 inserts its own genome into the chromosome of the host cell, near
the gene for a regulatory protein (the proto-oncogene). 3 Viral par-
ticles released from the infected cell sometimes “capture” a host gene,
in this case a proto-oncogene. 4 During several cycles of infection,
a mutation occurs in the viral proto-oncogene, converting it to an
oncogene. 5 When the virus subsequently infects a cell, it introduces
the oncogene into the cell’s DNA. Transcription of the oncogene leads
to the production of a defective regulatory protein that continuously
gives the signal for cell division, overriding normal regulatory mech-
anisms. Host cells infected with oncogene-carrying viruses undergo
unregulated cell division—they form tumors. Proto-oncogenes can
also undergo mutation to oncogenes without the intervention of a
retrovirus, as described in the text.
8885d_c12_471 2/20/04 1:29 PM Page 471 mac76 mac76:385_reb:
Some oncogenes encode surface receptors with de-
fective or missing signal-binding sites such that their in-
trinsic Tyr kinase activity is unregulated. For example,
the protein ErbB is essentially identical to the normal
receptor for epidermal growth factor, except that ErbB
lacks the amino-terminal domain that normally binds
EGF (Fig. 12–48) and as a result sends the “divide” sig-
nal whether EGF is present or not. Mutations in erbB2,
the gene for a receptor Tyr kinase related to ErbB, are
commonly associated with cancers of the glandular ep-
ithelium in breast, stomach, and ovary. (For an expla-
nation of the use of abbreviations in naming genes and
their products, see Chapter 25.)
Mutant forms of the G protein Ras are common in
tumor cells. The ras oncogene encodes a protein with
normal GTP binding but no GTPase activity. The mu-
tant Ras protein is therefore always in its activated
(GTP-bound) form, regardless of the signals arriving
through normal receptors. The result can be unregu-
lated growth. Mutations in ras are associated with 30%
to 50% of lung and colon carcinomas and more than 90%
of pancreatic carcinomas.
Defects in Tumor Suppressor Genes Remove Normal
Restraints on Cell Division
Tumor suppressor genes encode proteins that
normally restrain cell division. Mutation in one
or more of these genes can lead to tumor formation.
Unregulated growth due to defective tumor suppressor
genes, unlike that due to oncogenes, is genetically re-
cessive; tumors form only if both chromosomes of a pair
contain a defective gene. In a person who inherits one
correct copy and one defective copy, every cell has one
defective copy of the gene. If any one of those 10
12
so-
matic cells undergoes mutation in the one good copy, a
tumor may grow from that doubly mutant cell. Muta-
tions in both copies of the genes for pRb, p53, or p21
yield cells in which the normal restraint on cell division
is lost and a tumor forms.
Retinoblastoma is a cancer of the retina that occurs
in children who have two defective Rb alleles. Very
young children who develop retinoblastoma commonly
have multiple tumors in both eyes. Each tumor is de-
rived from a single retinal cell that has undergone a mu-
tation in its one good copy of the Rb gene. (A fetus with
two mutant alleles in every cell is nonviable.)
Retinoblastoma patients also have a high incidence of
cancers of the lung, prostate, and breast.
A far less likely event is that a person born with two
good copies of a gene will have two independent muta-
tions in the same gene in the same cell, but this does
occur. Some individuals develop retinoblastomas later in
childhood, usually with only one tumor in only one eye.
These individuals were presumably born with two good
copies of Rb in every cell, but both Rb genes in a single
retinal cell have undergone mutation, leading to a tumor.
Mutations in the gene for p53 also cause tumors; in
more than 90% of human cutaneous squamous cell car-
cinomas (skin cancers) and about 50% of all other hu-
man cancers, p53 is defective. Those very rare individ-
uals who inherit one defective copy of p53 commonly
have the Li-Fraumeni cancer syndrome, in which mul-
tiple cancers (of the breast, brain, bone, blood, lung, and
skin) occur at high frequency and at an early age. The
explanation for multiple tumors in this case is the same
as that for Rb mutations: an individual born with one
defective copy of p53 in every somatic cell is likely to
suffer a second p53 mutation in more than one cell in
his or her lifetime.
Mutations in oncogenes and tumor suppressor
genes do not have an all-or-none effect. In some can-
cers, perhaps in all, the progression from a normal cell
to a malignant tumor requires an accumulation of
mutations (sometimes over several decades), none of
which, alone, is responsible for the end effect. For ex-
ample, the development of colorectal cancer has several
recognizable stages, each associated with a mutation
(Fig. 12–49). If a normal epithelial cell in the colon un-
dergoes mutation of both copies of the tumor suppres-
sor gene APC (adenomatous polyposis coli), it begins to
divide faster than normal and produces a clone of itself,
a benign polyp (early adenoma). For reasons not yet
known, the APC mutation results in chromosomal in-
stability; whole regions of a chromosome are lost or re-
Chapter 12 Biosignaling472
EGF-binding
domain
EGF-binding site
empty; tyrosine
kinase is inactive.
Binding of EGF
activates
tyrosine kinase.
Tyrosine
kinase is
constantly
active.
Tyrosine kinase
domain
EGF
Extracellular space
Normal EGF receptor ErbB protein
FIGURE 12–48 Oncogene-encoded defective EGF receptor. The
product of the erbB oncogene (the ErbB protein) is a truncated ver-
sion of the normal receptor for epidermal growth factor (EGF). Its in-
tracellular domain has the structure normally induced by EGF bind-
ing, but the protein lacks the extracellular binding site for EGF.
Unregulated by EGF, ErbB continuously signals cell division.
8885d_c12_472 2/20/04 1:30 PM Page 472 mac76 mac76:385_reb:
arranged during cell division. This instability can lead to
another mutation, commonly in ras, that converts the
clone into an intermediate adenoma. A third mutation
(probably in the tumor suppressor gene DCC) leads to
a late adenoma. Only when both copies of p53 become
defective does this cell mass become a carcinoma, a ma-
lignant, life-threatening cancer. The full sequence there-
fore requires at least seven genetic “hits”: two on each
of three tumor suppressor genes (APC, DCC, and p53)
and one on the protooncogene ras. There are probably
several other routes to colorectal cancer as well, but the
principle that full malignancy results only from multiple
mutations is likely to hold. When a polyp is detected in
the early adenoma stage and the cells containing the
first mutations are removed surgically, late adenomas
and carcinomas will not develop; hence the importance
of early detection. ■
Apoptosis Is Programmed Cell Suicide
Many cells can precisely control the time of their own
death by the process of programmed cell death, or
apoptosis (appH11032-a-toeH11032-sis; from the Greek for “drop-
ping off,” as in leaves dropping in the fall). In the de-
velopment of an embryo, for example, some cells must
die. Carving fingers from stubby limb buds requires the
precisely timed death of cells between developing fin-
ger bones. During development of the nematode
Caenorhabditis elegans from a fertilized egg, exactly
131 cells (of a total of 1,090 somatic cells in the em-
bryo) must undergo programmed death in order to con-
struct the adult body.
Apoptosis also has roles in processes other than de-
velopment. When an antibody-producing cell begins to
make antibodies against an antigen normally present in
the body, that cell undergoes programmed death in the
thymus gland—an essential mechanism for eliminating
anti-self antibodies. The monthly sloughing of cells of
the uterine wall (menstruation) is another case of apop-
tosis mediating normal cell death. Sometimes cell sui-
cide is not programmed but occurs in response to bio-
logical circumstances that threaten the rest of the
organism. For example, a virus-infected cell that dies
before completion of the infection cycle prevents spread
of the virus to nearby cells. Severe stresses such as heat,
hyperosmolarity, UV light, and gamma irradiation also
trigger cell suicide; presumably the organism is better
off with aberrant cells dead.
The regulatory mechanisms that trigger apoptosis
involve some of the same proteins that regulate the cell
cycle. The signal for suicide often comes from outside,
through a surface receptor. Tumor necrosis factor
(TNF), produced by cells of the immune system, inter-
acts with cells through specific TNF receptors. These
receptors have TNF-binding sites on the outer face of
the plasma membrane and a “death domain” of about
80 amino acid residues that passes the self-destruct sig-
nal through the membrane to cytosolic proteins such as
TRADD (TNF receptor-associated death domain) (Fig.
12–50). Another receptor, Fas, has a similar death do-
main that allows it to interact with the cytosolic protein
FADD (Fas-associated death domain), which activates
a cytosolic protease called caspase 8. This enzyme be-
longs to a family of proteases that participate in apop-
tosis; all are synthesized as inactive proenzymes, all have
a critical Cys residue at the active site, and all hydrolyze
their target proteins on the carboxyl-terminal side of
specific Asp residues (hence the name caspase).
When caspase 8, an “initiator” caspase, is activated
by an apoptotic signal carried through FADD, it further
self-activates by cleaving its own proenzyme form. Mi-
tochondria are one target of active caspase 8. The pro-
tease causes the release of certain proteins contained
between the inner and outer mitochondrial membranes:
12.10 Oncogenes, Tumor Suppressor Genes, and Programmed Cell Death 473
APC
ras
DCC?
p53
?
?
Normal
colorectal
epithelium
Early
adenoma
Intermediate
adenoma
Advanced
adenoma
Colorectal
carcinoma
Invasive
carcinoma
Metastatic
carcinoma
Tumor suppressor gene
Oncogene
Unknown status
FIGURE 12–49 From normal epithelial cell to colorectal cancer. In
the colon, mutations in both copies of the tumor suppressor gene APC
lead to benign clusters of epithelial cells that multiply too rapidly (early
adenoma). If a cell already defective in APC suffers a second muta-
tion in the proto-oncogene ras, the doubly mutant cell gives rise to
an intermediate adenoma, forming a benign polyp of the colon. When
one of these cells undergoes further mutations in the tumor suppres-
sor genes DCC (probably) and p53, increasingly aggressive tumors
form. Finally, mutations in genes not yet characterized lead to a ma-
lignant tumor and finally to a metastatic tumor that can spread to other
tissues. Most malignant tumors probably result from a series of muta-
tions such as this.
8885d_c12_473 2/20/04 1:30 PM Page 473 mac76 mac76:385_reb:
cytochrome c (Chapter 19) and several “effector” cas-
pases. Cytochrome c binds to the proenzyme form of the
effector enzyme caspase 9 and stimulates its proteolytic
activation. The activated caspase 9 in turn catalyzes
wholesale destruction of cellular proteins—a major cause
of apoptotic cell death. One specific target of caspase
action is a caspase-activated deoxyribonuclease.
In apoptosis, the monomeric products of protein
and DNA degradation (amino acids and nucleotides) are
released in a controlled process that allows them to be
taken up and reused by neighboring cells. Apoptosis
thus allows the organism to eliminate a cell without
wasting its components.
SUMMARY 12.10 Oncogenes, Tumor Suppressor
Genes, and Programmed Cell Death
■ Oncogenes encode defective signaling proteins.
By continually giving the signal for cell division,
they lead to tumor formation. Oncogenes are
genetically dominant and may encode defective
growth factors, receptors, G proteins, protein
kinases, or nuclear regulators of transcription.
■ Tumor suppressor genes encode regulatory
proteins that normally inhibit cell division;
mutations in these genes are genetically
recessive but can lead to tumor formation.
■ Cancer is generally the result of an
accumulation of mutations in oncogenes and
tumor suppressor genes.
■ Apoptosis can be triggered by extracellular
signals such as TNF through plasma membrane
receptors.
Chapter 12 Biosignaling474
FIGURE 12–50 Initial events of apoptosis. Receptors in the plasma
membrane (Fas, TNF-R1) receive signals from outside the cell (the Fas
ligand or tumor necrosis factor (TNF), respectively). Activated recep-
tors foster interaction between the “death domain” (an 80 amino acid
sequence) in Fas or TNF-R1 and a similar death domain in the cy-
tosolic proteins FADD or TRADD. FADD activates a cytosolic pro-
tease, caspase 8, that proteolytically activates other cellular proteases.
TRADD also activates proteases. The resulting proteolysis is a primary
factor in cell death.
Fas
Fas
ligand
FADD
TNF-R1
TNF
TRADD
Mitochondrion
Plasma
membrane
Effector
caspases
Activation of
DNase
Cytochrome c
Death
domains
Protein
degradation
Cell death
Caspase 8
(initiator)
Key Terms
signal transduction 421
enzyme cascade 422
desensitization 422
ligand-gated receptor channel 426
voltage-gated ion channel 427
second messenger 428
autophosphorylation 429
SH2 domain 429
G proteins 429
MAPK cascade 430
receptor Tyr kinase 432
serpentine receptors 435
G protein–coupled receptors
(GPCR) 435
7 transmembrane segment (7tm)
receptors 435
stimulatory G protein (G
s
) 436
H9252-adrenergic receptor kinase
(H9252ARK) 441
H9252-arrestin (H9252arr; arrestin 2) 441
G protein–coupled receptor kinases
(GRKs) 441
scaffold proteins 441
inhibitory G protein (G
i
) 441
calmodulin (CaM) 444
Ca
2+
/calmodulin-dependent protein
kinases (CaM kinases I–IV) 444
two-component signaling
systems 452
receptor His kinase 452
response regulator 452
receptorlike kinase (RLK) 455
hormone response element
(HRE) 465
tamoxifen 465
RU486 466
cyclin 467
cyclin-dependent protein kinase (CDK)
467
ubiquitin 469
proteasome 469
growth factors 469
cytokine 469
retinoblastoma protein (pRb) 470
oncogene 471
tumor suppressor genes 472
programmed cell death 473
apoptosis 473
Terms in bold are defined in the glossary.
8885d_c12_474 2/20/04 1:30 PM Page 474 mac76 mac76:385_reb:
Chapter 12 Further Reading 475
Further Reading
General
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health and disease: the Sir Hans Krebs Medal Lecture. Eur. J.
Biochem. 268, 5001–5010.
An intermediate-level review of the role of protein kinases, their
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Cohen, P. (2000) The regulation of protein function by multisite
phosphorylation—a 25 year update. Trends Biochem. Sci. 25,
596–601.
Historical account of the developments in protein
phosphorylation.
Heilmeyer, L. & Friedrich, P. (eds) (2001) Protein Modules in
Cellular Signalling, IOS Press, Washington, DC.
Receptor Ion Channels
See also Chapter 11, Further Reading, Ion Channels.
Aidley, D.J. & Stanfield, P.R. (1996) Ion Channels: Molecules
in Action, Cambridge University Press, Cambridge.
Clear, concise introduction to the physics, chemistry, and
molecular biology used in research on ion channels; emphasis is
on molecular approaches.
Lehmann-Horn, F. & Jurkat-Rott, K. (1999) Voltage-gated ion
channels and hereditary disease. Physiol. Rev. 79, 1317–1372.
Advanced review of the structure and function of ion channels,
with special emphasis on cases in which ion-channel defects
produce human diseases.
Receptor Enzymes
Foster, D.C., Wedel, B.J., Robinson, S.W., & Garbers, D.L.
(1999) Mechanisms of regulation and functions of guanylyl
cyclases. Rev. Physiol. Biochem. Pharmacol. 135, 1–39.
Advanced review of the structure and function of signal-
transducing guanylyl cyclases.
Saltiel, A.R. & Pessin, J.E. (2002) Insulin signaling pathways in
time and space. Trends Biochem Sci. 12, 65–71.
Short, intermediate-level review.
Schaeffer, H.J. & Weber, M.J. (1999) Mitogen-activated protein
kinases: specific messages from ubiquitous messengers. Mol. Cell.
Biol. 19, 2435–2444.
Intermediate-level review of MAPKs and the basis for specific
signaling through these general signaling proteins.
Schlessinger, J. (2000) Cell signaling by receptor tyrosine
kinases. Cell 103, 211–225.
Shepherd, P.R., Withers, D.J., & Siddle, K. (1998) Phospho-
inositide 3-kinase: the key switch mechanism in insulin signalling.
Biochem. J. 333, 471–490.
Intermediate-level review of the importance of PKB and PI-3K
in metabolic regulation by insulin.
Shields, J.M., Pruitt, K., McFall, A., Shaub, A., & Der, C.J.
(2000) Understanding Ras: it ain’t over ’til it’s over. Trends Cell
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Intermediate-level review of the monomeric G protein Ras.
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kinase module from yeast to human. Physiol. Rev. 79, 143–180.
Advanced review of the roles of MAPKs in diverse organisms,
from yeast, slime mold, and nematode to vertebrates and plants.
Zajchowski, L.D. & Robbins, S.M. (2002) Lipid rafts and little
caves: compartmentalized signalling in membrane microdomains.
Eur. J. Biochem. 269, 737–752.
Serpentine Receptors
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sors for activated signaling proteins. Curr. Opin. Cell Biol. 14,
167–172.
Brief, intermediate-level review.
Hamm, H.E. (1998) The many faces of G protein signaling.
J. Biol. Chem. 273, 669–672.
Introduction to a series of short reviews on G proteins.
Lodowski, D.T., Pitcher, J.A., Capel, W.D., Lefkowitz, R.J., &
Tesmer, J.J.G. (2003) Keeping G proteins at bay: a complex be-
tween G protein–coupled receptor kinase 2 and G
H9252H9253
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Martin, T.F.J. (1998) Phosphoinositide lipids as signaling mole-
cules. Annu. Rev. Cell Dev. Biol. 14, 231–264.
Discussion of the roles of phosphatidylinositol derivatives in
signal transduction, cytoskeletal regulation, and membrane
trafficking.
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heptahelical receptor signaling and regulation. Trends Cell Biol.
12, 130–138.
Role of arrestins in serpentine receptor adaptation.
Pinna, L.A. & Ruzzene, M. (1996) How do protein kinases rec-
ognize their substrates? Biochim. Biophys. Acta 1314, 191–225.
Advanced review of the factors, including consensus sequences,
that give protein kinases their specificity.
Roach, P.J. (1991) Multisite and hierarchal protein phosphoryla-
tion. J. Biol. Chem. 266, 14,139–14,142.
Report on the importance of multiple phosphorylation sites in
the fine regulation of protein function.
Skiba, N.P. & Hamm, H.E. (1998) How G
sH9251
activates adenylyl
cyclase. Nat. Struct. Biol. 5, 88–92.
Intermediate-level review of the mechanism of G-protein action,
based on structural studies.
Zaccolo, M., De Giorgi, F., Cho, C.Y., Feng, L., Knapp, T.,
Negulescu, P.A., Taylor, S.S., Tsien, R.Y., & Pozzan, T. (2000)
A genetically encoded, fluorescent indicator for cyclic AMP in liv-
ing cells. Nat. Cell Biol. 2, 25–29.
Describes the technique shown in Figure 5 of Box 12–2.
Zhang, J., Campbell, R.E., Ting, A.Y., & Tsien, R.Y. (2002)
Creating new fluorescent probes for cell biology. Nat. Rev. Molec.
Cell Biol. 3, 906–918.
The basis for techniques such as those described in Box 12–2.
Scaffold Proteins and Membrane Rafts
See also Chapter 11, Further Reading, Membrane Dynamics.
Filipek, S., Stenkamp, R.E., Teller, D.C., & Palczewski, K.
(2003) G protein-coupled receptor rhodopsin: A prospectus.
Annu. Rev. Physiol. 65, 851–879.
Advanced review.
8885d_c12_475 2/20/04 1:30 PM Page 475 mac76 mac76:385_reb:
Chapter 12 Biosignaling476
Lim, W.A. (2002) The modular logic of signaling proteins: building
allosteric switches from simple binding domains. Curr. Opin.
Struct. Biol. 12, 61–68.
Michel, J.J.C. & Scott, J.D. (2002) AKAP mediated signal
transduction. Annu. Rev. Pharmacol. Toxicol. 42, 235–257.
Advanced review of the proteins that target PKA to subcellular
compartments.
Neel, B.G., Gu, H., & Pao, L. (2003) The “Shp”ing news: SH2
domain–containing tyrosine phosphatases in cell signaling. Trends
Biochem. Sci. 28, 284–293.
Pawson, T. & Nash, P. (2003) Assembly of cell regulatory systems
through protein interaction domains. Science 300, 445–452.
Intermediate-level review of multivalent proteins and the com-
plexes they form.
Saltiel, A.R. & Pessin, J.E. (2002) Insulin signaling pathways in
time and space. Trends Cell Biol. 12, 65–71.
Siegal, G. (1999) The surprisingly flexible PTB domain. Nat.
Struct. Biol. 6, 7–10.
Tsui-Pierchala, B.A., Encinas, M., Milbrandt, J., & Johnson,
E.M., Jr. (2002) Lipid rafts in neuronal signaling and function.
Trends Neurosci. 25, 412–417.
Yaffe, M.D. & Elia, A.E.H. (2001) Phosphoserine/threonine-
binding domains. Curr. Opin. Cell Biol. 13, 131–138.
Calcium Ions in Signaling
Berridge, M.J. (1993) Inositol triphosphate and calcium sig-
nalling. Nature 361, 315–325.
Classic description of the IP
3
signaling system.
Berridge, M.J., Lipp, P., & Bootman, M.D. (2000) The versatil-
ity and universality of calcium signaling. Nat. Rev. Molec. Cell
Biol. 1, 11–21.
Intermediate review.
Chin, D. & Means, A.R. (2000) Calmodulin: a prototypical
calcium receptor. Trends Cell Biol. 10, 322–328.
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signaling. J. Cell Sci. 115, 3715–3716.
A comprehensive poster of all elements of Ca
2H11001
signaling.
Takahashi, A., Camacho, P., Lechleiter, J.D., & Herman, B.
(1999) Measurement of intracellular calcium. Physiol. Rev. 79,
1089–1125.
Advanced review of methods for estimating intracellular Ca
2H11001
levels in real time.
Thomas, A.P., Bird, G.S.J., Hajnoczky, G., Robb-Gaspers,
L.D., & Putney, J.W., Jr. (1996) Spatial and temporal aspects of
cellular calcium signaling. FASEB J. 10, 1505–1517.
Signaling in Plants and Bacteria
Bakal, C.J. & Davies, J.E. (2000) No longer an exclusive club:
eukaryotic signaling domains in bacteria. Trends Cell Biol. 10,
32–38.
Intermediate review.
Becraft, P.W. (2002) Receptor kinase signaling in plant develop-
ment. Annu. Rev. Cell Dev. Biol. 18, 163–192.
Advanced review.
Chang, C. & Stadler, R. (2001) Ethylene hormone receptor
action in Arabidopsis. Bioessays 23, 619–627.
Cock, J.M., Vanoosthuyse, V., & Gaude, T. (2002) Receptor
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with mechanistic similarities. Curr. Opin. Cell Biol. 14, 230–236.
Hwang, I., Chen, H.-C., & Sheen, J. (2002) Two-component
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413, 383–389.
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Advanced review.
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Vision, Olfaction, and Gustation
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Chapter 12 Problems 477
1. Therapeutic Effects of Albuterol The respi-
ratory symptoms of asthma result from constriction
of the bronchi and bronchioles of the lungs due to contrac-
tion of the smooth muscle of their walls. This constriction can
be reversed by raising the [cAMP] in the smooth muscle. Ex-
plain the therapeutic effects of albuterol, a H9252-adrenergic ag-
onist taken (by inhalation) for asthma. Would you expect this
drug to have any side effects? How might one design a bet-
ter drug that did not have these effects?
2. Amplification of Hormonal Signals Describe all the
sources of amplification in the insulin receptor system.
3. Termination of Hormonal Signals Signals carried by
hormones must eventually be terminated. Describe several
different mechanisms for signal termination.
4. Specificity of a Signal for a Single Cell Type Dis-
cuss the validity of the following proposition. A signaling
molecule (hormone, growth factor, or neurotransmitter) elic-
its identical responses in different types of target cells if they
contain identical receptors.
5. Resting Membrane Potential A variety of unusual in-
vertebrates, including giant clams, mussels, and polychaete
worms, live on the fringes of hydrothermal vents on the ocean
bottom, where the temperature is 60 H11034C.
(a) The adductor muscle of a deep-sea giant clam has a
resting membrane potential of H1100295 mV. Given the intracellu-
lar and extracellular ionic compositions shown below, would
you have predicted this membrane potential? Why or why not?
(b) Assume that the adductor muscle membrane is per-
meable to only one of the ions listed above. Which ion could
determine the V
m
?
Concentration (mM)
Ion Intracellular Extracellular
Na
H11001
50 440
K
H11001
400 20
Cl
H11002
21 560
Ca
2H11001
0.4 10
Problems
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Apoptosis
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Chapter 12 Biosignaling478
6. Membrane Potentials in Frog Eggs Fertilization of
a frog oocyte by a sperm cell triggers ionic changes similar
to those observed in neurons (during movement of the
action potential) and initiates the events that result in cell
division and development of the embryo. Oocytes can be
stimulated to divide without fertilization by suspending them
in 80 mM KCl (normal pond water contains 9 mM KCl).
(a) Calculate how much the change in extracellular
[KCl] changes the resting membrane potential of the oocyte.
(Hint: Assume the oocyte contains 120 mM K
H11001
and is perme-
able only to K
H11001
.) Assume a temperature of 20 H11034C.
(b) When the experiment is repeated in Ca
2H11001
-free wa-
ter, elevated [KCl] has no effect. What does this suggest about
the mechanism of the KCl effect?
7. Excitation Triggered by Hyperpolarization In most
neurons, membrane depolarization leads to the opening of
voltage-dependent ion channels, generation of an action po-
tential, and ultimately an influx of Ca
2H11001
, which causes release
of neurotransmitter at the axon terminus. Devise a cellular
strategy by which hyperpolarization in rod cells could pro-
duce excitation of the visual pathway and passage of visual
signals to the brain. (Hint: The neuronal signaling pathway in
higher organisms consists of a series of neurons that relay
information to the brain (see Fig. 12–31). The signal released
by one neuron can be either excitatory or inhibitory to the
following, postsynaptic neuron.)
8. Hormone Experiments in Cell-Free Systems In the
1950s, Earl W. Sutherland, Jr., and his colleagues carried out
pioneering experiments to elucidate the mechanism of action
of epinephrine and glucagon. Given what you have learned in
this chapter about hormone action, interpret each of the ex-
periments described below. Identify substance X and indicate
the significance of the results.
(a) Addition of epinephrine to a homogenate of normal
liver resulted in an increase in the activity of glycogen phos-
phorylase. However, if the homogenate was first centrifuged
at a high speed and epinephrine or glucagon was added to
the clear supernatant fraction that contains phosphorylase,
no increase in the phosphorylase activity occurred.
(b) When the particulate fraction from the centrifuga-
tion in (a) was treated with epinephrine, substance X was
produced. The substance was isolated and purified. Unlike
epinephrine, substance X activated glycogen phosphorylase
when added to the clear supernatant fraction of the cen-
trifuged homogenate.
(c) Substance X was heat-stable; that is, heat treatment
did not affect its capacity to activate phosphorylase. (Hint:
Would this be the case if substance X were a protein?) Sub-
stance X was nearly identical to a compound obtained when
pure ATP was treated with barium hydroxide. (Fig. 8–6 will
be helpful.)
9. Effect of Cholera Toxin on Adenylyl Cyclase
The gram-negative bacterium Vibrio cholerae pro-
duces a protein, cholera toxin (M
r
90,000), that is responsi-
ble for the characteristic symptoms of cholera: extensive loss
of body water and Na
H11001
through continuous, debilitating di-
arrhea. If body fluids and Na
H11001
are not replaced, severe de-
hydration results; untreated, the disease is often fatal. When
the cholera toxin gains access to the human intestinal tract
it binds tightly to specific sites in the plasma membrane of
the epithelial cells lining the small intestine, causing adenyl-
yl cyclase to undergo prolonged activation (hours or days).
(a) What is the effect of cholera toxin on [cAMP] in the
intestinal cells?
(b) Based on the information above, suggest how cAMP
normally functions in intestinal epithelial cells.
(c) Suggest a possible treatment for cholera.
10. Effect of Dibutyryl cAMP versus cAMP on Intact
Cells The physiological effects of epinephrine should in
principle be mimicked by addition of cAMP to the target cells.
In practice, addition of cAMP to intact target cells elicits only
a minimal physiological response. Why? When the structurally
related derivative dibutyryl cAMP (shown below) is added to
intact cells, the expected physiological response is readily ap-
parent. Explain the basis for the difference in cellular re-
sponse to these two substances. Dibutyryl cAMP is widely
used in studies of cAMP function.
11. Nonhydrolyzable GTP Analogs Many enzymes can
hydrolyze GTP between the H9252 and H9253 phosphates. The GTP
analog H9252,H9253-imidoguanosine 5H11032-triphosphate Gpp(NH)p, shown
below, cannot be hydrolyzed between the H9252 and H9253 phosphates.
Predict the effect of microinjection of Gpp(NH)p into a myo-
cyte on the cell’s response to H9252-adrenergic stimulation.
12. G Protein Differences Compare the G proteins G
s
,
which acts in transducing the signal from H9252-adrenergic re-
ceptors, and Ras. What properties do they share? How do they
differ? What is the functional difference between G
s
and G
I
?
Gpp(NH)p
CH
2
O O
OH
H9253H9252( , -imidoguanosine 5H11032-triphosphate)
OH
H
HH
H
N
O
N
HN
H
2
N
N
O
H11002
P
O
O
O
H11002
P
O
H11002
O
P
O
H11002
O
H
N
Dibutyryl cAMP
(N
6
,O
2
H11032
-Dibutyryl adenosine 3H11032,5H11032-cyclic monophosphate)
(CH
2
)
2
CH
3
CH
2
C
(CH
2
)
2
CH
3
C
O
OO O
O
H
HH
H
N
NH
N
N
N
O
P
O
O
H11002
8885d_c12_478 2/20/04 2:03 PM Page 478 mac76 mac76:385_reb:
Chapter 12 Problems 479
13. EGTA Injection EGTA (ethylene glycol-bis(H9252-amino-
ethyl ether)-N,N,NH11032,NH11032-tetraacetic acid) is a chelating agent
with high affinity and specificity for Ca
2H11001
. By microinjecting
a cell with an appropriate Ca
2H11001
-EDTA solution, an experi-
menter can prevent cytosolic [Ca
2H11001
] from rising above 10
H110027
M.
How would EGTA microinjection affect a cell’s response to
vasopressin (see Table 12–5)? To glucagon?
14. Visual Desensitization Oguchi’s disease is an
inherited form of night blindness. Affected individu-
als are slow to recover vision after a flash of bright light
against a dark background, such as the headlights of a car on
the freeway. Suggest what the molecular defect(s) might be
in Oguchi’s disease. Explain in molecular terms how this de-
fect accounts for the night blindness.
15. Mutations in PKA Explain how mutations in the R or
C subunit of cAMP-dependent protein kinase (PKA) might
lead to (a) a constantly active PKA or (b) a constantly inac-
tive PKA.
16. Mechanisms for Regulating Protein Kinases Iden-
tify eight general types of protein kinases found in eukary-
otic cells, and explain what factor is directly responsible for
activating each type.
17. Mutations in Tumor Suppressor Genes and Onco-
genes Explain why mutations in tumor suppressor genes
are recessive (both copies of the gene must be defective for
the regulation of cell division to be defective) whereas mu-
tations in oncogenes are dominant.
18. Retinoblastoma in Children Explain why
some children with retinoblastoma develop multiple
tumors of the retina in both eyes, whereas others have a sin-
gle tumor in only one eye.
19. Mutations in ras How does a mutation in the ras
gene that leads to formation of a Ras protein with no GTPase
activity affect a cell’s response to insulin?
8885d_c12_479 2/20/04 1:31 PM Page 479 mac76 mac76:385_reb:
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