412 / CHAPTER 40
percentage of genes injected into a fertilized mouse ovum
will be incorporated into the genome and found in both
somatic and germ cells. Hundreds of transgenic animals
have been established, and these are useful for analysis of
tissue-specific effects on gene expression and effects of
overproduction of gene products (eg, those from the
growth hormone gene or oncogenes) and in discovering
genes involved in development—a process that hereto-
fore has been difficult to study. The transgenic approach
has recently been used to correct a genetic deficiency in
mice. Fertilized ova obtained from mice with genetic hy-
pogonadism were injected with DNA containing the
coding sequence for the gonadotropin-releasing hormone
(GnRH) precursor protein. This gene was expressed and
regulated normally in the hypothalamus of a certain
number of the resultant mice, and these animals were in
all respects normal. Their offspring also showed no evi-
dence of GnRH deficiency. This is, therefore, evidence
of somatic cell expression of the transgene and of its
maintenance in germ cells.
Targeted Gene Disruption or Knockout
In transgenic animals, one is adding one or more copies
of a gene to the genome, and there is no way to control
where that gene eventually resides. A complementary—
and much more difficult—approach involves the selec-
tive removal of a gene from the genome. Gene knock-
out animals (usually mice) are made by creating a
mutation that totally disrupts the function of a gene.
This is then used to replace one of the two genes in an
embryonic stem cell that can be used to create a het-
erozygous transgenic animal. The mating of two such
animals will, by mendelian genetics, result in a ho-
mozygous mutation in 25% of offspring. Several hun-
dred strains of mice with knockouts of specific genes
have been developed.
RNA Transcript & Protein Profiling
The “-omic” revolution of the last several years has cul-
minated in the determination of the nucleotide se-
quences of entire genomes, including those of budding
and fission yeasts, various bacteria, the fruit fly, the worm
Caenorhabditis elegans,the mouse and, most notably, hu-
mans. Additional genomes are being sequenced at an ac-
celerating pace. The availability of all of this DNA se-
quence information, coupled with engineering advances,
has lead to the development of several revolutionary
methodologies, most of which are based upon high-den-
sity microarray technology.We now have the ability to
deposit thousands of specific, known, definable DNA se-
quences (more typically now synthetic oligonucleotides)
on a glass microscope-style slide in the space of a few
square centimeters. By coupling such DNA microarrays
with highly sensitive detection of hybridized fluores-
cently labeled nucleic acid probes derived from mRNA,
investigators can rapidly and accurately generate profiles
of gene expression (eg, specific cellular mRNA content)
from cell and tissue samples as small as 1 gram or less.
Thus entire transcriptome information(the entire col-
lection of cellular mRNAs) for such cell or tissue sources
can readily be obtained in only a few days. Transcrip-
tome information allows one to predict the collection of
proteins that might be expressed in a particular cell, tis-
sue, or organ innormal and disease states based upon the
mRNAs present in those cells. Complementing this high-
throughput, transcript-profiling method is the recent de-
velopment of high-sensitivity, high-throughput mass
spectrometry of complex protein samples.Newer mass
spectrometry methods allow one to identify hundreds to
thousands of proteins in proteins extracted from very
small numbers of cells (< 1 g). This critical information
tells investigators which of the many mRNAs detected in
transcript microarray mapping studies are actually trans-
lated into protein, generally the ultimate dictator of phe-
notype. Microarray techniques and mass spectrometric
protein identification experiments both lead to the gen-
eration of huge amounts of data. Appropriate data man-
agement and interpretation of the deluge of information
forthcoming from such studies has relied upon statistical
methods; and this new technology, coupled with the
flood of DNA sequence information, has led to the de-
velopment of the field of bioinformatics,a new disci-
pline whose goal is to help manage, analyze, and inte-
grate this flood of biologically important information.
Future work at the intersection of bioinformatics and
transcript-protein profiling will revolutionize our under-
standing of biology and medicine.
• A variety of very sensitive techniques can now be ap-
plied to the isolation and characterization of genes
and to the quantitation of gene products.
• In DNA cloning, a particular segment of DNA is re-
moved from its normal environment using one of
many restriction endonucleases. This is then ligated
into one of several vectors in which the DNA seg-
ment can be amplified and produced in abundance.
• The cloned DNA can be used as a probe in one of
several types of hybridization reactions to detect
other related or adjacent pieces of DNA, or it can be
used to quantitate gene products such as mRNA.
• Manipulation of the DNA to change its structure, so-
called genetic engineering, is a key element in cloning
(eg, the construction of chimeric molecules) and can
MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY Y / 413
also be used to study the function of a certain frag-
ment of DNA and to analyze how genes are regulated.
• Chimeric DNA molecules are introduced into cells
to make transfected cells or into the fertilized oocyte
to make transgenic animals.
• Techniques involving cloned DNA are used to locate
genes to specific regions of chromosomes, to identify
the genes responsible for diseases, to study how faulty
gene regulation causes disease, to diagnose genetic
diseases, and increasingly to treat genetic diseases.
ARS:Autonomously replicating sequence; the ori-
gin of replication in yeast.
Autoradiography:The detection of radioactive
molecules (eg, DNA, RNA, protein) by visualiza-
tion of their effects on photographic film.
Bacteriophage:A virus that infects a bacterium.
Blunt-ended DNA:Two strands of a DNA duplex
having ends that are flush with each other.
cDNA:A single-stranded DNA molecule that is
complementary to an mRNA molecule and is syn-
thesized from it by the action of reverse transcrip-
Chimeric molecule:A molecule (eg, DNA, RNA,
protein) containing sequences derived from two
Clone:A large number of organisms, cells or mole-
cules that are identical with a single parental or-
ganism cell or molecule.
Cosmid:A plasmid into which the DNA sequences
from bacteriophage lambda that are necessary for
the packaging of DNA (cos sites) have been in-
serted; this permits the plasmid DNA to be pack-
aged in vitro.
Endonuclease:An enzyme that cleaves internal
bonds in DNA or RNA.
Excinuclease:The excision nuclease involved in nu-
cleotide exchange repair of DNA.
Exon:The sequence of a gene that is represented
(expressed) as mRNA.
Exonuclease:An enzyme that cleaves nucleotides
from either the 3′or 5′ends of DNA or RNA.
Fingerprinting:The use of RFLPs or repeat se-
quence DNA to establish a unique pattern of
DNA fragments for an individual.
Footprinting:DNA with protein bound is resistant
to digestion by DNase enzymes. When a sequenc-
ing reaction is performed using such DNA, a pro-
tected area, representing the “footprint” of the
bound protein, will be detected.
Hairpin:A double-helical stretch formed by base
pairing between neighboring complementary se-
quences of a single strand of DNA or RNA.
Hybridization:The specific reassociation of com-
plementary strands of nucleic acids (DNA with
DNA, DNA with RNA, or RNA with RNA).
Insert:An additional length of base pairs in DNA,
generally introduced by the techniques of recom-
binant DNA technology.
Intron:The sequence of a gene that is transcribed
but excised before translation.
Library:A collection of cloned fragments that rep-
resents the entire genome. Libraries may be either
genomic DNA (in which both introns and exons
are represented) or cDNA (in which only exons
Ligation:The enzyme-catalyzed joining in phos-
phodiester linkage of two stretches of DNA or
RNA into one; the respective enzymes are DNA
and RNA ligases.
Lines:Long interspersed repeat sequences.
Microsatellite polymorphism:Heterozygosity of a
certain microsatellite repeat in an individual.
Microsatellite repeat sequences: Dispersed or
group repeat sequences of 2–5 bp repeated up to
50 times. May occur at 50–100 thousand loca-
tions in the genome.
Nick translation:A technique for labeling DNA
based on the ability of the DNA polymerase from
E coli to degrade a strand of DNA that has been
nicked and then to resynthesize the strand; if a ra-
dioactive nucleoside triphosphate is employed, the
rebuilt strand becomes labeled and can be used as
a radioactive probe.
Northern blot:A method for transferring RNA
from an agarose gel to a nitrocellulose filter, on
which the RNA can be detected by a suitable
Oligonucleotide:A short, defined sequence of nu-
cleotides joined together in the typical phosphodi-
Ori:The origin of DNA replication.
PAC:A high capacity (70–95 kb) cloning vector
based upon the lytic E. colibacteriophage P1 that
replicates in bacteria as an extrachromosomal ele-
Palindrome:A sequence of duplex DNA that is the
same when the two strands are read in opposite di-
Plasmid:A small, extrachromosomal, circular mole-
cule of DNA that replicates independently of the
Polymerase chain reaction (PCR):An enzymatic
method for the repeated copying (and thus ampli-
fication) of the two strands of DNA that make up
a particular gene sequence.
414 / CHAPTER 40
Primosome:The mobile complex of helicase and
primase that is involved in DNA replication.
Probe:A molecule used to detect the presence of a
specific fragment of DNA or RNA in, for in-
stance, a bacterial colony that is formed from a ge-
netic library or during analysis by blot transfer
techniques; common probes are cDNA molecules,
synthetic oligodeoxynucleotides of defined se-
quence, or antibodies to specific proteins.
Proteome:The entire collection of expressed pro-
teins in an organism.
Pseudogene:An inactive segment of DNA arising
by mutation of a parental active gene.
Recombinant DNA:The altered DNA that results
from the insertion of a sequence of deoxynu-
cleotides not previously present into an existing
molecule of DNA by enzymatic or chemical
Restriction enzyme:An endodeoxynuclease that
causes cleavage of both strands of DNA at highly
specific sites dictated by the base sequence.
Reverse transcription:RNA-directed synthesis of
DNA, catalyzed by reverse transcriptase.
RT-PCR:A method used to quantitate mRNA lev-
els that relies upon a first step of cDNA copying of
mRNAs prior to PCR amplification and quantita-
Signal:The end product observed when a specific
sequence of DNA or RNA is detected by autoradi-
ography or some other method. Hybridization
with a complementary radioactive polynucleotide
(eg, by Southern or Northern blotting) is com-
monly used to generate the signal.
Sines:Short interspersed repeat sequences.
SNP:Single nucleotide polymorphism. Refers to
the fact that single nucleotide genetic variation in
genome sequence exists at discrete loci throughout
the chromosomes. Measurement of allelic SNP
differences is useful for gene mapping studies.
snRNA:Small nuclear RNA. This family of RNAs
is best known for its role in mRNA processing.
Southern blot:A method for transferring DNA
from an agarose gel to nitrocellulose filter, on
which the DNA can be detected by a suitable
probe (eg, complementary DNA or RNA).
Southwestern blot:A method for detecting pro-
tein-DNA interactions by applying a labeled DNA
probe to a transfer membrane that contains a rena-
Spliceosome:The macromolecular complex respon-
sible for precursor mRNA splicing. The spliceo-
some consists of at least five small nuclear RNAs
(snRNA; U1, U2, U4, U5, and U6) and many
Splicing:The removal of introns from RNA ac-
companied by the joining of its exons.
Sticky-ended DNA:Complementary single strands
of DNA that protrude from opposite ends of a
DNA duplex or from the ends of different duplex
molecules (see also Blunt-ended DNA, above).
Tandem:Used to describe multiple copies of the
same sequence (eg, DNA) that lie adjacent to one
Terminal transferase:An enzyme that adds nu-
cleotides of one type (eg, deoxyadenonucleotidyl
residues) to the 3′end of DNA strands.
Transcription:Template DNA-directed synthesis
of nucleic acids; typically DNA-directed synthesis
Transcriptome:The entire collection of expressed
mRNAs in an organism.
Transgenic:Describing the introduction of new
DNA into germ cells by its injection into the nu-
cleus of the ovum.
Translation:Synthesis of protein using mRNA as
Vector:A plasmid or bacteriophage into which for-
eign DNA can be introduced for the purposes of
Western blot:A method for transferring protein to
a nitrocellulose filter, on which the protein can be
detected by a suitable probe (eg, an antibody).
Lewin B: Genes VII.Oxford Univ Press, 1999.
Martin JB, Gusella JF: Huntington’s disease: pathogenesis and
management. N Engl J Med 1986:315:1267.
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Labora-
tory Manual.Cold Spring Harbor Laboratory Press, 1989.
Spector DL, Goldman RD, Leinwand LA: Cells: A Laboratory
Manual.Cold Spring Harbor Laboratory Press, 1998.
Watson JD et al: Recombinant DNA,2nd ed. Scientific American
Books. Freeman, 1992.
Weatherall DJ: The New Genetics and Clinical Practice,3rd ed. Ox-
ford Univ Press, 1991.
Membranes:Structure & Function
Robert K. Murray, MD, PhD, & Daryl K. Granner, MD
Biochemistry of Extracellular
& Intracellular Communication
Membranes are highly viscous, plastic structures.
Plasma membranes form closed compartments around
cellular protoplasm to separate one cell from another
and thus permit cellular individuality. The plasma
membrane has selective permeabilitiesand acts as a
barrier, thereby maintaining differences in composition
between the inside and outside of the cell. The selective
permeabilities are provided mainly by channels and
pumpsfor ions and substrates. The plasma membrane
also exchanges material with the extracellular environ-
ment by exocytosis and endocytosis, and there are spe-
cial areas of membrane structure—the gap junctions—
through which adjacent cells exchange material. In
addition, the plasma membrane plays key roles in cell-
cell interactionsand in transmembrane signaling.
Membranes also form specialized compartments
within the cell. Such intracellular membranes help
shape many of the morphologically distinguishable
structures (organelles), eg, mitochondria, ER, sarcoplas-
mic reticulum, Golgi complexes, secretory granules,
lysosomes, and the nuclear membrane. Membranes lo-
calize enzymes,function as integral elements in excita-
tion-response coupling, and provide sites of energy
transduction,such as in photosynthesis and oxidative
Changesin membrane structure (eg caused by is-
chemia) can affect water balance and ion flux and there-
fore every process within the cell. Specific deficiencies
or alterations of certain membrane components lead to
a variety of diseases(see Table 41–5). In short, normal
cellular function depends on normal membranes.
MAINTENANCE OF A NORMAL INTRA-
& EXTRACELLULAR ENVIRONMENT
IS FUNDAMENTAL TO LIFE
Life originated in an aqueous environment; enzyme re-
actions, cellular and subcellular processes, and so forth
have therefore evolved to work in this milieu. Since
mammals live in a gaseous environment, how is the
aqueous state maintained? Membranes accomplish this
by internalizing and compartmentalizing body water.
The Body’s Internal Water
Water makes up about 60% of the lean body mass of
the human body and is distributed in two large com-
This compartment constitutes two-thirds of total body
water and provides the environment for the cell (1) to
make, store, and utilize energy; (2) to repair itself;
(3)to replicate; and (4) to perform special functions.
This compartment contains about one-third of total
body water and is distributed between the plasma and
interstitial compartments. The extracellular fluid is a
delivery system. It brings to the cells nutrients (eg, glu-
cose, fatty acids, amino acids), oxygen, various ions and
trace minerals, and a variety of regulatory molecules
(hormones) that coordinate the functions of widely sep-
arated cells. Extracellular fluid removes CO
416 / CHAPTER 41
Ratio of protein to lipid
Ratio of protein to lipid in different
membranes. Proteins equal or exceed the quantity of
lipid in nearly all membranes. The outstanding excep-
tion is myelin, an electrical insulator found on many
products, and toxic or detoxified materials from the im-
mediate cellular environment.
The Ionic Compositions of Intracellular
& Extracellular Fluids Differ Greatly
As illustrated in Table 41–1, the internal environment
is rich in K
, and phosphate is its major
anion. Extracellular fluidis characterized by high Na
content, and Cl− is the major anion. Note
also that the concentration of glucose is higher in extra-
cellular fluid than in the cell, whereas the opposite is
true for proteins. Why is there such a difference? It is
thought that the primordial sea in which life originated
was rich in K
. It therefore follows that en-
zyme reactions and other biologic processes evolved to
function best in that environment—hence the high
concentration of these ions within cells. Cells were
faced with strong selection pressure as the sea gradually
changed to a composition rich in Na+ and Ca2
changes would have been required for evolution of a
completely new set of biochemical and physiologic ma-
chinery; instead, as it happened, cells developed barri-
ers—membranes with associated “pumps”—to main-
tain the internal microenvironment.
MEMBRANES ARE COMPLEX
STRUCTURES COMPOSED OF LIPIDS,
PROTEINS, & CARBOHYDRATES
We shall mainly discuss the membranes present in eu-
karyotic cells, although many of the principles de-
scribed also apply to the membranes of prokaryotes.
The various cellular membranes have different compo-
sitions, as reflected in the ratio of protein to lipid (Fig-
ure 41–1). This is not surprising, given their divergent
functions. Membranes are asymmetric sheet-like en-
closed structures with distinct inner and outer surfaces.
These sheet-like structures are noncovalent assemblies
that are thermodynamically stable and metabolically ac-
tive. Numerous proteins are located in membranes,
where they carry out specific functions of the organelle,
the cell, or the organism.
The Major Lipids in Mammalian
Membranes Are Phospholipids,
Glycosphingolipids, & Cholesterol
Of the two major phospholipid classes present in mem-
branes, phosphoglyceridesare the more common and
consist of a glycerol backbone to which are attached
two fatty acids in ester linkage and a phosphorylated al-
cohol (Figure 41–2). The fatty acid constituents are
usually even-numbered carbon molecules, most com-
monly containing 16 or 18 carbons. They are un-
branched and can be saturated or unsaturated. The sim-
plest phosphoglyceride is phosphatidic acid, which is
Table 41–1. . Comparison of the mean
concentrations of various molecules outside and
inside a mammalian cell.
MEMBRANES: STRUCTURE & FUNCTION N / 417
A phosphoglyceride showing the fatty
), glycerol, and phosphorylated alcohol
components. In phosphatidic acid, R
1,2-diacylglycerol 3-phosphate, a key intermediate in
the formation of all other phosphoglycerides (Chapter
24). In other phosphoglycerides, the 3-phosphate is es-
terified to an alcohol such as ethanolamine, choline,
serine, glycerol, or inositol (Chapter 14).
The second major class of phospholipids is com-
posed of sphingomyelin,which contains a sphingosine
backbone rather than glycerol. A fatty acid is attached
by an amide linkage to the amino group of sphingosine,
forming ceramide. The primary hydroxyl group of
sphingosine is esterified to phosphorylcholine. Sphin-
gomyelin, as the name implies, is prominent in myelin
The amounts and fatty acid compositions of the var-
ious phospholipids vary among the different cellular
The glycosphingolipids (GSLs) are sugar-containing
lipids built on a backbone of ceramide; they include
galactosyl- and glucosylceramide (cerebrosides) and the
gangliosides. Their structures are described in Chapter
14. They are mainly located in the plasma membranes
The most common sterol in membranes is cholesterol
(Chapter 14), which resides mainly in the plasma mem-
branes of mammalian cells but can also be found in
lesser quantities in mitochondria, Golgi complexes, and
nuclear membranes. Cholesterol intercalates among the
phospholipids of the membrane, with its hydroxyl
group at the aqueous interface and the remainder of the
molecule within the leaflet. Its effect on the fluidity of
membranes is discussed subsequently.
All of the above lipids can be separated from one an-
other by techniques such as column, thin layer, and
gas-liquid chromatography and their structures estab-
lished by mass spectrometry.
Each eukaryotic cell membrane has a somewhat dif-
ferent lipid composition, though phospholipids are the
major class in all.
Membrane Lipids Are Amphipathic
All major lipids in membranes contain both hydropho-
bic and hydrophilic regions and are therefore termed
“amphipathic.” Membranes themselves are thus am-
phipathic. If the hydrophobic regions were separated
from the rest of the molecule, it would be insoluble in
water but soluble in oil. Conversely, if the hydrophilic
region were separated from the rest of the molecule, it
would be insoluble in oil but soluble in water. The am-
phipathic nature of a phospholipid is represented in
Figure 41–3. Thus, the polar head groups of the phos-
pholipids and the hydroxyl group of cholesterol inter-
face with the aqueous environment; a similar situation
applies to the sugar moieties of the GSLs (see below).
Saturated fatty acids s have straight tails, whereas
unsaturated fatty acids,which generally exist in the cis
form in membranes, make kinked tails (Figure 41–3).
As more kinks are inserted in the tails, the membrane
becomes less tightly packed and therefore more fluid.
Detergentsare amphipathic molecules that are impor-
tant in biochemistry as well as in the household. The
molecular structure of a detergent is not unlike that of a
phospholipid. Certain detergents are widely used to sol-
ubilize membrane proteins as a first step in their purifi-
cation. The hydrophobic end of the detergent binds to
Polar head group
Apolar, hydrocarbon tails
Diagrammatic representation of a
phospholipid or other membrane lipid. The polar head
group is hydrophilic, and the hydrocarbon tails are hy-
drophobic or lipophilic. The fatty acids in the tails are
saturated (S) or unsaturated (U); the former are usually
attached to carbon 1 of glycerol and the latter to car-
bon 2. Note the kink in the tail of the unsaturated fatty
acid (U), which is important in conferring increased
418 / CHAPTER 41
Diagram of a section of a bilayer mem-
brane formed from phospholipid molecules. The unsat-
urated fatty acid tails are kinked and lead to more spac-
ing between the polar head groups, hence to more
room for movement. This in turn results in increased
membrane fluidity. (Slightly modified and reproduced,
with permission, from Stryer L: Biochemistry, 2nd ed. Free-
hydrophobic regions of the proteins, displacing most of
their bound lipids. The polar end of the detergent is
free, bringing the proteins into solution as detergent-
protein complexes, usually also containing some resid-
Membrane Lipids Form Bilayers
The amphipathic character of phospholipids suggests
that the two regions of the molecule have incompatible
solubilities; however, in a solvent such as water, phos-
pholipids organize themselves into a form that thermo-
dynamically serves the solubility requirements of both
regions. A micelle (Figure 41–4) is such a structure;
the hydrophobic regions are shielded from water, while
the hydrophilic polar groups are immersed in the aque-
ous environment. However, micelles are usually rela-
tively small in size (eg, approximately 200 nm) and
thus are limited in their potential to form membranes.
As was recognized in 1925 by Gorter and Grendel, a
bimolecular layer,or lipid bilayer,can also satisfy the
thermodynamic requirements of amphipathic mole-
cules in an aqueous environment. Bilayers, not mi-
celles, are indeed the key structures in biologic mem-
branes. A bilayer exists as a sheet in which the
hydrophobic regions of the phospholipids are protected
from the aqueous environment, while the hydrophilic
regions are immersed in water (Figure 41–5). Only the
ends or edges of the bilayer sheet are exposed to an un-
favorable environment, but even these exposed edges
can be eliminated by folding the sheet back upon itself
to form an enclosed vesicle with no edges. A bilayer can
extend over relatively large distances (eg, 1 mm). The
closed bilayer provides one of the most essential proper-
ties of membranes. It is impermeable to most water-
soluble molecules, since they would be insoluble in the
hydrophobic core of the bilayer.
Lipid bilayers are formed by self-assembly,driven
by the hydrophobic effect. When lipid molecules
come together in a bilayer, the entropy of the surround-
ing solvent molecules increases.
Two questions arise from consideration of the
above. First, how many biologic materials are lipid-
solubleand can therefore readily enter the cell? Gases
such as oxygen, CO
, and nitrogen—small molecules
with little interaction with solvents—readily diffuse
through the hydrophobic regions of the membrane.
The permeability coefficients of several ions and of a
number of other molecules in a lipid bilayer are shown
in Figure 41–6. The three electrolytes shown (Na
) cross the bilayer much more slowly than
water. In general, the permeability coefficients of small
molecules in a lipid bilayer correlate with their solubili-
ties in nonpolar solvents. For instance, steroids more
readily traverse the lipid bilayer compared with elec-
trolytes. The high permeability coefficient of water it-
self is surprising but is partly explained by its small size
and relative lack of charge.
The second question concerns molecules that are
not lipid-soluble:How are the transmembrane concen-
tration gradients for non-lipid-soluble molecules main-
tained? The answer is that membranes contain proteins,
Diagrammatic cross-section of a mi-
celle. The polar head groups are bathed in water,
whereas the hydrophobic hydrocarbon tails are sur-
rounded by other hydrocarbons and thereby pro-
tected from water. Micelles are relatively small (com-
pared with lipid bilayers) spherical structures.
MEMBRANES: STRUCTURE & FUNCTION N / 419
Permeability coefficient (cm/s)
Permeability coefficients of water,
some ions, and other small molecules in lipid bilayer
membranes. Molecules that move rapidly through a
given membrane are said to have a high permeability
coefficient. (Slightly modified and reproduced, with per-
mission, from Stryer L: Biochemistry,2nd ed. Freeman,
and proteins are also amphipathic molecules that can be
inserted into the correspondingly amphipathic lipid bi-
layer. Proteins form channels for the movement of ions
and small molecules and serve as transporters for larger
molecules that otherwise could not pass the bilayer.
These processes are described below.
Membrane Proteins Are Associated
With the Lipid Bilayer
Membrane phospholipidsact as a solvent for mem-
brane proteins, creating an environment in which the
latter can function. Of the 20 amino acids contributing
to the primary structure of proteins, the functional
groups attached to the αcarbon are strongly hydropho-
bic in six, weakly hydrophobic in a few, and hy-
drophilic in the remainder. As described in Chapter 5,
the α-helical structure of proteins minimizes the hy-
drophilic character of the peptide bonds themselves.
Thus, proteins can be amphipathic and form an inte-
gral part of the membrane by having hydrophilic re-
gions protruding at the inside and outside faces of the
membrane but connected by a hydrophobic region tra-
versing the hydrophobic core of the bilayer. In fact,
those portions of membrane proteins that traverse
membranes do contain substantial numbers of hy-
drophobic amino acids and almost invariably have ei-
ther a high α-helical or β-pleated sheet content. For
many membranes, a stretch of approximately 20 amino
acids in an αhelix will span the bilayer.
It is possible to calculate whether a particular se-
quence of amino acids present in a protein is consistent
with a transmembrane location.This can be done by
consulting a table that lists the hydrophobicities of each
of the 20 common amino acids and the free energy val-
ues for their transfer from the interior of a membrane
to water. Hydrophobic amino acids have positive val-
ues; polar amino acids have negative values. The total
free energy values for transferring successive sequences
of 20 amino acids in the protein are plotted, yielding a
so-called hydropathy plot.Values of over 20 kcal⋅mol
are consistent with—but do not prove—a transmem-
Another aspect of the interaction of lipids and pro-
teins is that some proteins are anchored to one leaflet or
another of the bilayer by covalent linkages to certain
lipids.Palmitate and myristate are fatty acids involved
in such linkages to specific proteins. A number of other
proteins (see Chapter 47) are linked to glycophos-
phatidylinositol (GPI) structures.
Different Membranes Have Different
The number of different proteins s in a membrane
varies from less than a dozen in the sarcoplasmic reticu-
lum to over 100 in the plasma membrane. Most mem-
brane proteins can be separated from one another using
sodium dodecyl sulfate polyacrylamide gel electro-
phoresis (SDS-PAGE), a technique that has revolution-
ized their study. In the absence of SDS, few membrane
proteins would remain soluble during electrophoresis.
Proteins are the major functional moleculesof mem-
branes and consist of enzymes, pumps and channels,
structural components, antigens (eg, for histocompati-
bility), and receptors for various molecules. Because
every membrane possesses a different complement of
proteins, there is no such thing as a typical membrane
structure. The enzymatic properties of several different
membranes are shown in Table 41–2.
Membranes Are Dynamic Structures
Membranes and their components are dynamic struc-
tures.The lipids and proteins in membranes undergo
turnover there just as they do in other compartments of
the cell. Different lipids have different turnover rates,
and the turnover rates of individual species of mem-
brane proteins may vary widely. The membrane itself
can turn over even more rapidly than any of its con-
stituents. This is discussed in more detail in the section
Membranes Are Asymmetric Structures
This asymmetry can be partially attributed to the irreg-
ular distribution of proteins within the membranes. An
inside-outside asymmetryis also provided by the ex-
ternal location of the carbohydrates attached to mem-
brane proteins. In addition, specific enzymes are lo-
420 / CHAPTER 41
Table 41–2. . Enzymatic markers of different
GlcNAc transferase I
Golgi mannosidase II
Inner mitochondrial membrane e ATP synthase
Membranes contain many proteins, some of which have enzy-
matic activity. Some of these enzymes are located only in certain
membranes and can therefore be used as markers to follow the
purification of these membranes.
TGN, trans golgi network.
cated exclusively on the outside or inside of mem-
branes, as in the mitochondrial and plasma membranes.
There are regional asymmetries in membranes.
Some, such as occur at the villous borders of mucosal
cells, are almost macroscopically visible. Others, such as
those at gap junctions, tight junctions, and synapses,
occupy much smaller regions of the membrane and
generate correspondingly smaller local asymmetries.
There is also inside-outside (transverse) asymmetry
of the phospholipids.The choline-containing phos-
pholipids (phosphatidylcholine and sphingomyelin)
are located mainly in the outer molecular layer; the
aminophospholipids (phosphatidylserine and phos-
phatidylethanolamine) are preferentially located in the
inner leaflet. Obviously, if this asymmetry is to exist at
all, there must be limited transverse mobility (flip-flop)
of the membrane phospholipids. In fact, phospholipids
in synthetic bilayers exhibit an extraordinarily slow
rate of flip-flop;the half-life of the asymmetry can be
measured in several weeks. However, when certain
membrane proteins such as the erythrocyte protein gly-
cophorin are inserted artificially into synthetic bilayers,
the frequency of phospholipid flip-flop may increase as
much as 100-fold.
The mechanisms involved in the establishment of
lipid asymmetry are not well understood. The enzymes
involved in the synthesis of phospholipids are located
on the cytoplasmic side of microsomal membrane vesi-
cles. Translocases (flippases)exist that transfer certain
phospholipids (eg, phosphatidylcholine) from the inner
to the outer leaflet. Specific proteins that preferen-
tially bindindividual phospholipids also appear to be
present in the two leaflets, contributing to the asym-
metric distribution of these lipid molecules. In addi-
tion, phospholipid exchange proteins recognize specific
phospholipids and transfer them from one membrane
(eg, the endoplasmic reticulum [ER]) to others (eg, mi-
tochondrial and peroxisomal). There is further asym-
metry with regard to GSLsand also glycoproteins;the
sugar moieties of these molecules all protrude outward
from the plasma membrane and are absent from its
Membranes Contain Integral
& Peripheral Proteins
It is useful to classify membrane proteins into two
types: integraland peripheral. Most membrane pro-
teins fall into the integral class, meaning that they inter-
act extensively with the phospholipids and require the
use of detergents for their solubilization. Also, they gen-
erally span the bilayer. Integral proteins are usually
globular and are themselves amphipathic. They consist
of two hydrophilic ends separated by an intervening hy-
drophobic region that traverses the hydrophobic core of
the bilayer. As the structures of integral membrane pro-
teins were being elucidated, it became apparent that
certain ones (eg, transporter molecules, various recep-
tors, and G proteins) span the bilayer many times (see
Figure 46–5). Integral proteins are also asymmetrically
distributed across the membrane bilayer. This asym-
metric orientation is conferred at the time of their in-
sertion in the lipid bilayer. The hydrophilic external re-
gion of an amphipathic protein, which is synthesized
on polyribosomes, must traverse the hydrophobic core
of its target membrane and eventually be found on the
outside of that membrane. The molecular mechanisms
involved in insertion of proteins into membranes and
the topic of membrane assembly are discussed in Chap-
Peripheral proteins do not interact directly with
the phospholipids in the bilayer and thus do not require
use of detergents for their release. They are weakly
bound to the hydrophilic regions of specific integral
proteins and can be released from them by treatment
with salt solutions of high ionic strength. For example,
ankyrin, a peripheral protein, is bound to the integral
protein “band 3” of erythrocyte membrane. Spectrin, a
cytoskeletal structure within the erythrocyte, is in turn
bound to ankyrin and thereby plays an important role
in maintenance of the biconcave shape of the erythro-
cyte. Many hormone receptor molecules are integral
proteins, and the specific polypeptide hormones that
bind to these receptor molecules may therefore be con-
sidered peripheral proteins. Peripheral proteins, such as
polypeptide hormones, may help organize the distribu-
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