Membranes membrane associated with an inside and an outside.

Membranes play a central role in both the structure
and function of all cells, prokaryotic and eukaryotic, plant and animal.
Membranes basically define compartments, each membrane associated with an
inside and an outside. If this were all they did, membranes would be
considerably less interesting than they are. But, membranes not only define
compartments, they also determine the nature of all communication between the
inside and outside. This may take the form of actual passage of ions or
molecules between the two compartments (in and out) or may be in the form of
information, transmitted through conformational changes induced in membrane
components. In addition, attached to membranes are many cellular enzymes. Some
of these enzymes catalyze transmembrane reactions, involving reactants on both
sides of the membrane or molecular transport. Others are involved in sequential
reactions involving a series of enzymes which are concentrated in the plane of
the membrane, thus facilitating efficient interactions. Still other enzymes
have membrane-bound substrates and/or are involved in the maintenance or
biosynthesis of the membrane. Most of the fundamental biochemical functions in
cells involve membranes at some point, including such diverse processes as
prokaryotic DNA replication  protein
biosynthesis, protein secretion, bioenergetics, and hormonal responses.

 (1) Plasma
membrane: The plasma membrane defines the boundaries of the cell and is the
point of contact between the cell and its environment. As such, the plasma
membrane contains specialized components involved in intercellular contacts and
communication, honnonal response, and transport of both small and large
molecules into and out of the cell. However, the plasma membrane is itself
divided into specialized regions in those cells which are simultaneously in
contact with different environments. Figure 1.2 shows the location of the
apical and basolateral plasma membrane domains for a hepatocyte and for a
polarized epithelial cell. The apical membrane is that which is in contact with
the “external” environment, such as the bile canaliculus in the case
of the liver cell or the gastrointestinal lumen for an epithelial cell in the
gut. The apical membrane can contain specialized structures such as the microvilli,
which can be organized to forrn the brush border membranes in some absorptive
cells. Microvilli greatly increase the effective surface area of the membrane
and facilitate efficient transport. The basolateral membrane is that which is
in contact with other cells (lateral or contiguous membrane) or blood sinusoids
(sinusoidal membrane). In the hepatocyte, the lateral and sinusoidal membranes
are morphologically and biochemically separable

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The Importance and Diversity of Membranes


 Figure 1.1.
Schematic showing organelles of eukaryotic animal and plant cells as revealed
by electron microscopy.


 Membrane Isolation

There has been a growing appreciation over the past 3
decades of the enormous number of cellular functions which are
membrane-dependent. Both plant and animal cells are highly compartmentalized,
and many of the cytoplasmic organelles are membranous, as outlined. In addition
to the organelles indicated for the generalized case, there are specialized
membranous systems such as the sarcoplasmic reticulum of muscle cells, the
myelin sheath around peripheral nerve axons, the chloroplast thylakoid
membrane, and the disk membranes of the rod cells in the retina. Prokaryotic
organisms also have membranes, although the internal elaborations found in the
eukaryotic systems are generally not present. Gram-positive bacteria such as
Bacillus subtilis have a single cytoplasmic membrane, whereas gram-negative
bacteria such as Escherichia coli have, in addition, an unusual outer membrane
external to a thin peptidoglycan cell wall Some specialized organelles are also
found in prokaryotic organisms, notably the chromatophore containing the
photosynthetic apparatus in purple non-sulfur bacteria such as Rhodobacter
sphaeroides. Some animal cell viruses, known as enveloped viruses, also are
surrounded by true membranes, and these have been quite useful systems for

Most studies on membranes require as a prerequisite
the purification of the particular membrane to be examined. Each system
presents unique preparative problems. For example, if one is interested in
studying the plasma membrane of a particular cell population (e.g., hepatocytes
of the liver) it is obviously advantageous to first isolate these cells from
the whole tissue. Then, one must consider the optimal procedures for cell
disruption and for physically separating the membrane of interest from the
other cellular components. An important consideration is the criterion used to
assess the purity of the membranes obtained.


It is desirable to select a procedure which will
effectively disrupt the cell without destroying the membrane structure to be
isolated. In the case of many animal cells, relatively gentle procedures can be
used, such as a Dounce or Potter—Elvehjem glass—Teflon type of tissue
homogenizer. This disrupts the cells by shear forces by forcing the suspension
through a narrow gap between a Teflon plunger and the glass wall of the
apparatus. This should strip off the plasma membrane and sever the connections
between the various organelles but still maintain the integrity of the
individual organelles. Specialized regions of the plasma membrane, such as the
basolateral and apical membranes of epithelial cells, can also be severed by
these procedures. It is usually desirable to work under conditions in which the
organelles remain intact to minimize release of hydrolytic enzymes (e.g., from
lysosomes) and to optimize subsequent separation procedures.

Harsher procedures are required to disrupt cells which
have walls, such as bacteria, fungi, and plant cells. Sometimes the cells are
pre-treated with degradative enzymes prior to physical disruption to assist in
breaking the cell wall. For example, Tris-EDTA and lysozyme treatments can be
used when disrupting E. coli (1102). The more harsh disruption techniques rely
on grinding, sonication, and extrusion. Grinding is usually done in the
presence of an abrasive such as sand, alumina, or glass beads. Small-scale work
can be done with a mortar and pestle, but mechanical devices can also be used.
Sonication is often used for breaking bacterial cells. Presumably, this
technique works by creating shear forces in solution produced by cavitation.
Shear forces are also produced by extruding the cell suspension through a small
orifice, e.g., as occurs with the French press. There are many variations on
these techniques and the choice will depend on the particular system being

It should be noted that disrupted and fragmented
membranes will usually spontaneously form vesicles. Examplesinclude (l)
microsomes derived from plasma membrane, endoplasmic reticulum, or specialized
systems such as the sarcoplasmic membrane of muscle cells; (2) submitochondrial
particles, from inner mitochondrial membrane; (3) synaptosomes, derived from
pinched-off nerve ends at synaptic junctions; and (4) bacterial membrane
vesicles (Kaback vesicles) from the cytoplasmic membrane of E. coli. Other
membrane systems such as Golgi also vesiculate. In most cases, the size of the
vesicles is critically dependent on the method used to disrupt the cells. Since
the vesicle size in large part determines the sedimentation rate (see next
section) and behavior in subsequent purification steps, the disruption step is
of obvious importance. Some membranes do not form vesicles, notably the lateral
or contiguous membranes of animal cells (see Figure 1.2), which are stripped
off as pairs of adjacent membranes derived from neighboring cells, held
together by junctions. The presence of these junctions prevents vesicle
formation, and these membranes are isolated as sheets or ribbon-like structures

The choice of medium used for cell disruption can also
be important. For example, in order to maintain the structure of sealed
membranous organelles, it is important to use a breakage medium which is
iso-osmotic with the organelle interior. Sucrose (0.25—0.30 M) is most commonly
used for this purpose, but sorbitol and mannitol are also utilized and in some
cases favored It should be noted that the subsequent preparative steps for
intact organelles are usually critically dependent on the maintenance of
isotonic conditions.

Composition of Membranes

The major components of membranes are proteins and
lipid. Carbohydrate may comprise as much as 10% of the weight of some
membranes, but the carbohydrate is invariably in the form of glycolipid or
glycoprotein. The relative amounts of protein and lipid vary significantly, ranging
from about 20% (dry weight) protein (myelin) to 80% protein (mitochondria).
Tables 1.3 and 1.4 summarize the com-


      Membranes           Major proteins      L/P (w/w)          Major


Basic protein










PS 8.5%
SM 8.5%
Ganglioside 26%

Disk membranes







PE 39%
Trace of cholesterol


Band 3


PC 25%




PE 22%












Cholesterol 25%

Rectal gland

Na+/K+-ATPase (1)


PC 50.4%

plasma membrane



PE 35.5%




PS 8.4%
PI 0.5%
SM 5.7%




PC 24%




PE 23%




PS 9.6%

(Torpedo marmorata)



Cholesterol 40%








PE 12.6%




PI 8.1%
Cholesterol 10%

E. coli











CL 3%

Purple membrane







Glycolipids 30%

Neutral lipids 6%


position of a number of membranes. The density of a
membrane is directly proportional to the amount of protein in the membrane.
Higher protein composition results in increased density as determined by
isopycnic centrifugation.

To some extent, the protein components associated with
a membrane will depend on the procedures used to isolate the membrane. A number
of proteins are not strongly associated with membranes and can easily be
removed by such procedures as washing at high or low ionic strength, washing at
alkaline pH, or including a chelator such as EDTA in the buffer. In some cases,
it is difficult to distinguish proteins which should properly be considered as
membrane components from cytoplasmic proteins which may bind adventitiously to the
membrane surface during the isolation procedures.

 Membrane Lipids

The most striking feature of membrane lipids is their
enormous diversity. The reason for the diversity is not at all clear, although
there is an increasing awareness of the multiple roles of lipids in membranes
(see Section 1.52). Certainly the major role of membrane lipids is to form the
bilayer matrix with which the proteins interact. The major lipid classes are
pictured in Figure 1.8 and are briefly discussed below.


These are the most commonly found membrane lipids. One
of the glycerol hydroxyls is linked to a polar phosphate-containing group and
the other two hydroxyls are linked to hydrophobic groups. Glyceride
nomenclature is often in terns of the stereospecific numbering (sn) system.
When the glycerol is drawn in a Fischer projection, with the hydroxyl in the
middle drawn to the left, the positions are numbered as shown in Figure 1.9,
and the prefix sn- is used before the name (e.g., sn-3 position). Several different
stereochemical conventions are used: sn, D/L, and R/S. Figure 1.9 also
illustrates the stereochemistry about carbon atom C-2 in the three conventions
(see ref. 604). Natural phospholipids generally have the R (or D)

Most phosphoglycerides have the phosphate at the sn-3
position of glycerol. The phosphate is usually linked to one of the several
groups as indicated in Figure 1.10, including choline, ethanolamine,
myo-inositol, serine, and glycerol.

The long-chain hydrocarbons attached to sn-l and sn-2
positive may be attached through ester or ether linkages. The chains themselves
vary widely in terms of length, branching, and degree of unsaturation.

(a)   1
,2-Diacylphosphoglycerides or phospholipids. These fatty acid esters of
glycerol are the predominant lipids in most eukaryotic and prokaryotic
membranes, excluding archaebacteria .Phosphatidylcholine is a major component
in animal cell membranes, and phosphatidylethanolamine is often a major
component in bacterial membranes. Table I .5 lists a number of the more common








—GlcNAc—Gal ganglioside

                                                        NeuNAc NeuNAc

            OH                                     sterol

Figure 1.8. The structures of some classes of membrane
lipids. The structures are drawn so as to emphasize the amphipathic nature of
the lipids, with nonpolar groups on the left and polar moieties on the right.
Gal, galactose; GIC, glucose; NeuNAc, N-acetylneuraminic acid



1 .52 Lipids Play Multiple Roles Within Membranes

Although the distribution of lipids in various
membranes does not seem to be random, there is no satisfying
explanation for the observed patterns. Any single membrane can contain well
over 100 unique lipid species. Why are there so many and why does each membrane
have a unique distribution of lipids? The biosynthesis of membrane lipids and
the mechanisms by which they are distributed to different membranes are
discussed in Section 10.4. However, the reasons for the heterogeneity are not
known, although lipids are increasingly being recognized as active participants
in membrane-associated processes. Several factors can be considered.

1.         Minimally
the lipid mixture must form a stable bilayer in which the proteins can
function. This is discussed in the next chapter.

2.         Some
lipids may be required because their shapes favor packing configurations that
may be necessary to stabilize regions of high curvature, junctions between
membranes, or optimal interactions with specific proteins This polymorphic
aspect of membrane lipids is discussed in the next chapter.

3.         Some
lipids are important as regulatory agents. Most notable are the derivatives of
phosphatidylinositol in the plasma membranes of eukaryotic cells

4.         Some
lipids participate in biosynthetic pathways. For example, in E. coli
phosphatidylglycerol provides the glycerol phosphate moiety in the biosynthesis
of periplasmic oligosaccharides).

5.         Specific
lipids may be required for optimal enzyme activity of particular enzymes. This
topic is addressed in.

6.         Gangliosides,
in particular, have been implicated as playing a role in the regulation of cell
growth, in binding to specific receptors in the plasma membrane, and in

7.         Other
lipid components are also known to play specialized roles. These include the
polyisoprenoids, such as dolichol, ubiquinones, menaquinones, and carotenoids,
and the platelet activating factor .

 Membrane Proteins

As shown in Tables 1.3 and 1.4, membranes contain
between 20% and 80% (w/w) protein. It is the proteins, of course, which are the
biochemically active components of the membrane and provide the diversity of
enzymes, transporters, receptors, pores, etc. which distinguishes each
particular membrane. Progress in our understanding of membrane proteins was
initiated when biochemists learned to use detergents to solubilize these
proteins from membranes in biochemically active forms. Initially, success was
with the enzyme complexes of the mitochondrial inner membrane. The realization
that membrane proteins were not predominantly ß-pleated sheet, as postulated to
best fit the Davson—Danielli—Robertson “unit membrane” model, but
contained significant amounts of a-helix, was a significant step forward. Also
important was the insight that membrane proteins extended deeply into or
completely through the lipid bilayer and were stabilized by hydrophobic
interactions. This thermodynamic argument was essentially an extension of the
principles of the “hydrophobic force” being developed to understand
protein structure, i.e., nonpolar hydrophobic interior and polar hydrophilic
exterior in contact with water.

As techniques for membrane protein purification were
developed, more membrane proteins were obtained in homogeneous form. The
insoluble characteristics of most membrane proteins and of the hydrophobic
peptides derived from them made primary structure determination difficult.
Progress on two membrane proteins, glycophorin and cytochrome 4, helped to
establish the structural themes which have been dominant since the mid-1970s.
The amino acid sequence of glycophorin, a sialoglycoprotein from the
erythrocyte membrane, indicated a short stretch of 23 nonpolar amino acids near
the middle of the molecule.Topological and other studies indicated that
glycophorin extended completely through the erythrocyte membrane and that this
hydrophobic stretch was in an a-helical form and was buried in the membrane .
This work contributed to the now firmly entrenched concept of membrane-spanning
ahelical domains in proteins. Electron microscopy image reconstruction studies
on bacteriorhodopsin in the purple membrane of Halobacterium halobium and X-ray
diffraction studies on bacterial photosynthetic reaction centers have provided
the highest resolution data available for membrane-spanning proteins. These
proteins consist of a series of a-helical segments traversing the bilayer

The major point is that membrane proteins are now
generally viewed as being folded so as to present a nonpolar hydrophobic
surface which can interact with the nonpolar portions of the lipid bilayer.
Polar or charged regions of the protein can interact with the lipid headgroups
at the surface of the bilayer. Many membrane proteins are transmembranous and
extend through the bilayer. Other membrane proteins are probably bound to the
membrane exclusively through interactions with other proteins.

Membrane proteins are generally bound to the membrane
through noncovalent forces, such as the hydrophobic force or electrostatic
interactions There is, however, a small but growing number of examples of
membrane proteins which are covalently bound to lipids. Many of the proteins in
plant or animal plasma membranes are glycoproteins, such as glycophorin. The
carbohydrate residues are always located on the extracytoplasmic side of the

Operationally, membrane proteins are classified as
extrinsic (or peripheral) or intrinsic (or integral). This generally denotes
the degree of harshness of the treatment required to release the protein from
the membrane. Extrinsic proteins are dislodged by washing the membranes in low
ionic strength buffer, in a buffer at low or high pH, and/or in the presence of
a divalent cation chelator such as EDTA (1438). Such proteins are thought to be
weakly bound to the membrane surface by electrostatic interactions either with
the lipid headgroups or with other proteins. It is often hard to distinguish an
extrinsic membrane protein from a cytoplasmic protein which has become
adventitiously bound to the membrane during the isolation procedure. As much as
30% of the proteins associated with the erythrocyte membrane is solubilized by
treatment at low ionic strength. Somewhat harsher treatments to release
extrinsic proteins involve the use of chaotropic agents such as C104- or SCN-
These reagents are, in some cases, strong enough to disrupt some
protein-protein interactions but are not sufficently strong to denature the
individual polypeptides. Chaotropics or “water structure breakers”
act by effectively reducing the magnitude of the hydrophobic force

Treatments involving detergents or, occasionally,
organic solvents are required to release intrinsic membrane proteins. The
detergents disrupt the lipid bilayer and presumably bind to the membrane
proteins at the nonpolar binding sites normally in contact with the bilayer
interior. Intrinsic membrane proteins require the continued presence of
detergents to remain in a soluble, monodisperse form. Removal of the detergent
invariably results in the formation of high-molecularweight protein aggregates
and, usually, precipitation.



The cell membrane is crucial to the survival of every
cell type. It provides a barrier between the inside and outside environment of
the cell, and plays an important role in cell to cell signaling and
communication. It is composed primarily of phospholipids and proteins, and the
exact composition of the cell membrane varies depending on the cell type.
Although there is a wide variety of cell types which exist in nature, one
uniting feature they share is the cell membrane.