Table Of ContentSurface Membranes of Specific
Cell Types
Edited by
G. A. Jamieson Ph.D., D.SC
Research Director
American Red Cross Blood Research Laboratory
Bethesda, Maryland, USA
and
Adjunct Professor of Biochemistry
Georgetown University Schools of Medicine and Dentistry
Washington, DC, USA
and
D. M. Robinson Ph.D.
Professor of Biology, Georgetown University
and
Member, Vincent T. Lombardi Cancer Research Center
Georgetown University Schools of Medicine and Dentistry
Washington, DC, USA
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First published 1977
© Butterworth & Co (Publishers) Ltd 1977
ISBN 0 408 70773 9
Library of Congress Cataloging in Publication Data (Revised)
Main entry under title:
Mammalian cell membranes.
Includes bibliographical references and index.
CONTENTS: v. 1. General concepts, v. 2. The
diversity of membranes, v. 3. Surface membranes of
specific cell types, v. 4. Membranes and cellular functions.
1. Mammals—Cytology. 2. Cell membranes.
1. Jamieson, Graham A., 1929- II. Robinson,
David Mason, 1932- [DNLM: 1. Cell membrane.
2. Mammals. QH601 M265]
QL739.15.M35 599/.08/75 75-33317
ISBN 0-408-70773-9
Filmset and printed Offset Litho in Great Britain by
Cox & Wyman Ltd, London, Fakenham and Reading
Contributors
C. R. AUSTIN
Physiological Laboratory, University of Cambridge, Cambridge, CB3 2EG,
England
LUIGI M. DE LUCA
Differentiation Control Section, Lung Cancer Branch, National Cancer
Institute, National Institutes of Health, Bethesda, Maryland 20014, USA
CATHERINE HICKEY-WILLIAMS
American National Red Cross Blood Research Laboratory, 9312 Old
Georgetown Road, Bethesda, Maryland 20014, USA
G. A. JAMIESON
American National Red Cross Blood Research Laboratory, 9312 Old
Georgetown Road, Bethesda, Maryland 20014, USA
MARTI JETT
American National Red Cross Blood Research Laboratory, 9312 Old
Georgetown Road, Bethesda, Maryland 20014, USA
ROBERT J. MCLEAN
Department of Biological Sciences, State University College, Brockport,
New York 14420, USA
WINIFRED G. NAYLER
Cardiothoracic Institute, 2 Beaumont Street, London, WIN 2DX, England
C. A. PASTERNAK
Department of Biochemistry, St George's Hospital Medical School,
University of London, Blackshaw Road, Tooting, London, SW17 0QT,
England
JERE P. SEGREST
Departments of Pathology and Biochemistry, University of Alabama in
Birmingham, The Medical Center, Birmingham, Alabama 35294, USA
DAVID F. SMITH
Biochemisches Institut der Universität Freiburg im Breisgau, D78 Freiburg
IBR, West Germany
LIST OF CONTRIBUTORS
G. R. STRICHARTZ
Department of Physiology and Biophysics, State University of New York at
Stony Brook, Stony Brook, New York 11794, USA
EARL F. WALBORG, JR.
Department of Biochemistry, The University of Texas System Cancer Center,
M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77025, USA
Preface
This series on 'MAMMALIAN CELL MEMBRANES' represents an attempt to bring
together broadly based reviews of specific areas so as to provide as compre
hensive a treatment of the subject as possible. We sought to avoid producing
another collection of raw experimental data on membranes, rather have we
encouraged authors to attempt interpretation, where possible, and to express
freely their views on controversial topics. Again, we have suggested that
authors should not pay too much attention to attempts to avoid all overlap
with fellow contributors in the hope that different points of view will provide
greater illumination of controversial topics. In these ways, we hope that
the series will prove readable for specialists and generalists alike.
The first volume, entitled General Concepts, served to introduce the subject
and covered the essential aspects of physical and chemical studies which
have contributed to our present knowledge of membrane structure and
function. The second volume, The Diversity of Membranes, was concerned
with specific types of intra- and extracellular membranes. This third
volume, Surface Membranes of Specific Cell Types, as its title indicates,
reviews the knowledge that we have of the surface membranes of the various
cell types which have been studied in any detail to this time. Membranes and
Cellular Functions will be covered in Volume 4, which will deal with ultra-
structural, biochemical and physiological aspects. Since the cell surface
represents the point of interaction with the cellular environment, Volume 5,
entitled Responses of Plasma Membranes, addresses itself to the way in
which external influences are mediated by the plasma membrane.
As editors, our approach to our responsibilities has been rather permissive.
With regard to nomenclature and useful abbreviations, we have used 'cell
surfaces' and 'plasma membranes' where appropriate rather than 'cell
membranes' since this last is nonspecific. Both British and American usage
and spelling have been utilized depending upon personal preference of the
authors and editors with, again, no attempt at rigid adherence to a particular
style.
While the title of the series is 'MAMMALIAN CELL MEMBRANES', we have
encouraged authors to introduce concepts and techniques from non-
mammalian systems which may be useful in their application to eukaryotic
cells. The aim of this series is to provide a background of information and,
hopefully, a stimulation of interest to those investigators working in, or
about to enter, this burgeoning field.
Finally, the editors would like to acknowledge the dedication and resource
fulness of their secretary and editorial assistant, Mrs Alice R. Scipio, in the
coordination and preparation of these volumes.
G. A. JAMIESON
D. M. ROBINSON
1
The erythrocyte: topomolecular
anatomy of MN-glycoprotein
Jere P. Segrest
Departments of Pathology and Biochemistry, University of Alabama
in Birmingham, The Medical Center, Birmingham, Alabama
1.1 INTRODUCTION
Biological membranes represent thin sheets of aqueous discontinuity that
provide a complex barrier to a variety of ionic and polar molecules. Lipids
and proteins, in approximately equal proportions, form the bulk of the dry
weight of most isolated membranes, such as red. cell ghosts (Korn, 1969).
Two key problems concerning membranes that are, as yet, only partially
resolved are the precise organization of the lipids and proteins of biological
molecules into aqueous barriers and the relationship of this organization to
membrane function.
1.1.1 Lipid organization in biological membranes
Artificial phospholipid bilayers have many of the properties of membranes.
It is reasonably clear now, on the basis of X-ray diffraction studies of lipid
model membrane systems (Levine, Bailey and Wilkins, 1968) and of mem
branes themselves, both in vivo and in vitro (Engelman, 1971), that phospho
lipid bilayers form a significant portion of the structure of most biological
membranes.
The formation of the phospholipid bilayer can be best understood by
consideration*of the closely related phenomenon of micelle formation.
Molecules such as sodium dodecyl sulfate which have both a polar or charged
end and a nonpolar or hydrophobic end (i.e. are amphipathic) in general
tend to form spherical- or ellipsoidal-shaped aggregates when suspended in
aqueous media (Tanford, 1974). These aggregates or micelles are composed
of the amphipathic molecules oriented with their hydrophobic ends directed
1
2 THE ERYTHROCYTE! TOPOMOLECULAR ANATOMY OF MN-GLYCOPROTEIN
toward the micelle center and away from the surrounding water, and their
polar ends directed outward and exposed to the water.
The driving forces in micelle formation are the hydrophobic interactions,
which are of considerable biological importance, being a major factor in
protein folding and the annealing of double-stranded DNA. Although our
understanding of this force is still basically qualitative, it appears that
hydrophobic interactions are the result of the propensity of water to seek
to minimize interfacial surfaces with anything other than itself or other
polar molecules (Tanford, 1973); water has a higher negative free energy
when interacting with itself or polar molecules than with nonpolar molecules.
For example, the aversion of water to an interaction with air produces surface
tension, i.e. a minimization of the water-air interfacial area.
Phospholipid molecules are amphipathic molecules. However, owing to
their geometry (their hydrophobic portions are equal to or greater in cross-
sectional area than the polar ends), they tend to form multilamellar structures
in aqueous solutions rather than simple micelles*. Each lamella or sheet
is formed of a bilayer of phospholipid molecules, with the nonpolar portion
oriented toward the middle away from the water to form an aqueous
discontinuity.
Electron spin resonance (ESR) (Kornberg and McConnell, 1971a), nuclear
magnetic resonance (NMR) (Chapman et ai, 1968) and differential thermal
calorimetry (Steim et #/., 1969), among other techniques, have provided
additional information about the physical state of membrane lipids.
The finding that the hydrocarbon interior of phospholipid bilayers, in
cluding those associated with many biological membranes, is often fluid
under appropriate conditions of temperature (McConnell, Wright and
McFarland, 1972) is having profound effects upon our understanding of
many membrane-associated phenomena, such as movement of receptor
molecules transverse to the cell surface (Frye and Edidin, 1970). Below a
certain critical temperature, called the liquid crystalline transition temperature
(Phillips, Ladbrooke and Chapman, 1970), these same bilayer interiors
become semicrystalline (i.e. have a wax-like consistency).
Factors which affect the transition temperature include the degree of
saturation and chain length of the phospholipid hydrocarbon chains. Other
lipids, especially cholesterol, also have a profound effect on the fluidity of
bilayers. From these facts alone, it is clear that the lipid composition of
membranes can profoundly influence their bulk properties [including, to
a small degree, ionic permeability (Papahadjopoulos, 1973)]. Because of the
extremely hydrophobic nature of the long hydrocarbon chains of phospho
lipid molecules, bilayers are quite stable structures.
Bretscher (1972) has proposed that the erythrocyte membrane has an
asymmetrical distribution of phospholipids across the plane of its bilayer.
In his model, phosphatidylcholine and sphingomyelin form the outer leaflet
of the erythrocyte membrane bilayer and phosphatidylethanolamine and
phosphatidylserine form the inner leaflet.
* The geometry of a sphere explains this phenomenon. The inward-directed portion of an
amphipathic molecule will pack much more easily if it has a small cross-sectional area com
pared with its polar end. A bilayer, on the other hand, should be most stable if both ends are
approximately of equal area. If lecithin is hydrolyzed to lysolecithin, with one-half the nonpolar
cross-sectional area of lecithin, micelles rather than bilayers are formed in water.
THE ERYTHROCYTE: TOPOMOLECULAR ANATOMY OF MN-GLYCOPROTEIN 3
It is reasonable to infer that the colligative properties of the phosphoHpid
molecules will largely determine the physical nature of the membrane as
a result of hydrophobic interactions, the strength of which is illustrated by
the low, almost negligible rate of phosphoHpid flip-flop (i.e. movement or
exchange of individual phosphoHpid molecules across a bilayer) observed
in bilayers by ESR studies (Kornberg and McConnell, 1971b).
1.1.2 Protein organization in biological membranes
It is apparent that knowledge of the gross physical state of lipids in biological
membranes cannot explain many of such important properties as mediation
of cell-cell interactions, the presence of specific receptors and antigenic
binding sites on cell surfaces, message transmission across membranes, and
active transport. Proteins, which represent close to 50 percent of the dry
weight of most membranes, must have an important role in these phenomena,
either directly (for example, as carrier molecules) or indirectly (for example,
by local organization of lipids). Therefore, the structure and organization
of membrane proteins relative to lipids may be the key to the understanding
of membrane function.
One approach to understanding this organization is to utilize the principle
of dominance of hydrophobic interactions. Nonpolar regions of membrane
proteins will have a strong tendency to be excluded from the aqueous phase
and to bury themselves in the membrane interior (Singer, 1971; Tanford,
1974). An even more useful principle is to assume that polar regions of
proteins, particularly if charged, will be excluded from the hydrophobic
region. Resistance to passage of charged amino acid residues into this region
of low dielectric constant will be significant even if the polar regions are
neutralized with counter-ions (Singer, 1971; Tanford, 1974). The low degree
of phosphoHpid flip-flop has its basis in this principle (Kornberg and
McConnell, 1971b). Use of these simple rules implies that knowledge of the
amino acid sequence of a membrane protein can provide important guidelines
as to the way in which it may be associated with the membrane lipids.
Although lipids may dominate the physical nature of membranes, it is
equally clear that proteins can modulate the organization, and, therefore,
the properties of the lipids of membranes and artificial bilayers. This modula
tion can be diffuse, in the way that cholesterol has a generalized effect on
phosphoHpid bilayers (Chapman, 1968), or it can be multifocal, in the way
that intramembranous particles seen by freeze-etch electron microscopy are
discrete (Branton, 1969).
An example of protein having a diffuse effect on phosphoHpid bilayer
structure is the interaction of the plasma apolipoproteins (delipidated
protein components of the Hpoproteins) with phosphoHpid vesicles in which
these vesicles undergo a morphological alteration upon addition of apo-
protein (Hoff, Morrisett and Gotto, 1973).
In a reciprocal fashion, the lipids of a lipoprotein complex such as a
membrane can affect the structure and properties of the protein moieties.
When plasma apolipoproteins (Lux et al., 1972) or the hydrophobic peptide
of the human erythrocyte membrane glycoprotein (to be discussed later)
interact with phosphoHpid vesicles, conformational changes are produced
4 THE ERYTHROCYTE: TOPOMOLECULAR ANATOMY OF MN-GLYCOPROTEIN
in these proteins. Similar conformational changes are probably involved in
the activation of enzymes such as cytochrome oxidase by association with
phospholipids (Vanderkooi et al, 1972).
The state of organization of the proteins of membranes has been studied
by a variety of physical techniques including X-ray diffraction (Blaurock,
1972), circular dichroism (CD) (Lenard and Singer, 1966), fluorescent probe
spectroscopy (Metcalfe, Metcalfe and Engelman, 1971), proton magnetic
resonance (PMR) (Glaser et al, 1970) and ESR (Tourtellotte, Branton and
Keith, 1970). In general these techniques have been unsuccessful in elucidat
ing precise details of protein organization in biological membranes because
of the statistical nature of the information gained, though they have been far
more successful in studies of membrane lipid organization.
Because of the heterogeneity and asymmetrical distribution of membrane
proteins, techniques providing average statistical values are basically un
suitable for studying most intact biological membranes. What is required to
study the topomolecular anatomy of membrane proteins in situ is the
reconstitution of membranes from well characterized, homogeneous con
stituents. This is the approach that has been utilized in the studies to be
described in this chapter.
1.1.3 Erythrocyte membrane
Membrane models generally assume a common topomolecular pattern for
all, or most, biological membranes. The accepted model for many years
was the unit membrane (Robertson, 1964), which was based in a large part
upon electron microscopic and X-ray diffraction studies of the myelin nerve
sheath. Currently the fluid-mosaic model of Singer and Nicolson (1972)
serves as the basic membrane paradigm. However, there is reason to suspect
that the topomolecular patterns of biological membranes vary, as for example
in the purple membrane (Blaurock and Stoeckenius, 1971).
It is important when investigating general principles of membrane organi
zation to select reasonably representative membranes for study. The erythro
cyte membrane, in addition to being convenient for study, has many of the
properties of the fluid-mosaic model. X-ray diffraction studies suggest that
a phospholipid bilayer is a major component of the erythrocyte membrane
(Wilkins, Blaurock and Engelman, 1971). Integral membrane proteins in the
erythrocyte can move laterally in the plane of the membrane under certain
conditions (Tillack, Scott and Marchesi, 1972).
Another advantage of using the erythrocyte membrane is that the classifica
tion and characterization of the major polypeptide chains associated with
it have begun. The classification of the major polypeptide chains of the
human erythrocyte ghost prepared by Fairbanks, Steck and Wallach (1971)
will be used here. The polypeptides are numbered from I to VI in the direction
of decreasing molecular weight as determined by polyacrylamide gel electro-
phoresis in sodium dodecyl sulfate.
Four of these proteins are designated peripheral proteins on the basis of
their solubilization by ionic manipulations, i.e. polypeptides I, II (mol. wt
in excess of 200000), polypeptide V (mol. wt 41000) and polypeptide VI
(mol. wt 36 000). Polypeptides I and II appear to be identical to the protein
THE ERYTHROCYTE: TOPOMOLECULAR ANATOMY OF MN-GLYCOPROTEIN 5
termed spectrin (Marchesi and Steers, 1968) or tektin (Masia and Ruby, 1968).
Polypeptide VI has subsequently been shown to be the enzyme glycer-
aldehyde-3-phosphate dehydrogenase (Carraway and Shin, 1972).
Three of the human erythrocyte membrane polypeptides are designated
integral proteins by Fairbanks, Steck and Wallach (1971) on the basis of
their tenacious binding to the membrane, requiring detergents for solubiliza-
tion. These are polypeptide HI (mol. wt 100000), polypeptide IV (mol. wt
77 000) and the major glycoprotein of the human erythrocyte membrane,
PAS-1 (mol. wt 30000), also known as glycophorin (Marchesi et al, 1972).
The latter glycoprotein will be referred to in this chapter as MN-glycoprotein
for reasons to be discussed.
All of the major polypeptide chains have been localized exclusively to the
inside surface of the erythrocyte membrane with the exception of polypeptide
III and MN-glycoprotein. Both of these latter appear, on the basis of labeling
(Bretscher, 1971a, b; Segrest et al, 1973), enzymatic degradation (Bender,
Garan and Berg, 1971; Kant and Steck, 1972) and amino acid sequence
analysis (Segrest et al, 1972), to span the membrane.
Studies with various cross-linking reagents have suggested that polypeptide
component III is present as a dimer (Yu and Steck, 1974) or even a tetramer
(Wang and Richards, 1974) in situ, presumably forming a portion of the
erythrocyte membrane intramembranous particles (seen by freeze-etch
electron microscopy) as the multimer penetrates the bilayer. Further, there
is evidence that one of the functions of component III is the transport of
chloride ions across the membrane (Cabantchik and Rothstein, 1972).
Polypeptide VI (glyceraldehyde-3-phosphate dehydrogenase) appears to
bind reversibly to the erythrocyte membrane (Kant and Steck, 1973). This
protein has been shown to bind in vitro to isolated component III (Yu and
Steck, 1974).
Polypeptides I and II (spectrin) have been shown to form actin-like
filaments under certain conditions in vitro (Steers and Marchesi, 1969) and
have been localized in situ to a heavy filamentous coat on the cytoplasmic
surface of the human erythrocyte membrane (Nicolson, Marchesi and Singer,
1971).
1.2 MN-GLYCOPROTEIN
The details of the topomolecular anatomic relationship of MN-glycoprotein
with the membrane are better characterized than for any other membrane
protein, integral or peripheral, including rhodopsin (Hong and Hubbell,
1972) and cytochrome b (Spatz and Strittmatter, 1971). Much of this char
5
acterization has been the result of work by Marchesi and co workers (Segrest
et al., 1972, 1973; Jackson et al., 1973). More recent work on this problem
will be discussed later in this chapter.
1.2.1 Isolation
A glycoprotein containing the MN blood group activity was independently
isolated by Winzler (1969) and Morawiecki (1964) from the human red cell
