© Elsevier/North- Holland Biomedical Press, 1981 All rights reserved No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior permission of the copyright owner ISBN for the series: 0444 80303 ISBN for the volume: 0444 80304 I Published by: Elsevier/North-Holland Biomedical Press 335, Jan van Galenstraat, P.O Box 211 Amsterdam, The Netherlands Sole distributors for the U.S.A and Canada: Elsevier/North-Holland Inc 52 Vanderbilt Avenue New York, NY loon Library of Congress Cataloging in Publication Data Main entry under title: New comprehensive biochemistry InclUdes bibliographies and indexes Contents: v , Membrane structure [etc.] Biological chemistry I Finean, J B II Michell, R H [DNLM: Membranes Anatomy and histology Wl NE372F v.l / QJ3 532.5 M3 M534] Q.D4l5.N48 574.19'2 81-3090 ISBN 0-444-80303-3 (Elsevier/North-Holland : set) AACR2 Printed in The Netherlands Membrane structure Editors J.B FINEAN and R.H MICHELL Birmingham 1981 ELSEVIER/NORTH-HOLLAND BIOMEDICAL PRESS AMSTERDAM· NEW YORK· OXFORD New Comprehensive Biochemistry Volume I General Editors A NEUBERGER London L.L.M van DEENEN Utrecht ELSEVIER/NORTH-HOLLAND BIOMEDICAL PRESS AMSTERDAM· NEW YORK OXFORD v Preface In the former series of Comprehensive Biochemistry the contributions of membranes to cellular biochemistry were considered in a volume entitled Cytochemistry (1964) in which the organelles of the cell were considered individually Since that time the study of membranes has formed one of the most rapidly expanding fields of biology, and this volume is devoted to a consideration of only one aspect of this progress, namely our current understanding of the relationship between membrane structure and function Other aspects of membrane biochemistry will be discussed in forthcoming volumes on Phospholipids and on Membrane Transport One of the outstanding features of recent research on membrane structure has been a transition from the marked polarisation of views that characterised the 1960s towards a general agreement during the 1970s that all membranes share one basic form of structural organisation The aims of this volume are to identify general features of membrane structure, to discuss in considerable detail some selected aspects that have been studied intensively in recent years, and to relate some of this molecular information to individual membrane functions We anticipate that most of our readers will already have a general knowledge of cell structure and of the roles of individual membranes and organelles in particular cell functions For those who lack this background information, we would recommend reference to brief monographs such as Membranes and their Cellular Functions (J.B Finean, R Coleman and R.H Michell, 2nd ed., 1978, Blackwell, Oxford), The Biochemistry of Cell Organelles, (R.A Reid and R.M Leech, 1980, Blackie, Glasgow and London) and Biological Membranes (R Harrison and G.G Lunt, 2nd ed., 1980, Blackie, Glasgow and London) J.B Finean R.H Michell Birmingham, August 1980 CHAPTER I Isolation, composition and general structure of membranes J.B FINEAN and R.H MICHELL Department of Biochemistry, University of Birmingham P.o Box 363, Birmingham B15 2TT, us: Historical introduction Awareness of the existence of a discrete plasma membrane at the surface of cells gradually emerged as cell biologists of the late nineteenth century observed a variety of plant cells and single cell organisms and probed their cell boundaries using both physical and chemical techniques [1-3] From his studies of the permeability of cells to a variety of non-electrolytes, Overton [3] was even able to speculate on the lipid nature of the permeability barrier The first significant chemical study of a membrane was not reported until 1925, when Gorter and Grendel [4] extracted lipid from erythrocytes and spread it as a monolayer at an air-water interface in order to compare the area that it might potentially cover with the total surface area of the original erythrocytes A fortuitous mutual cancellation of experimental errors allowed the correct conclusion that there was sufficient lipid to form a lipid bilayet over all or almost all of the surface of the cell The probability that the lipid of biological membranes exists predominantly in bilayer form was later reinforced by physical measurements (optical and electrical) made both on biological membranes and on isolated lipid (mainly phospholipid) systems [5] This has since remained the dominant theme in considerations of membrane structure The initial suggestion that protein would probably be closely associated with lipid in plasma membranes (and maybe also in other membranes) was again a speculative one based on surface tension measurements and on the spontaneous association of water-soluble proteins with monolayers of lipid spread at an air-water interface Although there was no relevant information on the protein components of membranes, Danielli and Davson proposed a general structural scheme [6] for cell membranes which featured a bilayer of lipid coated at its aqueous interfaces with layers of protein Their first suggestion that protein might penetrate into or through the lipid layer [7] was not based on any direct knowledge of membrane proteins, but was simply a speculative attempt to account for the occurrence of facilitated permeation of solutes through plasma membranes Early thoughts on membrane structure were confined to the plasma membrane Finean r Michell [eds.] Membrane structure © Elsevier/North-Holland Biomedical Press, 1981 P.B Finean and R.B Michell Fig Electron micrographs of liver cells (hepatocytes) isolated by the procedure of Seglen [37] (A) Lead citrate-stained section of cells fixed with 1% OS04 and 1% tannic acid X51000 (B) Freeze-fracture replica of unfixed cell preparation X57000 [8,9]: the more extensive elaboration of membrane-bounded compartments within the cell was not recognised until the 1950s when improvements in the preparation of thin sections of tissues for examination by electron microscopy indicated a similar Isolation, composition and general structure approximately IO~m Fig.2 Diagrammatic illustration of variety of organelles in plant and animal cells as revealed by electron microscopy (from [44]) general form for both plasma membrane and the membranes of cytoplasmic organelles (Figs I and 2) This emphasised the limitations of the Danielli and Davson membrane model [6] in accounting in structural terms for a much greater range of functions, and hence inspired the proposal of alternative structural arrange- Fig Diagram illustrating the chronological order in which the most influential models have been proposed Isolation, composition and general structure ments (Fig.3) In particular, it was realised that: (a) under appropriate conditions some lipids would adopt configurations other than a bilayer; (b) the fine detail of membrane structure as seen at high magnification in some electron micrographs appeared granular; and (c) membranes were dissociated into lipoprotein "particles" by detergent treatment This encouraged speculation, especially by biochemists, that membranes might consist of laterally aggregated arrays of globular lipoprotein "subunits" (e.g [10-12,14]) It has only been as a result of the relatively recent progress in characterisation of membrane proteins that substantial agreement on a general model of membrane structure has been reached In particular, the identification of membrane proteins in which substantial exposed regions are dominated by non-polar amino acid side chains led to the realisation that such regions would be likely to associate with hydrocarbon regions of the membrane lipid phase; parts of these proteins might therefore be inserted deep into the membrane interior This, together with an emphasis on disordered or fluid packing of the lipid hydrocarbon chains and on free lateral diffusion of membrane components, was then featured in a new membrane model, the "fluid mosaic" model proposed by Singer and Nicholson [17] in 1972 This has since been generally accepted as a more realistic expression of the general characteristics of membranes than any previous model It may well be the last of the generalisable membrane models, because experimental work on membranes has now advanced to the stage at which the structural patterns of individual membranes are being defined in some detail [18] As a result, we now know that individual membranes differ both in the spatial distributions of their molecular components and in the mobilities of these components Isolation of membranes Some studies of membrane structure can be made using membranes still organised into cells; such studies include microscopical examination of membrane organisation in cells and of membrane-cytoskeletal interactions, X-ray diffraction analysis of cells which possess ordered membrane arrays, some types of measurement of the mobilities of membrane components, and labelling experiments designed to probe the asymmetric orientations of surface membrane components However, purified membrane preparations are needed for most studies of the chemical composition or spatial organization of membranes (a) Criteria for assessing purity For studies of chemical composition, the chief criterion is that membrane preparations should be pure samples of a single type of membrane [19] but studies of membrane structure also demand that samples of isolated membranes should preserve the spatial interrelationships between different molecules that prevail in the intact, healthy cell These constraints upon the purity of membrane preparations F.B Finean and R.B Michell used for structural studies are often much more stringent than the requirements to be met by membrane preparations in which the attribute of interest is some organelle-specific function (e.g an enzyme activity) that can be adequately studied even when the membrane exhibiting it exists only as a component of a membrane mixture For membranes which contribute substantially to the total membrane complement of cells, achievement of homogeneity requires a purification of only a few-fold, and appropriate techniques may not be unduly complex or difficult to devise Many membranes, however, constitute only a very small proportion of the total mass of the parent cell, and in such cases very substantial purification (sometimes 50- to 100-fold, or even more) is required to yield a small amount of homogeneous material for analysis The monitoring of membrane purification basically consists of following the purification of the required membrane by monitoring some membrane-specific criterion, associated with the simultaneous measurement of a variety of additional criteria specific for all of the possible contaminant structures Occasionally the morphology of a particular membrane structure remains sufficiently distinctive, even after homogenisation, for electron microscopy and/or phase contrast microscopy to provide a reliable guide to purification (e.g mitochondria, rough endoplasmic reticulum, intestinal epithelial brush borders, secretory vesicles), but much more often the isolated membrane fragments not retain a morphology that is sufficiently characteristic for their unequivocal identification (e.g smooth membrane fragments may come from, among others, smooth endoplasmic reticulum, plasma membrane or Golgi complex) In most cases, therefore, the progress of the required membrane and of contaminants through a fractionation procedure is followed by the assay of a variety of membrane-specific or "marker" criteria; these are usually enzyme activities known to be confined to particular membranes in the cell under study (see, for example, [19] and [20], section of [21], Chapters 1-4 of [22]) A membrane preparation should only be adjudged "pure"; (a) when the purification achieved corresponds to that which would be estimated from consideration of the morphology of the parent cell, and (b) when the concentrations of all known contaminating membranes, as assessed by the activities of their characteristic marker enzymes, have been reduced to levelswhere it can confidently be calculated that they contribute very little of the mass of the isolated membrane preparation In going from an homogenate to an isolated subcellular fraction, such enrichment or depletion in terms of particular membranes is usually expressed in terms of Relative SpecificActivities (RSAs) of the chosen marker enzymes, these RSAs being the ratios which compare the specific activities in the final fraction(s) to the specific activities in the initial homogenates [23] In interpreting RSAs, it is essential to remember that the mass contributed by a particular stucture to an isolated fraction is a function both of the experimentally determined RSA and of the contribution of the particular organelle to 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receptor, acetylcholine a-N-acetylglucosaminidase in carbohydrate analysis 129-130 a- N-acetylhexosaminidase in carbohydrate analysis 129-130 fJ- N-acetylhexosaminidli.se in carbohydrate analysis 129-130 Acholeplasmalaidlawii accessibility to phospholipases 95 accessibility of proteins 104 lipid localization 104, 105 Iysophospholipase 97 open ghosts 91 proteolytic degradation 104 protein iodination 101, 104 temperature-sensitive ATPase 41 ai-acid glycoprotein carbohydrate structure 134 leucocyte origin 135 actin in lymphocyte 'caps' 73 molecular weight 16 in erythrocyte ghost preparations a-actinin in lymphocyte 'caps' 73 acyldextrans as FPR probes 53, 54 adenine nucleotide transporter 162 assembly 247, 248 spans membrane 217 adenylate cyclase, hormone-sensitive 164-165, 197-200 fJ-adrenergic stimulation 70 cholera toxin stimulation 199-200 collision coupling 200 glucagon stimulation 180-181, 197-200 GTP-binding component 119- 200 intestinal epithelial 75 lipid requirement 177, 180-181, 198-199 mobility of components 70, 180- 181, 198199 multiple protein components 198 temperature sensitivity 43, 180-181 transmembrane control 180-181, 198 variation in hibernation 180-181 affinity chromatography in receptor isolation 191 amino acid sequencing 216, 230 via DNA sequence 230 aminopeptidase, intestinal spans membrane 217,218 anion transporter, erythrocyte, see band ankyrin 16,17,222 A rchaebacteria, see also Halobacterium lipids 18,20 asialoglycoprotein receptor, see receptor, asialoglycoprotein ATP / ADP exchange protein, see adenine nucleotide transporter ATPase, Ca2+-, erythrocyte calmodulin control 12 energy supply 171- 172 subunit molecular weight 16 ATPase, Ca2+-, sarcoplasmic reticulum 18, 163 lipid environment 41-42 lipid requirement 177 purification 192 rotational diffusion 48,49 ATPase, F I-, see ATP synthase ATPase, Na+ /K+-, 163 in band 16,17 energy supply 171-172 erythrocyte 16,17 intestinal epithelial 75 lipid dependence 176 purification 192 reconstitution 195 spans membrane 217 variation during hibernation 180-181 ATP synthase, energy - coupling (coupling ATPase, Fj-ATPase) 207-210 assembly 247 asymmetric disposition 208-209 DCCD-sensitive proteolipid 209-210 electron microscopy of 28, 208 Fa 209-210 FI-ATPase 208-209 260 hydrophobic polypeptides 209- 210, 234 membrane sector 209-210 oligomeric structure 14, 167, 188,207-210 oligomycin-sensitivity conferring protein 209 reconstitution 210 reviews 207 and 'structural protein' 188 subunit composition 208-209 Bacillus amyloliquefaciens phospholipid disposition 108 Bacillus megaterium phospholipid disposition 97 108, 109 Bacillus subtilis phospholipase action 95 phospholipid disposition 97, 108 bacterial transformation 231 bacteriorhodopsin amino acid sequence 33, 226 233 crystal analysis 225 crystallization 230 a-helical rods 31-33, 225, 226 hexagonal packing 12 hydrophobic surfaces 233 lateral segregation 185 lipid environment 18 lipophilicity 13 molecular weight 225 proton channel 33, 234 retinal location 225 rotational diffusion 46-47 spans memhrane 217 structure 31-33, 224-225 Band I, see ankyrin Band ABO antigens 137 accessibility 112 aldolase binding 114 amino-terminus localization 246 anion transport activity 16 \7, 162 change in En(a-) cells 142, 152 crosslinking 1\2 cytoskeleton binding \6, 17, 222 degradation 1\2-114 disposition in erythrocyte membrane 112114 \7\ erythroglycan component 16 137 in glucose transport \6, 17 glyceraldehyde- 3- phosphate dehydrogenase binding 16, 17 114, 170-17\ Ii antigens 137 interaction with DIDS \7,88 intramembranous particles 113, 114, 220 large complex oligosaccharides 16, 137 lateral diffusion 67 molecular weight 16, \12-\\4 molecular heterogeneity 16 Na+ /K+-ATPase in 16,17 proteolytic fragments 112-114 rotational diffusion 48, 49 spans membrane 217 structure 113, 114 Band 4.\ association with spectrin:actin 222 in erythrocyte cytoskeleton 16,17,171 bile salts in enzyme reconstitution 194 in protein solubilization 189- \90, 191, 192 blood group antigens ABO glycolipids 140, 143, 148, 152 ABO glycoproteins 137, 143, 148 152 analysis using lectins \46- \48 in embryonal carcinoma \52 of foetal cells 152 Ii glycolipids and glycoproteins 140, 148 \52 Lewis glycolipids 140 MN glycoprotein 148 brush border hydrolases amino terminus hydrophobic 217- 218 proteolytic release 217-2\8 Ca2+ , see also A'TPase, Ca2+ and protein mobility 72 74 capping (of surface antigens) cytoskeletal control 68-69, 71-74, \42 of Forssmann antigen 142 of gangliosides \42 pharmacology of 71-74 carbohydrate membrane, see glycolipid, glycosphingolipid, glycoprotein, oligosaccharide and entries for individual glycoproteins carboline as fluorescent probe of lipids 45 cells, techniques for isolation adsorption by immunoglobulins adsorption by lectins from body fluids free-flow electrophoresis by tissue culture from tissues cellular organization diagrammatic representation centrifugation cerebroside 20 261 chaotropic ions effects on membranes II chlorosulpholipids 21 cholera toxin adenylate cyclase activation 199- 200 capping of ganglioside 142 receptors 147 cholesterol distribution 20- 21 in electron density profiles 30- 31 exchange 100 in Golgi membrane 20- 21 in influenza virus 100 and lipid lateral diffusion 53, 54 and lipid mobility 40, 43 and lipid phase behaviour 177 localization 100 in Mycoplasma gallisepticum 100, 105 in plasma membranes 20- 21 in secretory vesicle membranes 20- 21 structure 19 transbilayer movement 100 in Vesicular Stomatitis virus 100 colchicine, and capping 71- 74 coated pits 222 coat proteins of phages assembly and secretion 241 membrane-spanning sequences 232 span membrane 217 concanavalin A, see also receptor, concanavalin A as mitogen 148 chloroplast glyceroglycolipids 20 protein synthesis and assembly 234, 246, 248 ribulose bisphosphate carboxylase synthesis 246 thylakoid protein synthesis 248 clathrin binding to and 'release from vesicles 222- 223 of coated vesicles 222-223 cyclic AMP, see also adenylate cyclase binding to erythrocyte membrane 112 cytochalasin B and capping 71-74 inhibition of protein diffusion 73 cytochrome bs amphiphilic behaviour 204 crystallization 224 detergent solubility 204 extraction 223 hydrophobic 'tail' 222 insertion into lipid bilayer 223 interaction with detergent 204 intracellular distribution 203 lipid-binding domain 205 mobility 70, 187,205 molecular weight 222 protease solubilization 204 reconstitution 205-206 rotational diffusion 47 site of synthesis 248 cytochrome bs reductase amphiphilic behaviour 204 insertion into lipid vesicles 223 mobility 70, 187 molecular weight 222 reconstitution 205-207 site of synthesis 248 cytochrome c, see also cytochrome oxidase, ubiquinone: cytochrome c reductase assembly 247 asymmetry of interaction 184 as extrinsic protein 169 cytochrome oxidase assembly 247 crystals 230 disposition in membrane 182-184 immobilization of lipids 41 oligomeric structure 14 purification 227 rotational diffusion 47 subunit structure 183, 227, 228 cytochrome P450 167 hydrophobic amino terminus 248 lateral mobility 70, 187 procedure for purification rotational diffusion 47 site of synthesis 248 cytochrome P450 reductase in electron transport at endoplasmic reticulum 167, 187 cytoskeleton association with intrinsic proteins 12-13 of erythrocyte ghosts 7, 16, 17, 114,222 and protein mobility 63-69,71-74 Danielli and Davson model detergents binding to membrane proteins 14,24, 190 and Ca2+-ATPase activity 177 characteristics 189- 191 in enzyme reconstitution 194 in glycolipid degradation 131 262 for intrinsic protein isolation 12, 184- 193 diacylindocarbocyanine dyes (di I's) for estimating lipid mobility 52-58 integration into erythrocyte membrane 56-57 partition between lipid phases 54-55 as probes "for FPR 52-58 structures 53 Dictyostelium discoideum carbohydrate-binding proteins 144 differential thermal calorimetry 23 detection of phase transitions 177 differentiation of cells changes in carbohydrate 149-153 diffraction, electron; see also electron density distributions 23 of bacteriorhodopsin 32-33, 225 of ubiquinone:cytochrome c reductase 228 diffraction, neutron 23 diffraction, X-ray, see also electron density distributions assignment of phases 29-30 of chloroplast grana 28- 29 of erythrocyte membrane 24 limitations on crystal analysis 29- 30, 230 of myelin 28- 29, 31 of retinal rods 28, 29 of virus membrane 101 diphenylhexatriene (DPH) as fluorescent probe of lipids 44 diphosphatidylglycerol in Gram-positive bacteria 108 localization in mitochondrial membrane 118, 119,120 structure 19 DNA, cloning and sequencing 231 eggs, fertilization and lipid mobility 58 lipid mobility 43, 52, 57, 58 protein mobility 64, 65 electron diffraction, see diffraction, electron electron density distributions effects of cholesterol 30- 31 from electron microscopy 28 of gap junction 31 general form in membranes 29 of myelin 31 of sarcoplasmic reticulum 31 from X-ray diffraction 28-32 electron microscopy of ATP synthase 28,208 of coated vesicles 223 cytochemical application 169 of erythrocyte membranes 27, 96, 113 Fourier synthesis from 28, 32 freeze-fracture 2, 26, 27, 28, 96 of gap junction membrane 28, 225, 227 intramembranous particles 25-27, 220 of lipid bilayer 25 of Micrococcus lysodeikticus 108 negative stain 25, 26, 28, 225 after sphingomyelinase treatment 96-97 transmission 2, 25, 26 of urinary bladder membrane 26 electron spin resonance spectroscopy, see EPR spectroscopy electron transfer chains, see also mitochondrial inner membrane, endoplasmic reticulum, fatty acid desaturase, ubiquinone:cytochrome c reductase and entries under cytochromes disposition in mitochondrial membrane 182184 in drug oxidation and hydroxylation 167-168 in energy coupling 167 in fatty acid desaturation 203-207 endo-a- N-acetylgalactosaminidase attack on O-glycosidic oligosaccharides 130 in carbohydrate analysis 129- 131 endo-fJ-N-acetylglucosaminidase in carbohydrate analysis 129-131 endo-fJ-galactosidase in carbohydrate analysis 129-131 attack on ABO antigens 130 attack on band oligosaccharide 137 attack on Ii antigens 130 attack on macroglycolipid 141 resistance of H antigens 130 endoplasmic reticulum, see also entries for individual components cytochrome bs 168, 187,203-207 cytochrome bs reductase 168, 187,203-207 electron transfer chains 167-168, 186-188 epoxide hydratase 168, 187 fatty acid desaturase 187, 203- 207 glucose 6-phosphatase 163 glutathione S-transferase 168, 187 lateral segregation of proteins 187 in secretory protein synthesis 166 in synthesis of membrane proteins 143, 165, 235-245 UDP-glucuronyltransferase 168, 187 enzymes, membrane-bound, see also entries for individual enzymes 161-214 allotopy (changes upon solubilization) 174 annular lipid 186 263 in asymmetric bilayer 177-181 and information transfer 163-165 latency 172 kinetic characterisation 173-174 lateral segregation 184-187 lipid dependence 175- 177 and lipid phase state 177- 181 in membrane biosynthesis 166 as multienzyme complexes 166-168 oligomeric nature 186 problems of assay 172 purification 191-192 reconstitution 193-195 reviews 162 solubilization 174-175, 188-191 at specialized catalytic surface 165- 166 and translocation of solutes 162-163 transverse asymmetry 181- 184 variation in activation energies 177- 181 eosin as fluorescent label for proteins 46, 47-48 epiglycanin O-linked oligosaccharides 138 epoxide hydratase 168, 187 EPR spectroscopy in assessing lipid mobility 38- 52 reviews 38 saturation-transfer 46, 49 timescale 39 ergosterol 21 erythrocyte membrane, see a/so actin, ankyrin, bands and 4.1, blood group antigens, glycophorin A, glycosphingolipids, glycerophospholipids etc adsorbed cytoplasmic components 11, 169172 detergent fractionation 12 extrinsic proteins II glycoproteins 15, 16 interaction with cytoskeleton 73 isolation in different media lipid/protein ratio 10 31 [ plNMR 98 phospholipase treatment 96, 97 phospholipid disposition 115, 116 phospholipid exchange 99 protein components 15, 16, 112-114, 169-172 transbilayer movements 115 structural water 24 vesiculation II erythroglycan 16, 17 erythroleukaemic cell line K562 changes during differentiation 152 Escherichia coli accessibility of phospholipid 110 Braun's lipoprotein 110 matrix protein 225, 227 membranes 109 membrane proteins of 110, III structure of outer membrane 110 ESR spectroscopy, see EPR spectroscopy fatty acid desaturase molecular weight 222 multienzyme sequence 187,203-207 site of synthesis 248 fibroblasts, see also fibronectin effects of viral transformation 142, 149 lipid mobility 57 protein mobility 65 surface glycoprotein 142 fibronectin association with cytoskeleton 73 decrease after transformation 142 influence of tunicamycin 154 lateral diffusion 65, 71 structure of oligosaccharides 132-135 'fluid mosaic' membrane model 5, 37 fluorescence correlation spectroscopy (FeS) 5052 assessment of lipid mobility 50-52 fluorescence polarisation in assessing lipid mobility 38- 52 fluorescent antibodies, as FPR probes 61, 62, 65 fluorescent lectins, as FPR probes 61,65 FPR (fluorescence photobleaching and recovery) 52-65 flying spot variant 63 for lipid lateral mobility 52- 58 in lipid models 52-56 in natural membranes 56-65 for protein lateral mobility 58-66 pattern photobleaching 63 theory 62-63 free-flow electrophoresis of cells of membranes freeze-fracture, see electron microscopy e-fucosidase in carbohydrate analysis 129-130 G protein (of YSV) 217 assembly in vitro 238 glycosylation 245 264 modifications during transport 249 signal sequence 242 spanning sequence 232, 233 synthesis 242 galactose oxidase in carbohydrate localization 92 on Semliki Forest glycoprotein 102, 142 a-galactosidase in carbohydrate analysis 129-130 p-galactosidase in carbohydrate analysis 129-130 and signal sequence 242 galactosylcerarnide 20 structure 19 gangliosides capping of 142 as cholera toxin receptor 147 structures 19, 138, 139 as tetanus toxin receptor 147 in TSH receptor 148 gap junctions electron density profile 31 structure from electron microscopy 28, 225, 227 water channel 33 glucose oxidase in carbohydrate localization 92 glucose-e-phosphatase transport function 163 a-glucosidase in glycoprotein biosynthesis 143 P-glucuronidase uptake by liver 146 glutathione S-transferase 168, 187 glyceraldehyde-3-phosphate dehydrogenase associated with erythrocyte membrane 8, 17, 112, 170-172 effect of ouabain on binding 172 interaction with band 114, 170-171 molecular weight 16 glyceroglycolipids of Archaebacteria 20 of blue-green algae 20 of chloroplasts 20 of Halobacteria 20 glycerol cryoprotectant for freeze-fracture 28 glycerophospholipids, see also entries for individual lipids 18- 20 structures 19 glycolipids, see also glycosphingolipids localization by enzymic modification 92 localization by lectin binding 92 glycophorin A (PAS I) absence from En(a-) cells 142, 152 accessibility 112 as agglutinin 145 amino acid sequence 13 disposition in membrane 17 hydrophobic domain 13 and intramembranous particles 113 and lipid order 42 molecular weight 16 MN antigenicity 148 N-linked oligosaccharide structure 132, 135 O-linked oligosaccharide structure 137-138 spans membrane 217 spanning sequence 232, 233 tunicamycin and synthesis 154 glycoproteins, intrinsic, see also entries for individual glycoproteins 127-160 accommodation in lipid bilayer 24 biosynthesis 143, 165,238-241,245,249-251 detergent binding 14,24 general characteristics 10- 18 in intracellular membranes 142 localization by enzymatic modification 92 localization by lectin binding 92 oligosaccharides of alditol acetates 129, 131 alkaline borohydride treatment 129 analysis by GLC-mass spectrometry 131 chromium trioxide oxidation 131 'complex'-type oligosaccharides 129, 132135 digestion with endoglycosidases 128, 129131 digestion with exoglycosidases 129-130 hybrid-type oligosaccharides 137 hydrazinolysis 128 large complex oligosaccharides 129, 135137 N-linked 131-137 O-linked 137-138 periodate oxidation 131 permethylation 130-131 proteolytic release 128 trimethylsilylation 129 glycosphingolipids 20, 127-159 an tigenici ty 20 biosynthesis 143 as blood group antigens 140 changes in malignancy 127 detection with antibodies 142 extemallocation 127, 141-143 gangliosides, structures 19, 138, 139 265 large 'complex' 141 neutral, structures 138-140 in plasma membranes 20 variety 10, 137-140 glycosyltransferases in algae 165 in bacterial lipopolysaccharide synthesis 202203 in cell-cell interactions 144, 164-165 in contact inhibition 165 in embryonic tissues 165 in Golgi complex 143 in membrane protein synthesis 143, 166 in platelet aggregation 165 in secretory protein synthesis 166 glyoxysomes protein assembly 234 Golgi membranes cholesterol content 20-21 glycosylation of membrane proteins 143 glycosylation of secretory proteins 166 lactose synthetase 12 gramicidin as FPR probe 53, 54 Gram-negative bacteria 109-111 channels in outer membrane 110, 227 immunochemicallabelling III lipoprotein in outer membrane 110, III Gram-positive bacteria electrophoresis of proteins 106 phospholipid disposition 108 protein localization 106 protoplast formation 105 group translocation 162, 195- 197 growth regulation role of carbohydrate 148-149 GTP-binding protein GTPase activity 199- 200 role in adenylate cyclase 199- 200 h-2, see histocompatibility antigens haemagglutinins, viral assembly in vitro 238 crystallization and structural analysis 224 spanning sequences 232 Halobacterium halobium, see also bacteriorhodopsin, purple membrane dihydrophytyllipids 18, 22 electron diffraction and microscopy 32-33 lateral heterogeneity of membrane 185 purple membrane 10, 225 structural synthesis 31- 33 X-ray diffraction 31-32 Hansenula wingei mannan agglutination factor 143-144 hamster liver membrane enzymes during hibernation 179-181 hepatocytes, see also liver electron micrographs isolation plasma membrane enzymes 179- 181 histocompatibility antigens (H-2 and HLA) 220 assembly in vitro 238-241 association with ,82-microglobulin 220, 238 cleavage of signal sequences 239 disposition in bilayer 239 hydrophobic domain 20 intestinal epithelial 75 papain digestion 240 precursors 238 protease digestion 240 rates of diffusion 66- 67, 73 shedding into medium 73 span membrane 217 tunicamycin and biosynthesis 239, 240 HLA antigens, see histocompatibility antigens hydrophobicity, definition 23 ,8-hydroxybutyrate dehydrogenase 175 lipid requirement 175, 193 immunoglobulins amino-terminal extension 235 amino-terminal sequences 235 on B-Iymphocytes 220 in cell separation Fe portion 220 hydrophobic domain 218, 220 light chain precursor 235, 236 in membrane isolation oligosaccharide of IgG 135 spanning sequences 232 synthesis 235 transfer into endoplasmic reticulum 236 intestinal epithelium aminopeptidase 217,218 brush border hydrolases 218 glycosphingolipids 20 protein segregation in plasma membrane 7475,185 inositol lipids, see phosphatidylinositol, phosphosphingolipids intracellular organelles, see also chloroplasts, endoplasmic reticulum, glyoxysomes, Golgi complex, microsomes, mitochondria and their individual components 266 asymmetric architecture 116 orientation of isolated membrane vesicles 117 phospholipid disposition 117, 118 protein disposition 117, 118 ionic strength, and cytochrome c binding 169 influence on membrane composition 7, II, 169-170 isolation of membranes 5-9 assessment of purification 5- cen trifugal electrophoretic by phase partition by selective adsorption isoprenoid alcohol phosphokinase 200- 202 detergent activation 200- 20 I lipid selectivity 20 I lipophilicity 20 I molecular weight 201 purification in butanol 189,201 reconstitution 202 kidney tubule epithelium protein segregation in plasma membrane 75 lactoperoxidase-mediated iodination 88 of A laidlawii 105 of lipids and proteins 10I of Newcastle disease virus 102 lactose synthetase role of a-lactalbumin 12 latency, enzyme 172 as probe for asymmetric disposition 182 lectins, see also concanavalin A binding to transformed cells 147 in blood grouping 146-148 in carbohydrate analysis 92, 137, 146-148 in cell separation in glycoprotein localization 92 mammalian 145 in membrane isolation as mitogens 148 lipid, see also lipid bilayer and entries for-indioidual lipid classes annuli around proteins 186 asymmetric disposition 22, 93 in virus membranes 93 in erythrocyte membranes 114-16 bilayer, see lipid bilayer boundary layer around proteins 41-42, 186 classification and diversity 18- 22 effects on enzyme activities 175-181 environmentally induced variations 40-41, 179-191 fluid 22, 37, 168 hydrocarbon chain oscillation 40 immunochemicallocalization 101 lateral mobility 50-58, 69, 142, 168 lateral phase separation 177-180 localization by NMR 101 long range order 42 mobility assessment by NMR 38, 39, 40 assessment by EPR 38, 39 assessment by fluorescence methods 38, 40 gradient 40 perturbation by proteins 41-44 variation with temperature 43 modifications 84 chemical 92 enzymic 101 phospholipases 94 specific requirements 23, 168 techniques for localization 84 lipid bilayer, black lipid membranes for functional reconstitution 195 as membrane model 22-23 in liposomes 22-23 in lipid multibilayers 22- 23 non-bilayer lipid configurations 22, 120 in natural membranes 1,21-23,85 transbilayer movement 85 of cholesterol 100 in erythrocyte membrane 115, 116 induced by membrane modification 86 in intracellular membranes 120 of phospholipids 100 in protoplasts 109 lipophilicity, definition 23 lipopolysaccharide, bacterial biosynthesis 166,202-203 immunochemicallocalization 101 reconstitution of biosynthesising complexes 202-203 liposomes 22, 23 definition 23 lipid mobility in 50- 55 as membrane models 22-23 for reconstitution of enzymes 193- 195 liver, see also hepatocytes asialoglycoprotein receptor 134-135 glycoprotein uptake 145 protein segregation in endoplasmic reticulum 185-188 267 protein segregation in plasma membrane 7475, 185 lymphocyte carbohydrate of B- and T- 149 glycolipids during differentiation 153 membrane agglutinin 145 mitogenic transformation 148-150 lysosomal hydrolases role of mannose 6-phosphate 145-146 uptake by liver 145-146 malignant transformation and fibroblast glycolipids 142, 149 and fibronectin content 142 maltose-binding protein signal sequence 243 a-mannosidase in carbohydrate analysis 129-130 in glycoprotein synthesis 143 ,8-mannosidase in carbohydrate analysis 129-130 mast cells lipid mobility 57 protein mobility 65 matrix protein (E coli) 225- 227 amino acid sequence 233 crystallization 230 hydrophobic sequences 233 membrane-bound compartments 215 exchange between 215, 221 membrane structure, models of Danielli and Davson 'fluid mosaic' historical summary 1-5 lipoprotein subunit micelles, definition 23 lipid-detergent 190 Micrococcus lysodeikticus antigens 108 ferritin-labelled anti-ATPase 108 freeze-fracture microscopy 108 immunological analysis 106, 107 lactoperoxidase-mediated iodination 108 lipid exchange 100 membrane-bound enzymes 108 protein disposition 106, 107 ,82 -microglobulin assembly 238, 241 in histocompatibility antigens 238 N-terrninal extension 238 precursor 238 synthesis 237, 238 microsomes, see also endoplasmic reticulum phospholipid disposition 119 mitochondria protein coding and assembly 234, 246 mitochondrial inner membrane, see also cytochrome c, cytochrome oxidase, NADH oxidase, ATP synthase, ubiquinone:cytochrome c reductase, adenine nucleotide transporter, electron transfer chains, etc ATP synthase 207-210 crystallized F\-ATPase 224 cytochrome oxidase subunits 227 in hibernation 179- 181 lateral distribution of proteins 186 lipid:protein ration 10, II phospholipid disposition 118, 119 transverse disposition of respiratory chain 182-184 multienzyme sequences 166-168 enhanced catalytic efficiency 166 Mycoplasma gallisepticum cholesterol disposition 100 Mycoplasma hominis phospholipase resistance 95 myelin basic protein 13 electron density profile 31 glycosphingolipids 20 lipid:protein ratio 10, II myoblasts lipid mobility 57 protein mobility 65 myosin in lymphocyte 'caps' 73 myotubes acetylcholine receptor mobility 75 lipid mobility 57 NADH diaphorase erythrocyte membrane 112 NADH oxidase, mitochondrial lipid requirement 176 transverse disposition 182-184 neuraminidase in carbohydrate analysis 129-130 in carbohydrate localization 92 crystallization of viral 224 in glycolipid localization 103 versus SFV glycoprotein 102 structure of viral 224 268 neuroblastoma cells lipid mobility during differentiation 57, 58 NAD nucleosidase erythrocyte membrane 112 NMR spectroscopy in assessing lipid mobility 38-52 chemical shift 98, 101 of erythrocyte membranes 98, 99 of intracellular membranes 120 and lipid disposition 101 peak broadening 101 reviews 38 timescale 39 5'-nucleotidase on bile canaliculi 75 during hibernation 180 Ochromonas danica chlorosulpholipids 21 oligosaccharides, structure and analysis, see entries under glycoproteins and glycosphingolipids organic solvents in enzyme solubilization 189 orosomucoid, see aI-acid glycoprotein ovalbumin mannose-rich oligosaccharides 136 oxidoreductases cytochemical localization 169 PAS-I, see glycophorin A PEP:sugar phospho transferase 195-197 components 196 phosphatidylglycerol requirement 197 reconstitution 197 vectorial phosphorylation by 196 periodic acid-Schiff stain 14 peroxisomes protein assembly 234 phagolysosomes 117, 118 phosphatase, alkaline intestinal epithelial 75 phosphatases cytochemical localization 169 phosphatidylcholine associated immobilized water 24 in bacteriorhodopsin:PC vesicles 47 diffusion of diI's in 53 diffusion of gramicidin and phage protein in 53 cholesterol and lateral diffusion 53 exchangeability in membranes 99 exchange protein 100 FPR experiments in 63 gel-liquid crystal phase transition 54, 55, 63 hydrolysis by phospholipase 97 lateral diffusion 54, 55, 57 localization in erythrocyte 115, 116 localization in eukaryotic membranes 118, 119 localization by [13 ClNMR 10I localization in Semliki Forest virus 103 probe in plasma membranes 57 required by ,B- hydroxybutyrate dehydrogenase 175, 193 and sarcoplasmic reticulum ATPase 49 structure 19 transbilayer movement 115 phosphatidylethanolamine accessibility in Semliki Forest virus 103 in bacteria 109 and bacterial lipopolysaccharide synthesis 202-203 induced transbilayer movement 98 labelling 93, 94 localization in bacteria 97, 100, 108, 109 localization in erythrocyte membranes 115, 116 localization in eukaryotic membranes 118, 119 localization in viruses 100 localization using exchange protein 100 probes for 57, 92 structure 19 phosphatidylglycerol aminoacy Iderivatives 18 of Archaebacteria 18,20 antibodies against 10I, 108 in bacteria 108 localization in Acholeplasma 104, 105 localization in Micrococcus 100 localization in PM2 phage 92 localization in viruses 100 localization using exchange protein 100 required for sugar phosphotransferase 197 transbilayer movement 104 phosphatidylinositol antibodies against 10I exchange protein 100 and hormone responses 22 localization ux-Micrococcus 108 localization in microsomal membranes 100, 101, 119 mannosylated derivatives 18 and Na+ /K+·ATPase 176 phosphorylated derivatives 18 structure 19 269 phosphatidylserine labelling 93, 94 localization in erythrocyte 115, 116 localization in eukaryote membranes 118, 119 localization in Micrococcus 100 localization in virus 100 localization using exchange protein 100 and Na+ /K+-ATPase 176 probes for 92 structure 19 phosphoglycerate kinase with erythrocyte membrane 170 phospholipases A and C 94 access to phospholipids 95 action of 94 action on Acholeplasma 104, 105 action on B megaterium 109 action on B subtilis 95 action on erythrocyte membranes 114 action on intracellular membranes 120 action on Mycoplasma 95 effects of hydrolysis products 97 in enzyme solubilization 189 membrane lysis by 97 sphingomyelinase 94 action on erythrocyte membranes 96 and transbilayer movement 97, 98 phospholipids, see also lipid and entries for individual phospholipids action of phospholipases 94 asymmetry in viruses 93 disposition in Acholeplasma 104, 105 disposition in erythrocyte 115-116 disposition in intracellular membranes 1l7, 118 disposition in Micrococcus 108 disposition in microsomal membranes 119 disposition in mitochondrial inner membrane 118, 119 disposition in sarcoplasmic reticulum 119 exchange 99, 100, 118, 120 immunochemicallocalization 101 labelling 92, 93, 94 monolayer:TNBS reaction 93, 94 transport 118 phosphosphingolipids, see also sphingomyelin 20 plasma membranes, see also ATPase, Ca2+ and Na +/K + , acetylcholinesterase, band 3, blood group and histocompatibility antigens, erythrocyte membranes, glycophorin A, myelin, spike proteins, viral envelopes, etc carbohydrate disposition 127, 141-143 cholesterol in 20- 21, 30- 31, 100, 105 in differentiation 149- 153 glycolipids 20, 138-143, 146-147 in growth 148-153 isolation 5-9 lipid disposition 101-120 lipid mobility 39-41 lipid lateral diffusion 56-58 protein lateral diffusion 64- 70 protein lateral segregation 68- 75, 184- 185 protein rotation 45-49 in protein synthesis 241-246 sphingomyelin in 20 promyelocytic cell line (HL60) differentiation in DMSO 150-152 protein, phospholipid exchange 92, 99, 100, 221 PC-specific 100 PI/PC-specific 100 Protein, membrane, see also entries for individual proteins and glycoproteins accommodation in lipid bilayer 24 ambiquity of assignment 11-12, 169-172 amino acid sequencing 216,230,252 assembly 215, 234-248 assembly after synthesis 246 assembly during synthesis 235 association between intrinsic and extrinsic 14 asymmetric disposition 83, 216 definition 221 detergent binding 14, 24, 190 disposition of amino terminus 246 of erythrocyte 15-18, 112-114 extrinsic 11- 12 general characteristics 10- 18 glycosylation, see glycoproteins identification 88 intracellular transport 249- 251 intrinsic 12-14 lateral diffusion by FPR 58-66 by interdiffusion 66-67 liberation by detergents 12, 188-192 lipophilic domains 12, 13 mobility 45, 55-75, 168 modification reagents 84 non-specific labelling 88 oligomeric 14, 166-168 restraints upon mobility 65- 75 release from membranes 11-12 rotational diffusion 45-49 spanning sequences 217, 221, 232, 233, 245, 251 270 techniques for localization, see proteins, techniques for localization in membranes that not span 221 that span once or more 216, 217, 220, 233 unique locations 222 proteins, techniques for localization in membranes bifunctional reagents 88, 89 chemical modification 86 chemical reagents 87 enzyme activity measurements 89, 90 with non-permeant substrates 89, 90 in closed vesicles 90, 91 and vesicle orientation 90 immunochemical 90 crossed immunoelectrophoresis 90, 106 with non-permeating reagents 87, 91 photochemical techniques 86, 88 proteolytic methods 89 combined with chemical probe 89 combined with enzyme assay 89 reactive groups 86 protein kinase Ca2+ jcalmodulin-stimulated 12 erythrocyte membrane 112 purple membrane, see also bacteriorhodopsin and Halobacterium halobium 224, 225 lipid:protein ratio 10, 11,18 structural organization 31-33 pyruvate kinase association with erythrocyte membrane 170 pyruvate oxidase lipid requirement 176 receptors acetylcholine clustering on myotubes 7,5 intracellular transport 251 lateral diffusion 65, 68, 74 spans membrane 217 a-adrenergic control of adenylate cyclase 70 asialoglycoprotein carbohydrate structure 134- 135 and pinocytosis of proteins 145 cholera toxin 147 capping 142 control of adenylate cyclase 199- 200 concanavalin A lateral diffusion 65, 67-68, 71 glucagon, see also adenylate cyclase variation during hibernation 181 control of adenylate cyclase 180, 181, 199 for hormones and neurotransmitters 163, 164 affinity chromatography of 191 ricin ,B-galactosyl sites 147 lateral diffusion 68 restriction mapping 231 retinal rods, see also rhodopsin lipid:protein ratio 10 protein diffusion 47-48, 59-60, 65 rhodopsin biosynthesis 241 intracellular transport 25I lateral diffusion 59-60, 65 and lipid order 42 location of amino terminus 246 mannose-rich oligosaccharide 135-137 rotational diffusion 47-48 spans membrane 217 ribulose bisphosphate carboxylase amino terminal extension 246 biosynthesis 246 Salmonella typhimurium accessibility of phospholipids 110 membranes of 109 sarcoplasmic reticulum, see also ATPase, Ca2+ electron density profile 31 lipid environment 41-42 lipid:protein ratio 10 phospholipid disposition 119 vesicles I 17 saturation transfer EPR for assessing protein rotation 49 SDS polyacrylamide gel electrophoresis 14-5 2-dimensional 14-5 or erythrocyte membranes 15, 16 secretory proteins synthesis and processing 166 Semliki Forest virus budding 219 crystallization 224 external carbohydrate 142 spike glycoproteins 135, 136,217,218 tunicamycin and synthesis 153-154 signal hypothesis 235-246 signal sequences 242-246 cleavage 244 genetic analysis 242 in G protein of VSV 242 of maltose-binding protein 243 mutations 242, 243, 244 271 signal sequences (cont'd) recognition 244 of spike glycoprotein of SFV 242 transfer 244, 245 signal peptidases effect on polypeptide transfer 245 location 245 Sindbis virus glycoprotein 135, 136 and lipid order 42 Singer and Nicolson, see 'fluid mosaic' model sitosterol 21 spectrin molecular weight 16 tetramers 17 in various erythrocyte ghost preparations spectrin:actin cytoskeleton 222 binding to band 114 sphingomyelin action of sphingomyelinase 94, 97 and non-specific exchange proteins 100 localization in erythrocyte membranes 115, 116 localization in eukaryotic membranes 118, 119 localization in Micrococcus 100 localization in viruses 100, 103 in plasma membranes structure 19 Spike glycoproteins assembly in vitro 238 cleavage 249 Semliki Forest virus 135, 136,217,218 Sindbis virus 133, 136 spanning sequences 232, 233 synthesis 242, 243 stigmasterol 21 sterols, see also individual entries 20- 21 subcellular fractionation general principles 5- isolation media marker enzymes relative specific activities succinate dehydrogenase effects of temperature 180 lipid requirement 176 transverse disposition 182-184 sucrase-isomaltase complex synthesis and cleavage 249 SV5 paramyxovirus glycoprotein carbohydrate structure 134-135 sulphate in carbohydrate 135 syndein 222 tetrahymanol 21 Tetrahymena 21 triplet states in measuring protein rotation 46, 47-48 Triton X-IOO erythrocyte membrane dissolution 12, 17 in protein solubilization 191 tunicamycin inhibitor of N·glycosylation 153-154, 239, 240 ubiquinone:cytochrome c reductase assembly 247 biochemical dissection 228, 229 electron diffraction of 227, 228 purification 227 structure 229 UDP-glucuronyItransferase 168, 187 urinary bladder membrane structure 26 cis-vaccenic acid and membrane fluidity 70 vesicular stomatitis virus (VSV) G protein carbohydrate 134- 135 G protein synthesis and assembly 242 virus envelopes budding 102, 218, 219 derivatives of host plasma membranes 117, 219 glycolipid disposition 103 glycoproteins (spike) disposition 102 galactose oxidase susceptibility 102 proteolytic digestion 103 span bilayer 103 M-protein 102 localization 103 Newcastle disease virus 102 phospholipid disposition 93, 103 PM2 phosphatidylglycerol disposition 92 proteins 103 Semliki Forest virus 102, 103 vesicular stomatitis virus, see also G protein 102 Water, organisation of 24 'membrane-bound' 24 non-freezable 24 NMR of immobilized 24 yeast mannan 143-144 sterols 21 .. .Membrane structure Editors J.B FINEAN and R.H MICHELL Birmingham 1981 ELSEVIER/NORTH-HOLLAND BIOMEDICAL PRESS AMSTERDAM· NEW YORK· OXFORD New Comprehensive Biochemistry Volume... of membranes Some studies of membrane structure can be made using membranes still organised into cells; such studies include microscopical examination of membrane organisation in cells and of membrane- cytoskeletal... of the relationship between membrane structure and function Other aspects of membrane biochemistry will be discussed in forthcoming volumes on Phospholipids and on Membrane Transport One of the