New comprehensive biochemistry vol 31 biochemistry of lipids, lipoproteins and membranes 3rd edition

Subsequent observations that such bilayers are fluid, allowing rapid lateral diffusion of lipid and protein in the plane of the membrane, and that membrane proteins are often inserted

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LIPOPROTEINS AND MEMBRANES

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New Comprehensive Biochemistry

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Biochemistry of Lipids,

Lipoproteins and Membranes

Editors

Lipid and Lipoprotein Research Group, Faculty of Medicine,

328 Heritage Medical Research Centre, Edmonton, Alberta, Canada T6G 2S2

1996 ELSEVIER Amsterdam - Lausanne - NewYork - Oxford - Shannon - Tokyo

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Sara Burgerhartstraat 25

P.O Box 21 1, 1000 AE Amsterdam, The Netherlands

L i b r a r y o f C o n g r e s s C a t a l o g i n g - i n - P u b l i c a t i o n D a t a

B i o c h e m i s t r y o f l i p i d s , l i p o p r o t e i n s , and membranes / e d i t o r s , Dennis

E Vance and J e a n E Vance

The cover illustration, which originally uppeured in the Journul of Biological Chemistrv,

is reproduced with the kind permission of Dr E.A Dennis

ISBN 0 444 82359 X (hardbound)

ISBN 0 444 82364 6 (paperback)

ISBN 0 444 80303 3 (series)

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 written permission of the publisher, Elsevier Science B.V., Copyright and Permissions Department, P.O.Box 521, 1000 AM Amsterdam, The Netherlands

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Preface

This is the third edition of this advanced textbook which has been written with two major objectives in mind One is to provide an advanced textbook covering the major areas in the fields of lipid, lipoprotein and membrane biochemistry and molecular biology The chapters within this volume are written for students who have already taken an introduc- tory course in biochemistry, who are familiar with basic concepts and principles of biochemistry and have a general background knowledge in the area of lipid metabolism This book should therefore provide the basis for an advanced course for students in the biochemistry of lipids, lipoproteins and membranes

The second objective of this book is to provide a clear summary of these research areas for scientists presently working in, or about to enter, these and related fields This book should satisfy the need for a general reference and review book for scientists studying lipids, lipoproteins and membranes Excellent up-to-date reviews are available

on the various topics covered by this book, and many of these reviews are cited in the individual chapters However, this book remains unique in that it is not a series of ex- haustive reviews of the various topics, but rather is a current, readable and critical summary of these areas of research This book should allow scientists to become familiar with recent developments related to their own research interests, and should also help clinical researchers and medical students keep abreast of developments in basic science that are important for subsequent clinical advances

All the chapters have been extensively revised since the last edition and up-to-date information is included Three new chapters have been included to take into account sub- stantial new insights into the roles of glycerolipids in signal transduction, lipid metabolism in adipose tissue, and lipid metabolism in plants We have not attempted to cover in detail the structure and function of biological membranes since that subject is covered already in a number of excellent books However, the first chapter does contain a summary of the principles of membrane structure as a basis for the subsequent chapters

We have limited the number of references cited and emphasized review articles How- ever, some readers may wish access to the primary literature in some instances Thus, we have introduced a novel approach to literature citation suggested by Charles Sweeley In some of the chapters reference has been made to published work by citing the name of the senior author and the year in which the work was published This should allow the reader to find the original citation via a computer search

The editors and contributors assume full responsibility for the content of the various chapters and we would be pleased to receive comments and suggestions for future edi- tions of this book

We are indebted to many other people who have made this book possible In particular we extend our thanks to Brad Hillgartner, Deborah Hodge, Laura Petrosky, Ten-ching Lee and Shirley Poston

Dennis and Jean Vance Edmonton, Alberta, Canada

March 1996

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Department of Molecular Genetics, University and Biocenter Vienna, Dr Bohr - Game

912, A-1030 Vienna, Austria

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Contents

Preface

List of contributors

Chapter 1 Physical properties and functional roles of lipids in membranes P R Cullis D B Fenske and M J Hope

1 2 3 4 5 6 7 8 9 Introduction and overview

Lipid diversity and distribution 2.1 Chemical diversity of lipids

2.2 Membrane lipid composi 2.3 Transbilayer lipid asymmetry

Model membrane systems

3.1 Lipid isolation and purifi

3.2 Techniques for making model membrane vesicles

3.3 Techniques for making planar bilayers and monolayers

3.4 Reconstitution of integral membrane proteins into vesicles

Physical properties of lipids

4.2 Lipid polymorphism

4.3 Factors which modulate lipid polymorphism 4.4 The physical basis of lipid polymorphism

Lipids and the permeability properties of membranes

5.1 Theoretical considerations

5.2 Permeability of water and non-electrolytes

4.1 Gel-liquid-crystalline phase behavior

5.3 Permeability of ions

Lipid-protein interactions

6.2 Intrinsic proteins

Lipids and membrane fusion

Fusion of biological membranes

Model membranes and drug delivery

6.1 Extrinsic proteins

7.1 7.2 Future directions

Fusion of model systems

References

Chapter 2 Lipid metabolism in prokaiyotes C.O Rock S Jackowski and J.E Cronan Jr

1 The study of bacterial lipid metabolism

2 Historical introduction

3 4 5 Membrane systems of E coli

6 An overview of lipid metabolism in E coli

Genetic analysis of lipid metabolism

Lipid biosynthetic pathways in E coli

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6.1

6.2

6.3

6.4

6.5

6.6

6.7

6.8

Acyl carrier protein (ACP)

Acetyl-CoA carboxylase ,

, '

6.4.3 3-Hydroxyacyl e ~

6.4.4 Enoyl-ACP reductase

Product diversification

Transfer to the membrane

7 Lipopolysaccharide biosynthesis

8.2 Thioesterases

9 Phospholipid turnover

10 Inhibitors of lipid metabolism ,

9.1 The diacylglycerol cycle 11.3 Transcriptional regulation of the genes of fatty acid synthesis

1 1.4 Regulation of phospholipid headgroup composition

11 .5 Coupling of fatty acid synthesis to phospholipid synthesis

1 1.6 Coordination of phospholipid and macromolecular synthesis

12 Lipid metabolism in bacteria other than E Cali

12.1 12.2 Bacteria containing phosphatidylcholine

12.3 Bacteria synthesizing unsaturated fatty acids by an aerobic pathway

12.4 Bacteria with a multifunctional fatty acid s 12.6 Other bacterial oddities

12.7 13 Future directions

Bacteria lacking unsaturated fatty acids ,

12.5 Bacteria with intracytoplasmic membranes

Lipids of non-bacterial (but related) organisns

' I

Chapter 3 Oxidation of fatty acids H Schulz ~

1 2 3 The pathway of /%oxidation: a historical account

Uptake and activation of fatty acids in animal Fatty acid oxidation in mitochondria I ,

3.2 Enzymes ofa-oxidation in mitochondr 3.3 P-Oxidation of unsaturated and odd-ch 3.1 Mitochondria1 uptake of fatty acids *

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3.4

3.5

Regulation of fatty acid oxidation in mitochondria

Inhibitors of mitochondrial fatty acid oxidation

4 5 Fatty acid oxidation in E coli

6 7 Future directions

References

/?-Oxidation in peroxisomes

Inherited diseases of fatty acid oxidation

Chapter 4 Fatty acid synthesis in eukaryotes L.M Salati and A.G Goodridge

1 2 3 4 5 6 7 8 Introduction

Signals in blood that mediate the effects of diet on fatty acid synthesis Which enzymes regulate fatty acid synthesis?

4.1 Production of pyru 4.2 Production of citra 4.3 Production of NADPH

Regulation of substrate supply

Regulation of the catalytic effici 5.1 A key regulatory reaction

5.2 5.3 Regulation by citrate 5.4 Regulation by long-c Fatty acid synthase

6.1 6.3 Structure and reaction mechanism

Animal fatty acid synthase: the component reactions Animal fatty acid synthase: structural organization

Regulation of enzyme concentration

Regulation of the expression of the lipogenic enzymes

7.3.1 Pre-adipocyte cell lines

7.1 7.3 Regulation in cells in culture Future directions

Acknowledgements

References

Chapter 5 Fatty acid desaturation and chain elongation in eukaryotes H W Cook

1 Introduction

2 Historical background

3 Chain elongation of long chain fatty acids

3.1 The endoplasmic reticulum elongation system

3.2 The mitochondrial elongation system

3.3 Functions of elongation systems

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4.1 Nomenclature to describe double bonds

4.2 Characteristics of monoene-forming des es

4.3 Modification of A9 desaturase activities in vitro

4.4 Age-related, dietary and hormonal regulation of A9 desaturase

Formation of polyunsaturated fatty acids

5.1 Characteristics in animal systems

5.2 Essential fatty acids: a contribution of plant systems 5.3 Families of fatty acids and their metabolism

5.3.1 The (n-6) family 5.3.2 The (n-3) family

5.3.3 The (n-9) family

5 5.3.4 The (n-7) family

5.4 Age-related, dietary and ho 6 Unsaturated fatty acids with trans 7 Abnormal patterns of distr rated fatty acids _

7.1 7.2 Zinc deficiency

7.4 Other clinical disorders ions of polyunsaturated acid synthesis

Essential fatty acid deficiency

7.3 Relationships to plasma c

8 Future

References

Chapter 6 Glycerolipid biosynthesis in eukaryotes D.E Vance

,

2 Phosphatidic acid bios 2.1 Glycerol-3-P acyltransferase 2.2 1 -Acylglycerol- 2.3 Dihydroxyaceto 2.4 Phosphatidic acid phosphohydrolase

3.1 Historical background

3.2 Choline transport and oxidation 3.3 Choline kinase

3.4 CTP:phosphoch 3.5 CDP-choline: 1,2-diacylglycerol cholinephosphotransferase

3.6 Phosphatidylethanolamine N-methyltransferase

Regulation of phosphatidylcholine biosynthesis

4.1 The rate-limiting reaction 4.2 The translocation 4.3 Regulation of phos 4.4 4.5 4.6 3 Phosphatidylcholine biosynthesis

4 s

Phosphorylation of cytidylyltransferase

Expression of cytidylyltransferase is also regulated Interrelationships among phosphatidylethanolamine methylation, the CDP-

choline pathway, hepatoma cell division and liver tumor suppression

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5.1 Historical background and biosynthetic pathways

5.2 Enzymes of the CDP-ethanolamine pathway 5.3 Regulation of the CDP-ethanolamine pathway

Triacylglycerol biosynthesis _ _

Phosphatidylserine biosynthesis

7.1 Historical developments and bi 5.4 Phosphatidylserine decarboxylase

6 7 7.2 Chinese hamster ovary cell mutants and regulation

8.1 Historical developments

8 Inositol phospholipids

8.2 Biosynthetic enzymes

9 I Historical developm 10 Remodeling of the acyl substituents of phospholipids

11 12 Glycosyl phosphatidylinositols for attachment of cell surface proteins

13 Future directions

9 Polyglycerophospholipids , ,

9.2 Enzymes and subcel Regulation of gene expression in yeast

n ,

Chapter 7, Ether-linked lipids and their bioactive species: occurrence, chemistry, metabolism, regulation, and function F Snyder

1 2 Introduction

Synopsis of historical developments

5 Natural occurrence

6 Biologically-active ether lipids

6.2 Receptors and antagonists

7.3.3 PAF transacetylase

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8 Catabolic enzymes

8 I Ether lipid precursors

8.1.1 Fatty alcohols , , ,

8.2.3 Phospholipases and lipases

8.3 PAF and related bioactive species

9 Metabolic regulation

11 Future directions Acknowledgements

References , ,

Chapter 8 Phospholipases M Waite

1 Overview

1 l 1.3 Definition of phospholipases

Interaction of phospholipases with interfaces

1.3.1 1.3.2 1.3.3 1.3.4 Conformational change 1.3.5 1.2 Assay of phospholipases ,

Increased effective substrate concentration

Orientation of the phospholipid molecule at the interface

Enhanced diffusion of the products from the enzyme

Nature of the aggregated lipid 2 The phospholipases

2.1 Phospholipase A,

2.1.1 Escherichia coli phosph 2.1.2 Lysosomal phospholipase A,

2.1.3 Lipases with phospholipase A1 activity

Phospholipase B and lysophospholipases

2.2.1 Penicillium notatum phospholipase B

Phospholipase A2

2.3.1 Groups 1-111 phospholipases A2

2.3.2 Group IV (cytosolic) phospholipases A,

2.3.3 Ca2+-independent and other phospholipases A2

2.4 Phospholipase C

2.4.1 Bacterial phospholipases C

2.4.2 Mammalian phospholipases C

2.5 Phospholipase D

3 Future directions

References

2.2 2.2.2 Mammalian lysophospholipases A, 2.3

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J.D Lambeth and S.H Ryu

1 2 Phosphatidylinositol cycle

Introduction: glycerolipids as a source of bioactive molecules

2.1 2.2 Inositol phosphate metab f intracellular calcium levels

2.3 Phosphatidylinositol-phospholipase C isoforms: occurrence and regulation

3 Diacylglycerols

3.1 Protein kinase C and its regulation by diacylglycerol

3.2 Evidence for novel mechanisms of diradylglycerol generation

Phosphatidylcholine hydrolysis and phospholipase D

4.1 Phosphatidylcholine hydrolysis as a source of signaling lipids 4.2 Phosphatidic acid as a signaling molecule

4.3 Receptor-coupled activation of phospholipase D 4.4 Molecular nature and mechanism of regulation o 4.5 A model for recept nvolving a phospholipase cascade

5 Phospholipid kinases and The discovery of the pho

4

5.1 Phosphatidylinositol4,5-bisphosphate 5.2 trisphosphate as potential signal molecules _

Phosphatidylinositol 3-kinase: its structure, regulation and biological relev 6 Future directions

References

Chapter 10 Adipose tissue and lipid metabolism D.A Bemlohr and M.A Simpson

1 Introduction , ,

2 Adipose development

2.1 2.2 Development of white and brown adipose tissue in vivo

In situ models of adipose conversion

3.1 Lipid delivery to adipose tissue

3.2 Fatty acid uptake and

3.5.2 Glucagon

3.6 Brown fat lipid metabolism

3.6.1 Triacylglycerol synthesis and

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4.1

4.2

4.3

Energy balance and basal metabolic rate

The hypothalamus-adipocyte circuit and the ob gene Cytokine control of adipose lipid metabolism

5 Future directions

Chapter 11 The eicosanoids: cyclooqgenase lipoxygenase and epoxygenase pathways W.L Smith and F.A Fitzpatrick

2 Prostanoid biosynthesis 2.3 2.4 2.6 2.7 Prostanoid catabolism and mechanisms of action

3.1 Prostanoid catabolism

3.2 Prostaglandin endoperoxide H2 (PGH2) formation

2.5 PGH synthases and non-steroidal anti-inflamma

PGH synthase active site

Regulation of PGHS-1 and PGHS-2 gene expression

2.8 PGH2 metabolism

Physico-chemical properties of PGH synthases 3 Physiological actions of prostanoids 3.3 Prostanoid receptors

4 Hydroxy- and hydroperoxy-eicosaenoic acids a trienes

4.1 Introduction and overview

4.2 Mechanism of leukotriene biosynthesis in human neutrophils

4.3 The enzymes of the 5-lipoxygenase pathway

4.4 Regulation of leukotriene synthesis

4.6 Biological activities of leukotrienes

5.1 Introduction

5.2 Structures, nomenclature, and biosynthesis 5.3 Occurrence of epoxyeicosatrienoic acids

5 Epoxygenase products

5.5 Biological actions of epoxygenase-derived EpETrEs and HE

6 Future directions

References

Chapter 12 Sphingolipids: metabolism and cell signalling AM Merrill Jr and C.C Sweeley

1 Introduction

Biological significance of sphingolipids

Structures and nomenclature of sphingolipids

1.1

1.2

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2 Chemistry and distribution

2.2 Ceramides

2.3 Phosphosphingolipids

2.1 Sphingoid bases

3.3 3.4 Gangliosides

3.5 Sulfatoglycosphingolipids

4 Sphingolipid catabolism

4.1 Sphingomyelin

4.3 Ceramide Neutral glycosphingolipids _

5 Regulation of sphingolipid metabolism 5.1 Embryogenesis

5.2 Neural development and function

5.3 Physiology (and pathophysiology) of the intestinal tract

5.4 Male-female differences in kidney sphingolipids

5.5 Leukocyte differentiation

6.2 Hydrolysis to bioactive lipid backbones

6.2.1 Ceramide 6.2.2 Sphingoid bases , ._

6.2.3 Sphingosine 1-phosphate

,

7 Future directions

References

Chapter 13 Isoprenoids, sterols and bile acids P.A Edwards and R Davis

1 Introduction _

1.1 The sterol biosynthetic pathway

2.1 Non-sterols

2.2 Sterols

2.2.2 Bile acids

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2.2.3 Steroid hormones

Cholesterol and bile acid synthesis

3.2 Mutations in the human chole 3.3 Regulation of cellular cholesterol homeostasis; an overview

3.5 3 3.1 Enzyme compartmentalization

Post-transcriptional regulation of HMG-Co reductase

4 Oxysterols

7 Regulation of bile acid synthesis

8 Isoprenylation of proteins

9 Future directions

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344 344 344 346 346 348 35 1 353 353 354 355 355 356 357 357 359 360 Chapter 14 Lipid metabolism in plants K.M Schmid and J.B Ohlrogge 363

1 2 3 4 5 6 7 8 9

3.2 Desaturation of acyl-ACPs

3.3 Acyl- ACP thioesterases

5.3 Traffic between prokaryotic and eukaryotic pathways: 16:3 and 18:3 plants

Glycerolipid synthesis pathways

6.1 Glycerolipids as substrates for desaturation

Sterol, isoprenoid and Lipid storage in plants

8.1 8.2 Seed triacylglyc 8.3 The pathway of 8.4 Progress in plant lipid 9.1 Mutants in lipid Lipid body structure and biogenesis Challenges in triacylglycerol synthesis

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9.2

10 Design of new plant oils

10.1 10.1.1 Improvements in nutritional value and stability 10.1.2 Alternatives to hydrogenated vegetable oils

10.2 Design of new industrial oils

10.2.1 High lauric oils

Arabidopsis mutants have allowed cloning of desaturases and elongases

Design of new edible oils

11 Future prospects

References Chapter IS Lipid assembly into cell membranes D.R Voelker

1 2 The diversity of lipids Introduction ,

3 Methods to study intra- and inter-membrane lipid transport *

3.1 Fluorescent probes 3.2 Spin labeled analogs

3.3 3.4 Phospholipid transfer proteins

3.5 3.6 4.1 4.2 Asymmetric chemical modification of membranes

Rapid plasma membrane isolation

4 Lipid transport processes

Organelle specific lipid metabolism ,

Intramembrane lipid translocation and model membranes

Intramembrane lipid translocation and biological membranes

4.2.2 Eukaryotes

4.3 I Transport in prokaryotes

References

Chapter 16 Assembly of proteins into membranes R A F Reithmeier _ _ , ,

1 Organization of membrane proteins

1.1 1.2 1.3 Secretion of proteins and the signal hypothesis

2.2 2.4

Classification of membrane proteins I

Membrane protein structure and energetics

Assembly of membrane proteins

2 2.1 The Palade secretion pathway

2.3 In vitro translation and translocation systems

signal sequence

The Blobel signal hypothesis

The Milstein experiment: secreted proteins are made with an amino-terminal

2.5 Signal sequences

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3.3

3.5

Biosynthesis of type I membrane proteins

4.1 4.2 VSV glycoprotein

Translocation components

Signal peptidase

IgM and the relationship between the biosynthesis of secreted proteins and single span TM proteins

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5.2 Asialoglycoprotein receptor

5.3 Sucrase-isomaltase ,

7.6 Cleaved signal sequences in multi-span membrane proteins

Glycosylation of proteins

8.1 N-Glycosylation

8.2 Processing of the oligosaccharide chain

8.3 9.1 Fatty acylation _._

10 Protein folding and exit from the ER

8 0-Glycosylation * ~

10.3 Assembly of multisubunit systems

10.4 Exit from the ER

11 Vesicular transport and targeting of proteins ,

1 I 1 Vesicles move proteins between organelles

11.2 Role of GTP-binding proteins

1 1.3 KDEL, an ER localization signal

11.4 Golgi localization I

1 1.5 Lysosomal targeting

12 Future directions

References 1 1.6 Protein sorting in epithelial cells

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R.A Davis and J.E Vance

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Overview: structure and function of plasma lipoproteins Assembly and secretion of apolipoprotein B-containing lipoprote

2.1 Apoproteins of VLDLs and chylomicrons

2.2 Intracellular route of apo B secretion

2.3.1 Apo B is an unusually large amphipathic protein

2.3.2 Motifs shared with vitellogenin, a primordial apolipoprotein

Transcriptional regulation of apo B synthesis

2.4.1 Tissue specificity of expression of apo B

2.4.2 The apo B gene: transcription regulatory elements

Regulation of apo B secretion by translocational efficiency

Chapter 18 Dynamics of lipoprotein transport in the human circulatory system

P E Fielding and C J Fielding

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2 Lipoprotein lipase and the metabolism of lipop

2.1 Initial events

2.2 The structure of lipoprotein lipase

2.3 Synthesis, regulation and transport of

2.4 Structure of the LPL-substrate complex at the vascular surface

2.5 Kinetics of the LPL reaction and the role of albumin

2.6 Later metabolism of chylomicron

2.7 Physiological regulation of LPL

HDL and plasma cholesterol metabolism

3.2 The structure of apo A1

3.4 Structure/function relations in LCAT

3.5 Substrate specificity of LCAT

3.6 Hepatic lipase and its role in HDL metabolism

3.7 Evidence from transgenic mice on the functions of apo A1

2.8 Congenital lipoprotein lipase deficiency

3.1 The apo A1 cycle

3.3 Origin of 1ecithin:cholesterol acyltransferase

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3.9 Congenital LCAT defici

4 Reactions linking metabolism i

4.2

4.3

Phospholipid transfer protein (PLTP)

Cholesteryl ester transfer protein (CETP)

5 Summary and future directions

The LDL receptor pathway

Familial hypercholesterolemia: biochemical basis and clinical consequences of

LDL receptor dysfunction

2.3.1 Biosynthesis and structure of the LDL receptor

Molecular defects in LDL receptors of patients with familial

The gene for the human LDL receptor

Four groups of LDL receptor mutations

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CHAPTER 1

PIETER R CULLIS1,2, DAVID B FENSKE' and MICHAEL J HOPE2,3

'Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, B.C., V6T 123, Canada, 21nex Pharmaceuticals Corp., 1779 W 75th Avenue, Vancouver, B.C., V6P 6P2, Canada and 'Division of Dermatology, Faculty of Medicine, University of British Columbia,

Vancouver, B.C., V5Z IL7, Canada

1 Introduction and overview

Biological membranes contain an astonishing variety of lipids As detailed throughout this book, generation of this diversity requires elaborate metabolic pathways The lipid compounds representing the end products of these pathways must bestow significant evolutionary advantages to the cellular or multicellular systems in which they reside, implying particular functional roles for each component However, clarification of the functional roles of individual lipid species has proven a difficult problem Here we present a synopsis of the physical properties of lipid systems and indicate how they may relate to the functional capacities of biological membranes

The major role of membrane lipids has been understood in broad outline since the early experiments of Gorter and Grendell [I], who extracted lipids from the erythrocyte membrane and measured the areas these lipids were able to cover as a monolayer at

an air-water interface This work led to the conclusion that the erythrocytes contained sufficient lipid to provide a bilayer lipid matrix surrounding the red blood cell This bilayer lipid organization, which provides a permeability barrier between exterior and interior compartments, has remained a dominant theme in our understanding of the organization and function of biological membranes Subsequent observations that such bilayers are fluid, allowing rapid lateral diffusion of lipid and protein in the plane of the membrane, and that membrane proteins are often inserted into and through the lipid matrix, have further contributed to our present understanding of membranes, resulting in the Singer and Nicholson [2] fluid mosaic model, a refined version of which is shown in Fig

1

The ability of lipids to assume the basic bilayer organization is dictated by a unifying characteristic of membrane lipids namely, their amphipathic character, which is indicated

by the presence of a polar or hydrophilic (water loving) head group region and non-polar

or hydrophobic (water hating) region The chemical nature of these hydrophilic and hydrophobic sections can vary substantially However, the lowest-energy macromolecular organizations assumed in the presence of water have similar characteristics, where the polar regions tend to orient towards the aqueous phase, while the hydrophobic sections are sequestered from water In addition to the familiar bilayer phase, a number of other

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C W Fig 1 The topography of membrane protein, lipid and carbohydrate in the fluid mosaic model of a typical eukaryotic plasma membrane Phospholipid asymmetry results in the preferential location of PE and PS in the cytosolic monolayer Carbohydrate moieties on lipids and proteins face the extracellular space AV represents

the transmembrane potential, negative inside the cell

macromolecular structures are compatible with these constraints It is of particular interest that many naturally occurring lipids prefer non-bilayer structures in isolation

The fluidity of membranes depends on the nature of the acyl chain region comprising the hydrophobic domain of most membrane lipids Most lipid species in isolation can undergo a transition from a very viscous gel (frozen) state to the fluid (melted) liquid- crystalline state as the temperature is increased This transition has been studied inten- sively, since the local fluidity, as dictated by the gel or liquid-crystalline nature of membrane lipids, may regulate membrane-mediated processes However, at physiological temperatures most, and usually all, membrane lipids are fluid; thus, the major emphasis

of this chapter concerns the properties of liquid-crystalline lipid systems As indicated later, the melted nature of the acyl chains depends on the presence of cis double bonds, which can dramatically lower the transition temperature from the gel to the liquid- crystalline state for a given lipid species

The ability of lipids to self-assemble into fluid bilayer structures is consistent with two major roles in membranes: establishing a permeability barrier and providing a matrix with which membrane proteins are associated Roles of individual lipid components may therefore relate to establishing appropriate permeability characteristics, satisfying insertion and packing requirements in the region of integral proteins (which penetrate into or through the bilayer), as well as allowing the surface association of peripheral proteins via electrostatic interactions All these demands are clearly critical An intact permeability

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electrochemical gradients which give rise to a membrane potential and drive other membrane-mediated transport processes In addition, the lipid in the region of membrane protein must seal the protein into the bilayer so that non-specific leakage is prevented and an environment appropriate to a functional protein conformation is provided

More extended discussions of biomembranes and the roles of lipids can be found in the excellent text by Gennis [3]

2 Lipid diversity and distribution

The general definition of a lipid is a biological material soluble in organic solvents, such

as ether or chloroform Here we discuss the diverse chemistry of the sub-class of lipids which are found in membranes This excludes other lipids which are poorly soluble in bilayer membrane systems, such as triacylglycerols and cholesteryl esters

2.1 Chemical diversity of lipids

The major classes of lipids found in biological membranes are summarized in Fig 2 In eukaryotic membranes the glycerol-based phospholipids are predominant, including phosphatidylcholine PC, phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and cardiolipin Sphingosine-based lipids, including sphingomyelin and the glycosphingolipids, also constitute a major fraction The glycolipids, which can also include carbohydrate-containing glycerol-based lipids (found particularly in plants), play major roles as cell-surface-associated antigens and recognition factors in eukaryotes (Chapter 12) Cholesterol is also a major component of eukaryotic membranes, particularly in mammalian plasma membranes, where it may be present in equimolar propor- tions with phospholipid

In most prokaryotic membranes, PC is not usually present (Chapter 2), whereas the major phospholipids observed are PE, phosphatidylglycerol, and cardiolipin In plant membranes on the other hand, lipids such as monogalactosyl and digalactosyl diacylglycerols can form the majority components of membranes such as the chloroplast membrane (Chapter 14)

These observations give some impression of the lipid diversity in membranes, but it must be emphasized that this diversity is much more complex Minority species such as sulfolipids, phosphatidylinositols, and lysolipids abound Furthermore, each lipid species exhibits a characteristic fatty acid composition In the case of glycerol-based phospholipids, for example, it is usual to find a saturated fatty acid esterified at the 1-position of the glycerol backbone and an unsaturated fatty acid at the 2-position Also, in eukaryotic membranes it is usual to find that PE and PS, for example, are more unsaturated than other phospholipids In order to give a true impression of the molecular diversity of phospholipids in a single membrane, we list in Table I the fatty acid composition of phospholipids found in the human erythrocyte membrane From this table it is clear that the number of molecular species of phospholipids in a membrane can easily exceed 100

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diolipin (diphosphatidylglycerol) is esterified to two phosphatidic acid molecules

2.2 Membrane lipid compositions

The lipid compositions of several mammalian membrane systems are given in Table I1 (see also Chapter 15) Dramatic differences are observed for the cholesterol contents Plasma membranes such as those of myelin or the erythrocyte contain approximately equimolar quantities of cholesterol and phospholipid, whereas the organelle membranes

of endoplasmic reticulum or the inner mitochondria1 membrane contain only small

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TABLE I

Gas chromatographic analyses of the fatty acid chains in human red cell phospholipid

genor (Ed.), The Red Blood Cell, Academic Press, New York, pp 147-213

“This code indicates the number of carbon atoms in the chain and the number of double bonds

amounts of cholesterol This cholesterol distribution correlates well with the distribution

of sphingomyelin Cholesterol may have a ‘fluidizing’ role in membranes containing sphingomyelin, which is relatively saturated Cardiolipin is almost exclusively localized

TABLE I1

The lipid composition of various biological membranes

(inner and outer reticulumb membrane)

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>2

aAverage number of double bonds per phospholipid molecule

to the inner mitochondria1 membrane, and it has been suggested that cardiolipin is required for the activity of cytochrome c oxidase, the terminal member of the respiratory electron-transfer chain In general, the lipids of more metabolically active membranes are considerably more unsaturated, as indicated in Table 111

The lipid composition of the same membrane system in different species can also vary significantly The rat erythrocyte membrane, for example, contains lower levels of sphingomyelin and elevated levels of PC compared to the human erythrocyte In the bovine erythrocyte, this distribution is reversed, with high sphingomyelin and low PC contents

2.3 Transbilayer lipid asymmetry

The inner and outer leaflets of membrane bilayers may exhibit different lipid compositions [4] The plasma membrane of human erythrocytes is the most thoroughly investi- gated The results obtained indicate that most membranes display some degree of lipid asymmetry The use of impermeable probes that react with the primary amines of PE and phosphatidylserine on only one side of the membrane has shown that the majority of the amino-containing phospholipids of the erythrocyte are located on the inner monolayer Combinations of chemical probes and phospholipase treatments indicate that in a normal red blood cell all the phosphatidylserine is located in the inner monolayer, whereas approximately 20% of the PE can be detected at the outer surface, with 80% confined to the inner monolayer The outer monolayer consists predominantly of PC, sphingomyelin, and glycolipids Figure 3 summarizes the transbilayer lipid distributions obtained for various mammalian cell membranes and viral membranes derived from animal-cell plasma membranes A common feature is that the amino-containing phospholipids are

chiefly limited to the cytosolic side of plasma membranes It is interesting that the infor-

mation available for organelle membranes suggests that PE and PS are also oriented towards the cytosol A general feature of plasma membrane asymmetry is that the majority

of phospholipids that exhibit a net negative charge at physiological pH (PS and PI; PE is only weakly anionic) are limited to the cytosolic half of the bilayer Certain proteins ap- pear to be involved in maintaining this asymmetry (Chapter 15) Treatment of erythrocytes with diamide, which induces cross-linking of the cytoskeletal protein spectrin, results in the appearance of PS in the outer monolayer Red blood cells known to have le- sions associated with cytoskeletal proteins also exhibit a partial breakdown of asymme-

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VSV envelope derived from hamster kidney BHK-21 cells

try, with an increased exposure of PS and PE on the outer half of the bilayer and an equivalent transfer of PC to the inner monolayer

These experiments suggest a possible interaction between cytoskeletal proteins and membrane phospholipids to generate and maintain asymmetry However, some phospholipids will redistribute across the bilayer of protein-free model membrane systems in re- sponse to transmembrane pH gradients Phosphatidylglycerol and phosphatidic acid, for example, will diffuse to the inner monolayer of large unilamellar vesicles that exhibit an interior pH that is basic with respect to the external pH [5] Similar responses to transmembrane proton gradients would be expected to occur in vivo On the other hand, an aminophospholipid translocase (see also Chapter 15) has been identified in a number of plasma membranes which appears to be responsible for the movement of PE and PS across the bilayer [4] This ATP-dependent ‘lipid pump’ activity has also been found in organelle membranes but oriented such that the aminophospholipids are transported from the inner monolayer to the outer monolayer, which is consistent with their phospholipid asymmetry

The functional importance of lipid asymmetry is not clear but could be related to pre- vention of exposure of PS at the outer surface of a normal cell, which has been suggested

to be a signal of senescence [6] Alternatively, PE and PS may be required to maintain a fusion competent surface for endocytosis and organelle fusion (see Fig 10 and [4])

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3 Model membrane systems

The physical properties and functional roles of individual lipid species in membranes are exceedingly difficult to ascertain in an intact biological membrane due to the complex lipid composition In order to gain insight into the roles of individual components, it is necessary to construct model membrane systems that contain the lipid species of interest This requires three steps, namely isolation or chemical synthesis of a given lipid, con- struction of an appropriate model system containing that lipid, and subsequent incorporation of a particular protein if understanding the influence of a particular lipid on protein function is desired By this method specific models of biological membranes can be achieved in which the properties of individual lipid components can be well characterized

3.1 Lipid isolation and purification

Although a wide range of synthetic and natural lipids are now commercially available, a

variety of techniques have been developed for isolation of lipids from membranes [ 7 ] In

the preparation of erythrocyte phospholipids, the first step involves disruption of the membrane in a solvent which denatures and precipitates most of the protein and solubi- lizes the lipid component The Bligh and Dyer procedure is perhaps most often employed and involves incubation of the membrane in a chloroform-methanol-water (1 :2:0.8 v/v/v) mixture, which forms a one-phase system The subsequent addition of chloroform and water to the mixture containing the extracted lipids results in a two-phase system where the lower (chloroform) phase contains most membrane lipids

Column chromatography is usually subsequently employed for isolation of individual lipid species A solid phase such as silicic acid, DEAE cellulose, aluminum oxide, or carboxymethyl cellulose is used, depending upon the lipid being isolated, and lipids are eluted using mixtures of solvents with different polarities, such as chloroform and methanol Thin-layer chromatography is generally used for lipid identification, small scale isolation, and for ascertaining purity All these separation techniques rely upon the different partitioning characteristics of lipids between the stationary phase surface and mobile solvent phase for different solvent polarities The exact nature of the binding of lipid to the solid phase is not well understood but appears to involve both electrostatic and hydrophobic interactions Carboxymethyl cellulose and DEAE cellulose are often used for separation of anionic lipids

High-pressure liquid chromatography enables the rapid purification of large quantities

of natural lipids Analytical high pressure liquid chromatography techniques are well- developed for the rapid separation of phospholipids by headgroup and acyl chain composition Reversed-phase chromatography, where the stationary phase is hydrophobic and the mobile phase hydrophilic, is particularly useful The solid support is usually coated with hydrocarbon chains of a defined length (and consequently of regulated hy- drophobicity), and the mobile phase is hydrophilic This technique is particularly useful for separating single lipid classes according to their acyl chain length and degree of unsaturation

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3.2 Techniques for making model membrane vesicles

Preparation of the simplest model system involves the straightforward hydration of a lipid film by mechanical agitation, such as vortex mixing In the case of bilayer-forming lipids, this hydration results in a macromolecular structure which is composed of a series

of concentric bilayers separated by narrow aqueous spaces Such structures are usually referred to as liposomes or multilamellar vesicles (MLVs) and have been used for many years as models for the bilayer matrix of biological membranes Their use is mostly restricted to physical studies on bilayer organization and the motional properties of individual lipids within a membrane structure MLVs are not ideal models for the study of other aspects of lipids in membrane structure and function, mainly because as little as 10% of the total lipid of a MLV is contained in the outermost bilayer As a result, methods have been sought by which unilamellar (single bilayer) model membranes can be obtained either directly or from MLVs [8]

Small unilamellar vesicles (SUVs) can be made from MLVs by subjecting the MLVs

to ultrasonic irradiation or by passage through a French press However, their small size limits their use in model membrane studies Typically, diameters in the range 2 5 4 0 nm are observed The radius of curvature experienced by the bilayer in S U V s is so small (Fig 4) that the ratio of lipid in the outer monolayer to lipid in the inner monolayer can

be as large as 2:l As a result of this curvature, the packing constraints experienced by the lipids perturb their physical properties which restricts the use of SUVs for physical studies on the properties of membrane lipid Moreover, the aqueous volume enclosed by the SUV membrane is often too small to allow studies of permeability or ion distributions between the internal and external aqueous compartments

A more useful membrane model is the large unilamellar vesicle (LUV) system, where the mean diameter is larger, and the distribution of lipid between the outer and inner monolayers is closer to 1 : 1 The most common procedures for producing LUVs result in unilamellar vesicles with diameters ranging from 50 to 500 nm These preparative procedures often include the use of detergents or organic solvents, although L W s can be pro- duced directly from MLVs

The most popular technique for making LUVs involves the direct extrusion of MLVs under moderate pressures (5500 psi) through polycarbonate filters of defined pore size This process can generate LUVs with size distributions in the range of 50-200 nm depending on the pore size of the filter employed [8] Extrusion does not require detergents

or solvents, which are difficult to remove, and it can be applied to all lipids which adopt liquid crystalline bilayer structures, including long chain saturated lipids The technique

is rapid, straightforward and convenient, allowing LUVs to be prepared in 10 min or less

3.3 Techniques for making planar bilayers and monolayers

Planar bilayers (also known as black lipid membranes) are favorite model membranes of electrophysiologists interested in current flow across a bilayer They are formed by dis- solving phospholipids in a hydrocarbon solvent and painting them across a small aperture (approximately 2 mm in diameter) which separates two aqueous compartments The sol-

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suv

25 nm

LUV

Trap No phospholipid No vesicles

molecules per per pmol

of lipid (mole ratio) (pl per pmol)

as small as 25 nm in diameter The radius of curvature for each vesicle size is shown in proportion The ratio

of lipid in the inner monolayer (IM) compared with lipid in the outer monolayer (OM) gives an indication of

the packing restrictions in bilayers with a small radius of curvature The trapped volume refers to the volume

of aqueous medium enclosed per micromole of phospholipid The calculations were made assuming a

bilayer thickness of 4 nm and a surface area per phospholipid molecule of 0.6 nm2

vent collects at the perimeter of the aperture, leaving a bilayer film across the center The electrical properties of the barrier are readily measured by employing electrodes in the two buffered compartments It is also possible to incorporate some membrane proteins into the film, if the protein can be solubilized by the hydrocarbon With this technique, ion channels have been reconstituted and voltage-dependent ion fluxes recorded The most serious problem of black lipid membranes is the presence of the hydrocarbon solvent, which may change the normal properties of the lipid bilayer being studied More recent techniques avoid some of these problems [9]

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Another planar bilayer model is the oriented multibilayer, which consists of membrane lamellae sandwiched between glass plates These models are primarily utilized in biophysical NMR studies of membrane lipid structure and motion The lipid mixture of choice is dissolved in an organic solvent and streaked onto glass plates, which are then stacked and placed under high vacuum to remove residual solvent Hydration of the lipid and formation of the multibilayers occurs during a 24 h incubation in a humid atmos- phere

In monolayer systems, amphipathic lipids orient at an air-water interface The result is

a monolayer film which, in the case of phospholipids, represents half of a bilayer, where the polar regions are in the aqueous phase and the acyl chains extend above the buffer surface Such films can be compressed and their resistance to compression measured The study of compression pressure versus surface area occupied by the film yields information on molecular packing of lipids and lipid-protein interactions Perhaps the best- known result of monolayer studies is the condensation effect of cholesterol and phospholipid, in which the area occupied by a typical membrane phospholipid molecule and a cholesterol molecule in a monolayer is less than the sum of their molecular areas in isolation This phenomenon provides a strong indication of a specific interaction between this sterol and membrane phospholipids [ 101

3.4 Reconstitution of integral membrane proteins into vesicles

An important step, both for the study of membrane protein function and for the building

of simple but more representative biological membranes, is the insertion of purified integral membrane proteins into well-defined lipid model membranes A large variety of membrane proteins have been reconstituted [ I] For the purpose of discussing the sali-

ent features of reconstitution techniques, we shall use the example of cytochrome c oxi-

dase from bovine heart mitochondria This integral membrane protein spans the inner

mitochondria1 membrane and oxidizes cytochrome c in the terminal reaction of the elec-

(Chapter 16), the protein in reconstituted systems is not necessarily inserted with a well-

defined asymmetric orientation In the case of reconstituted cytochrome oxidase systems, for example, oxidase-containing vesicles can exhibit protein orientations in which the

cytochrome c binding sites are on the outside or the inside Asymmetric protein orienta-

tion can be achieved by reconstitution at low protein to lipid ratios such that most vesicles contain one or zero protein molecules Populations containing only one oxidase

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cytochrome

oxidase cytochrome C

Fig 5 Unidirectionally shadowed freeze-fracture micrographs of cytochrome c oxidase reconstituted with dioleoyl-PC The protein to lipid ratio is <1:5000 (wlw) The vesicle diameter is approximately 100 nm Each particle represents one dimer of cytochrome c oxidase and is approximately 10 nm in diameter [T.D Madden,

19881 The orientation of the reconstituted protein is shown in the diagram below

molecule per vesicle with well-defined transmembrane orientations of the oxidase can subsequently be achieved by ion-exchange or affinity column chromatography, as illustrated in Fig 5

In some cases asymmetric incorporation of other proteins can be achieved by different procedures Erythrocyte glycophorin, for example, has a large carbohydrate-containing region which is normally localized on the exterior of the red cell Reconstituted systems

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can be obtained by hydrating a dried film of lipid and glycophorin, resulting in asymmetric vesicles in which more than 80% of the carbohydrate groups are on the vesicle exterior This is presumably due to the small size of the reconstituted vesicle, which limits the fraction of the bulky carbohydrate-containing groups that can pack into the interior volume

Alternative reconstitution techniques involving protein insertion into preformed vesicles have achieved some success in obtaining asymmetric incorporation One of these asymmetric insertion techniques utilizes the detergent octylglucoside It is possible to form vesicles in the presence of relatively high detergent concentrations (approximately

20 mM) which are sufficient to solubilize the spike protein of Semliki Forest virus [12] The spike protein consists of a hydrophilic spike and a smaller hydrophobic anchor portion of the molecule The anchor portion is solubilized by a coat of detergent, and this domain of the molecule can insert into the preformed bilayer upon dialysis

In summary, a large variety of sophisticated and well-defined model membrane systems are available The incorporation of protein, with well-defined lipid to protein ratios and asymmetric transmembrane protein orientations, is becoming more feasible Prob- lems remain, however, both in removing the last traces of detergent in reconstituted systems and in generating the lipid asymmetry observed in biological membrane systems

4 Physical properties of lipids

4.1 Gel-liquid-crystalline phase behavior

As indicated previously, membrane lipids can exist in a frozen gel state or fluid liquid-

crystalline state, depending on the temperature [ 131, as illustrated in Fig 6 Transitions

between the gel and liquid-crystalline phases can be monitored by a variety of techniques, including nuclear magnetic resonance (NMR), electron spin resonance, and fluo- rescence Differential scanning calorimetry, which measures the heat absorbed (or re- leased) by a sample as it undergoes an endothermic (or exothermic) phase transition, is

particularly useful A representative scan of dipalmitoyl-PC, which exhibits a gel to liq-

uid-crystalline transition temperature (T,) of 4loC, is illustrated in Fig 6 Three parame-

ters of interest in such traces are the area under the transition peak, which is proportional

to the enthalpy of the transition; the width of the transition, which gives a measure of the

‘cooperativity’ of the transition; and the transition temperature T, itself The enthalpy of

the transition reflects the energy required to melt the acyl chains, whereas cooperativity reflects the number of molecules that undergo a transition simultaneously

It is worth emphasizing two general points First, gel-state lipids always assume an overall bilayer organization, presumably because the interactions between the crystalline

acyl chains are maximized Thus, the non-bilayer hexagonal (H,) or other phases dis-

cussed in the following section are not available to gel-state systems Second, species of naturally occurring lipids exhibit broad non-cooperative transitions due to the heteroge- neity in the acyl chain composition Thus, sharp gel-liquid-crystal transitions, indicating highly cooperative behavior, are observed only for aqueous dispersions of molecularly well-defined species of lipid

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+ 32 mol%

Temperature (K) Fig 6 The phospholipid gel-liquid-crystalline phase transition and the effect of cholesterol (A) Phospholip- ids, when fully hydrated, can exist in the gel, crystalline form (Lb) or in the fluid, liquid-crystalline state (La)

In bilayers of gel-state PC, the molecules can be packed such that the acyl chains are tilted with respect to the bilayer normal (Lp state) (B) Raising the temperature converts the crystalline state into the liquid-ctystalline phase as detected by differential scanning calorimetry For dipalmitoyl-PC the onset of the main transition

occurs at approximately 41°C The pretransition represents a small endothermic reorganization in the packing

of the gel-state lipid molecules prior to melting (C) Influence of cholesterol The enthalpy of the phase transition (represented by the area under the endotherm) is dramatically reduced At greater than 30 mol% choles-

terol, the lipid phase transition is effectively eliminated

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‘The code denotes the number of carbons per acyl chain and the number of double bonds A gives the position

of the double bond, c denotes cis

bPC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PG, phosphatidylglycerol;

PA, phosphatidic acid

The calorimetric behavior of a variety of synthetic phospholipids is given in Table IV There are three points of interest First, for the representative phospholipid species, PC, there is an increase in T, by approximately 20°C as each two-carbon unit is added and a corresponding increase in enthalpy (2-3 kcallmol) Second, inclusion of a cis double bond at C-9 results in a remarkable decrease in T,, which is further lowered as the degree

of unsaturation is increased Inclusion of only one cis-unsaturated fatty acid at the sn-1 or sn-2 position of the glycerol backbone is sufficient to lower T, from 41°C for dipalmitoyl-PC to -5°C for the palmitoyl-oleoyl species, a major molecular subspecies of PC in biological membranes A final point is that the T, and enthalpy are also sensitive to the head-group constituent For example, molecular species of PE commonly exhibit T, values 20°C higher than corresponding species of PC The data of Table IV have some pre- dictive value in that approximate values of T, can be estimated for other molecular species of lipids

The calorimetric behavior of individual lipid species cannot be directly related to the behavior of the complex lipid mixtures found in biological systems; therefore, consider- able attention has been devoted to the properties of mixtures of pure lipid species Two general features have emerged First, when all component lipids are liquid crystalline (that is, T > T,), the lipid systems exhibit characteristics consistent with complete mixing

of the various lipids Second, at temperatures below the T, of one of the constituents, separation of the component with the highest melting temperature into crystalline domains (lateral phase separation) can occur under certain conditions For example, equimolar mixtures of two saturated PCs differing by four carbon units or more (ATc > 2OOC)

can exhibit lateral phase separation (indicated by calorimetric and freeze-fracture studies)

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Further studies of the calorimetric behavior of lipid systems have emphasized the remarkable physical properties of cholesterol [ 10,141 This lipid has the ability to inhibit the crystallization of lipids to form gel-state systems, as illustrated for dipalmitoyl-PC

in Fig 6C The enthalpy of the transition is progressively reduced as the cholesterol content is increased, until for PC:cholesterol molar ratios of 2:l and less, no transition is observable Such mixtures exist in the ‘liquid-ordered’ phase, in which the membranes are ‘fluid’ but highly ordered (as characterized by 2H-NMR and DSC measurements)

Gel-liquid crystalline transitions profoundly influence the motional properties of lipids and therefore are readily detected by NMR techniques In the liquid crystalline phase, lipids can rotate rapidly about their long axis and diffuse rapidly in the plane of the bilayer In the gel phase, such motions are inhibited 2H-NMR is of particular utility for characterizing both structure and motion in the hydrocarbon region of liquid crystalline bilayers The extent of molecular motion of any C-2H bond can be quantified by an order parameter S, derived from the width of 2H-NMR spectra, where S = 1 indicates a fully ordered system and S = 0 indicates isotropic (completely disordered) motion where the 2H nucleus is able to assume all possible orientations with respect to the magnetic field within -lo4 s A plot of the order parameter values for each position of an acyl chain, referred to as an order profile, can be generated employing phospholipids labeled spe- cifically in the acyl chain region or, more conveniently, by employing phospholipids containing perdeuterated fatty acids [ 141 Hydrocarbon regions of bilayer systems exhibit

a characteristic order profile with a ‘plateau’ region near the headgroup, after which the order decreases rapidly towards the center of the bilayer [14] Hydrocarbon order can be modulated by a variety of factors such as cholesterol or increased acyl chain saturation, both of which lead to larger order parameters

The relation between the gel-liquid crystalline properties of lipids and the roles of lipids in biological membranes remains obscure Suggestions that particular lipids may segregate into gel domains within a biological membrane, with possible effects on protein function (due to restricted mobility) or membrane permeability (due to packing defects), suffer in two respects First, there is simply no evidence for the presence of gel- state lipid components at physiological temperatures in eukaryotic membranes, although this has been suggested for certain prokaryotic systems Second, there is no obvious mechanism whereby lateral segregation of lipid into crystalline domains might be regulated Clearly, an organism cannot regulate fluidity by regulating temperature; thus, any such mechanism would require physiological factors that would isothermally modulate the local lipid composition The presence of such factors has not been unambiguously demonstrated

The theme that membranes do not require the presence of gel-state lipids is easily developed for eukaryotic membrane systems, such as the well-characterized erythrocyte membrane Of the erythrocyte membrane lipids, only sphingomyelin (with a T, close to physiological temperatures) could possibility form local crystalline domains However, the presence of equimolar levels of cholesterol would be expected to inhibit such formation, in agreement with the observation that no reversible phase transition is observable

in the intact erythrocyte (ghost) membrane by calorimetric or other techniques In other membranes which contain little or no cholesterol, such as the membranes of various sub-

~ 4 1

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cellular organelles, the absence of gel-state domains is indicated by the absence of relatively saturated lipid species, such as sphingomyelin, as well as by the increased unsaturation of other lipids present

In summary, available evidence indicates that membranes require a fluid bilayer matrix for function and that modulation of local fluidity and function by formation of crystalline domains is unlikely to be a general phenomenon The requirement for a liquid crystalline lipid matrix is more likely related to the consequent ability of lipids and proteins to diffuse rapidly in the plane of the membrane Liquid crystalline lipids exhibit

lateral diffusion rates ( D J of cm2/s or larger, whereas membrane proteins have D,

values of cm2/s or smaller This relates the average distance d a molecule can dif-

fuse in a time At via the relation d2 = 4D,At Thus, a liquid-crystalline lipid in a cell of

1 0 p m diameter would be able to diffuse the length of the cell within 25 s

H, structure Lipids which form micellar structures, such as lyso-PC, are minority components of membranes Second, the HU phase, which consists of a hydrocarbon matrix penetrated by hexagonally packed aqueous cylinders with diameters of about 2 nm, is not compatible with maintenance of a permeability barrier between external and internal compartments This immediately raises questions concerning the functional role of lipids which preferentially adopt this structure in isolation Finally, in contrast with the situa- tion for gel-state (crystalline) lipids, all biological membranes contain an appreciable fraction (up to 40 mol%) of lipid species which prefer the H, arrangement

The ability of lipids to adopt different structures on hydration is commonly referred to

as lipid polymorphism Three techniques which have been extensively employed to monitor lipid polymorphism are X-ray diffraction, 31P- and 2H-NMR, and freeze-fracture procedures X-Ray diffraction is the classical technique, allowing the detailed nature of the phase structure to be elucidated The use of 31P-NMR for identification of polymor- phic phase characteristics of phospholipids relies on the different motional averaging mechanisms available to phospholipids in different structures and provides a convenient and reliable diagnostic technique Freeze-fracture electron microscopy allows visualiza- tion of local structure which need not be arranged in a regular lattice, yielding information not available from X-ray or NMR techniques

The 31P-NMR and freeze-fracture characteristics of bilayer and Hn phase phospholipid systems are illustrated in Fig 7 Bilayer systems exhibit broad, asymmetric 31P- NMR spectra with a low-field shoulder and high-field peak separated by about 40 ppm, whereas H, phase systems exhibit spectra with reversed asymmetry which are narrower

by a factor of two The difference between bilayer and H, phase 31P-NMR spectra arises from the ability of H, phase phospholipids to diffuse laterally around the aqueous channels Freeze-fracture techniques show flat, featureless fracture planes for bilayer systems,

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