Manual on Membrane Lipids PDF
Preview Manual on Membrane Lipids
SPRINGER LAB MANUAL Springer-Verlag Berlin Heidelberg GmbH R. Prasad (Ed.) Manual on Membrane Lipids With 46 Figures Springer PROF. DR. RAJENDRA PRASAD Jawaharlal Nehru University School of Life Sciences 110067 New Delhi India Library of Congress Cataloging-in-Publication Manual on membrane lipids [edited by R. Prasad]. p. cm.-(Springer lab manual) lncludes bibliographical rcferences ISBN 978-3-642-48970-9 ISBN 978-3-642-79837-5 (eBook) DOI 10.1007/978-3-642-79837-5 1. Membrane lipids - Laboratory manuals. 1. Prasad, R. (Rajendra). 1947-. Il. Series. QP752.M45M36 1996 95-25142 574.87' 5-dc20 This work is subject to copyright. Ali rights are rcserved, whether the whole or pan of the material is concemed. specifically the rights of translation, rcprinting, reuse of illustrations, recitation. broadcasting. reproduction on microfilm or in any other way. and storage in data banks. Duplication of this publication or parts thcreof is permitted only under the provisions of thc German Copyright Law of September 9, 1965, in its current version, and permissions for usc must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. Product liability: The publishers cannot guarantee the accuray of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. © Springer-Verlag Berlin Heide1berg 1996 Originally published by Springer-Verlag Berlin Heide1berg New York in 1996 The use of general descriptive names. registered namcs, trademarks, etc. in this publication does not imply. cven in the absence of a specific statement. that such names arc exempt from the relevant protective laws and regulations and thercforc free for general use. Covcr design: Struve & Partner. Heidelberg Typesetting:Best-Set Typesetters, Hong Kong SPIN 10123274 39/3137-5 4 3 2 1 O-Printed on acid-frce paper Membrane research has reached a point where interdiscipli~ nary collaboration is the only pragmatic strategy to resolve the mysteries of structure and function of supermolecular mem brane organisation. The efforts of membrane biologists in the last few decades have concentrated in characterising the two major components of the membrane, i.e. protein and lipids, encompassing fields like pure chemistry, biochemistry and biophysics. It should, however, be acknowledged that mem brane proteins have received considerably more attention compared with membrane lipids. This was partly owing to the fact that initially, the role of membrane lipids was thought only to be the provision of the hydrophobic core of the lipid bilayer. The reason for the presence of a variety of membrane lipids in a lipid bilayer was frequently questioned, especially when o ne type of lipid, e.g. phosphatidylcholine, alone can form a lipid bilayer. Such questions have since been answered. The dis covery of a signal transduction mechanism, involving not only polyphosphatidylinositides but also phosphatidylcholines and sphingomyelins etc., has opened an en tirely new dimension. In addition, several other emerging lines of evidence concerning roles of membrane lipids are providing further evidence for their multifaceted characteristics. These recognitions led to a renewed interest in lipid research, and have resulted in the publication of several excellent volumes dealing with the vari ous and diverse roles oflipids. However, none ofthese is meth odologically oriented to study membrane lipids. The present volume is a systematic attempt to fill that gap, and has there fore been carefully planned and presented as a "user-friendly" VI Preface book. The protocol of a given method is preceded by the relevant background of each method, and is described in such a manner that it can be adapted to most laboratory situations. The final selection of the methods routinely used to study membrane lipids was extremely difficult, since no manual can claim to contain all methods employed to study membrane lipid behaviour. This manual provides only a selection of the methods which are frequently followed in laboratories to study structure and function of membrane lipids. All the methods presented in this manual have been contributed by investiga tors who use them routinely. The protocols described here also include minor details to help researchers. Wherever possible, various tips, tricks and precautions etc. are discussed. In addi tion, certain protocols are also highlighted at relevant places in order to emphasise the instructions or tips at that particular step. I have included a brief introductory chapter (Chap. I) on lipids, so that the researcher keen to follow the suggested methods has easy access to the details of common membrane lipids. Before identifying and characterising membrane lipids, the first prerequisite is to have pure membrane fractions as starting material. Chapter II deals exclusively with some well tried methods of membrane isolation. The limited availability of the pure membrane fraction has been the biggest handicap, and as a result, in several instances lipids of pure membrane fractions are still not known. Obviously, isolation methods of all subcellular membranes of different origin could not be listed. Chapter II describes only selected protocols of isolation of a few types of membranes. One must, however, consult relevant references, especially in Methods in Enzymology, for different methods of isolation of membranes. Chapters III and IV describe step by step instructions to isolate, identify and quantify the membrane lipids. Again, protocols listed in these chapters are described only as examples which can be adapted to individual needs. Chapters V to IX deal with common approaches used to study asymmetry, phase transition, lipo- Preface VII some formation and reconstitution, turnover and signal transduction of membrane lipids. These chapters are written by an expert in the respective field. As far as possible, methods have been described in steps that can be easily followed. How ever, in cases where this has not been possible, the method is discussed in the form of advice and examples. It is sincerely hoped that this volume will help those who are seeking common methods of membrane lipid isolation, characterisa tion, and the study of membrane phenomena, compiled in a single manual. Spring 1995 Rajendra Prasad New Delhi Acknowledgements My deepest gratitude is to my wife, Vibha, whose endurance has no parallel. She has been very supportive of this project, and very generous in allowing me the time it required. I am grateful to all my students, especially Pranab and Anjni, for offering me their unqualified support during the preparation of various drafts of this Volume. Without them, it would have been impossible to meet this academic and physical challenge. I am indebted to Ali Bhai, Jyotsna, Archana, Sanjeev, Krishnamurthy, Deepa and Raj Kishore, who read part of the text and offered helpful suggestions. I would also like to thank all my contributors who readily helped in producing the manual, and translating the concept into reality. Finally, I acknowledge the countless fond memories of my darling daughter, Juhi, who suddenly left us exactly two years ago, but who remains a constant companion and encourages me to keep going. Rajendra Prasad Spring 1995 New Delhi Contents Preface................ .... ............. ........ v Chapter I Structure and Distribution of Membrane Lipids RAJENDRA PRASAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 Fatty Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 Phosphoglycerides . . . . . . . . . . . . . . . . . . . . . . . . . 5 4 Sphingolipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 5 Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Chapter II Isolation of Pure Membrane Fractions for Lipid Analysis LEV A. 0KOROKOV, R.A. ZVYAGILSKAYA, L.P. LICHKO, T.V. KULAKOVSKAYA, V.P. YURINA AND M.S. 0DINTSOVA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 1 Background............................... 16 2 Isolation of Yeast Plasma Membrane by Mechanical Disruption . . . . . . . . . . . . . . . . . . . . . 17 3 Isolation of Yeast Plasma Membrane by Spheroplast Formation . . . . . . . . . . . . . . . . . . . . . 20 4 Isolation of Con A-Modified Plasma Membranes ................ .............. . 24 5 Isolation of Intracellular Organelles of Yeast and Their Membranes ..................... . 25 6 Fractionation of Membranes on a Sucrose Density Gradient ................. ........ . 27
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