New comprehensive biochemistry vol 04 phospholipids

PHOSPHOLIPIDS


New Comprehensive Biochemistry

Volume 4

General Editors

A. NEUBERGER
London

L.L.M. van DEENEN
Utrecht

ELSEVIER BIOMEDICAL PRESS
AMSTERDAM-NEWYORK-OXFORD


Phospholipids
Editors

J.N. HAWTHORNE and G.B. ANSELL
Nottingham and Birmingham

1982

ELSEVIER BIOMEDICAL PRESS
AMSTERDAM. NEW YORK*OXFORD

0 Elsevier Biomedical Press, 1982
All rights reserved. N o 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 3
ISBN for the volume: 0444 80427-7

Published by:

Elsevier Biomedical Press
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1000 BM Amsterdam, The Netherlands
Sole distributors for the LI.S.A.and Canada:
Elsevier Science Publishing Company Inc.
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Library of Congress Cataloging in Publication Data
Main entry under title:
Phospholipids.
(New comprehensive biochemistry; v. 4)
Includes bibliographical references and index.
1. Phospholipids. 2. phospholipids-Metabolism.
I. Hawthorne, J.N. (John Nigel) 11. Ansell, G.B.
(Gordon Brian) 111. Series.
QD41S.N48 VOI.4 574.19'2s [574.19'214] 82-18382
[QP752.P53]
ISBN 0-444-80427-7 (U.S.)

Printed in The Netherlands


To the memory of Maurice Gray (1930-1980),
a good friend and dedicated lipid
biochemist.


This Page Intentionally Left Blank


Preface
In the general preface to the original series of volumes entitled Comprehensive
Biochemistry, Florkin and Stotz stated: “The Editors are keenly aware that the
literature of biochemistry is already very large”. Even so, the chemistry of the
phospholipids formed only part of Vol. 6 (1965) and the whole of lipid metabolism
was covered in Vol. 18 published in 1970, of which only a small part was concerned
with phospholipid metabolism. For the present series, therefore, we were charged by
the General Editors to produce a volume on phospholipids which was to emphasise
metabolic aspects since their structural role in membranes was covered in Vol. 3. We
had to ensure coverage of developments in the last decade while, at the same time,
summarising essential findings of earlier periods.
There are various ways in which the book could have been organised. As will be
seen, we finally decided to devote separate chapters to individual or closely related
phospholipids in which the essential chemistry is first described followed by an
account of the metabolism, due regard being paid to the pioneering work of the past.
We have included a chapter on phospholipases in general and one on phospholipase
A2 since its structure and the mechanism of its action have been investigated in
greater detail than any other phospholipid metabolising enzyme. The increasingly

important topic of phospholipid exchange proteins is also treated separately. Furthermore, since the use of biochemically defined mutants shows great promise for
the better understanding of phospholipid biosynthesis and function, a chapter has
been devoted to genetic control of the enzymes involved.
This book is intended for advanced students and research workers and we believe
that it gives a comprehensive, though not exhaustive, account of phospholipid
biochemistry, Throughout, the reader will discover how advances in techniques have
added to our knowledge of the ever-expanding field. Though it is difficult sometimes
to avoid the impression that all research work is confined to the liver we hope that
key references to other organs and other organisms will enable those whose interest
lies outside the peritoneal cavity to be satisfied.
If the contents of the book belie the general title of the series, the responsibility
lies with the editors not the authors and we would appreciate comments on errors
and omissions.
We are grateful to Mrs. J. Paxton for her help in the preparation of the subject
index.
J.N. Hawthorne
G.B . Ansell

Nottingham and Birmingham, August 1982


Contents
Preface
Chapter I . Phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, by G.B. Ansell and
S. Spanner

vii

i

1
1
1
4
4

1. lntroduction
2. Discovery and chemistry
(a) Phosphatidylcholine and lysophosphatidylcholine
(b) Phosphatidylethanolamine
(c) Phosphatidylserine
3. Determination and distribution in animal tissues
4. Biosynthesis
(a) Phosphatidylserine
(i) Base-exchange
(ii) Other reactions
(b) Phosphatidylethanolamine
(i) Decarboxylation of phosphatidylserine
(ii) Cytidine pathway
(iii) Base-exchange reaction
(iv) Acylation of lysophosphatidylethanolamine
(v) General comments on phosphatidylethanolamine synthesis
(c) Phosphatidylcholine
(i) Stepwise methylation
(ii) Cytidine pathway
(iii) Base-exchange
(iv) Acylation of lysophosphatidylcholine
(v) Transacylation of lysophosphatidylcholine
(vi) Metabolism of phosphatidylcholine in the lung
5. Catabolic pathways

6. Aspects of sub-cellular metabolism
7. Transport in the body
(a) Absorption and the formation of chylomicrons
(b) High-density lipoproteins
(c) The liver and the production of phospholipids for bile and plasma
(d) Metabolism in amniotic fluid
8. The effects of drugs and other agents on metabolism
(a) Some effects on biosynthesis
(b) The modulation of methylation and decarboxylation by drugs and neurotransmitters
(c) Phosphatidylcholine and acetylcholine synthesis in the brain
(d) Roles of phosphatidylserine
9. Conclusion
References

6
7
7
8
9
9
12
12
12
13
14
14
16
17
17
18

20
23
28
28
29
29
33
33
34
34
39
40
41
41

Chapter 2. Plasmalogens and 0-alkyl glycerophospholipids, by L.A. Horrocks and M.Sharma

51

1. Introduction
2. Nomenclature

5

6

51
51

3. Discovery and structure
4. Methods and chemical properties

52
53
55

5. Chemical synthesis
6. Content and composition
(a) Bacteria
(i) Phytanyl ethers
(ii) Plasmalogens
(b) Protozoa. fungi, and plants
(c) Invertebrates
(d) Fish
(e) Mammals and birds
(i) Heart and skeletal muscle
(ii) Nervous system
(iii) Other organs
(0 Neoplasms
7. Biosynthetic pathways
(a) Synthesis of long-chain alcohols
(b) Synthesis of 0-alkyl bonds
(c) Synthesis of plasmalogens
8. Catabolic pathways
9. Turnover of ether-linked glycerophospholipids
10. Platelet activation factor
11. Function and biological role
References

56
56
56
58
60
60
61
62
63
63
68
71
72
72
73
75
79
81
81
83
85

Chapter 3. Phosphonolipids, by T. Hori and Y. Nozawa

95

1. Historical introduction and classification
2. Methods of isolation and characterization
(a) Isolation and purification
(b) Characterization

(i) Infrared spectrometry of intact phospholipids
(ii) Gas-liquid chromatography and mass spectrometry
(iii) Nuclear magnetic resonance spectroscopy
3. Occurrence and distribution
(a) Qualitative and quantitative distribution of phosphonolipids
(b) Fatty acid and sphingosine base compositions
4. Metabolism
(a) Biosynthesis
(i) 2-Aminoethylphosphonic acid (AEPn)
(ii) Glycerophosphonolipids (GPnL)
(iii) Sphingophosphonolipids (SPnL)
(b) Degradation
5 . Phosphonolipids and membranes of Tetrahymena
(a) Intracellular distribution
(b) Mechanism for enrichment of GPnL in the surface membranes
(c) Roles in membrane lipid adaptation
(i) Temperature
(ii) Nutrition
(iii) Alcohols
(iv) Aging
6. Other possible physiological functions
References

95
97
97
98
98
98
99

99
99
103
107
107
107
107
111

111
112
112
115
115

1 I7
121

124
124
125
125


Chapter 4. Sphingomyelin: metabolism, chemical synthesis, chemical and physical properties, by ,Y.
129
Barenholz and S. Gatt

(a) Sphingomyelin composition
2. Total and partial chemical synthesis of sphingomyelin

(a) Complete chemical synthesis of sphingomyelin
(i) Synthesis of LCB
(ii) Synthesis of ceramide
(iii) Synthesis of sphingomyelin
(b) Partial chemical synthesis of sphingomyelin
(c) Determination of sphingomyelin stereospecificity
3. Metabolic pathways of biosynthesis and degradation
(a) Biosynthesis of sphingomyelin
(b) Enzymic degradation of sphingomyelin
(c) Niemann-Pick disease
4. Physical properties of sphingomyelin
(a) Atom numbering
(b) Molecular structure of sphingomyelin
(c) Studies on monomolecular films
(d) Solubility in organic solvents
(e) Thermotropic behaviour
(f) Molecular motions of sphingomyelin in bilayers
5. Interactions of sphingomyelin with other lipids
(a) Interaction of sphingomyelin with phosphatidylcholine
(b) Interaction of sphingomyelin with cholesterol
6. Interaction of sphingomyelin with detergents
(a) Interaction with Triton X-100
(b) Interaction of sphingomyelin with bile salts
7. Interaction of sphingomyelin with proteins
8. Sphingomyelin in biological systems
(a) Distribution
(b) Membrane asymmetry
(c) Changes in sphingomyelin distribution associated with aging and pathological conditions
(d) Membrane integrity and membrane properties
(i) Membrane integrity

(ii) Mechanical properties and apparent microviscosity
(iii) Permeability and transport in membranes
9. Summary and conclusions
References

129
129
130
131
131
131
132
132
132
133
133
134
136
137
137
137
140
141
141
149
149
150
151
153
153

155
155
159
159
161
161
164
164
165
165
166
168

Chapter 5. Phosphatide metabolism and its relation to triacylgt'ycerol biosynthesis, by D.N. Brindley
and R. G. Sturton

179

1. Introduction

1. Introduction
2. Biosynthesis of phosphatidate

(a) From glycerophosphate
(b) From dihydroxyacetone phosphate
(c) From monoacylglycerols and diacylglycerols
3. The relative contribution of the glycerophosphate and dihydroxyacetone phosphate pathways
to the synthesis of glycerolipids
4. Control of phosphatidate synthesis
5. Conversion of phosphatidate to CDP-diacylglycerol

179
179
179
183
184
185
187
194


6. Conversion of phosphatidate to diacylglycerol
7. Deacylation of phosphatidate
8. Effects of ions in the direction of phosphatidate metabolism
9. Physiological control of PAP activity and triacylglycerol synthesis
10. Conclusion
References

194
197
198
20 I
206
207

Chapter 6. Polyglycerophospholipids: phosphatidylglycerol, diphosphatidvlglycerol and bis(monoacylglycero)phosphate, by K.Y. Hosretler

215

1. Introduction

2. Discovery of polyglycerophospholipids
(a) Diphosphatidylglycerol
(b) Phosphatidylglycerol
(c) Eis(monoacylg1ycero)phosphate
3. Structural and stereochemical investigations
(a) Diphosphatidylglycerol
(b) Phosphatidylglycerol
(c) Eis(monoacylg1ycero)phosphate and related compounds
4. Distribution and properties of polyglycerophosphatides in animals, plants and microorganisms
(a) Distribution in nature
(b) Fatty acid compositions of polyglycerophosphatides from some mammalian sources
5. Biosynthesis of the polyglycerophospholipids
(a) Phosphatidylglycerol synthesis
(b) Phosphatidylglycerophosphatase
(c) Diphosphatidylglycerol biosynthesis
(d) Biosynthesis of bis(monoacylg1ycero)phosphate and acylphosphatidylglycerol
6. Degradation of polyglycerophospholipids
(a) Phosphatidylglycerol
(b) Diphosphatidylglycerol
(c) Eis(monoacy1glycero)phosphate
7. The subcellular localization of polyglycerophospholipids and their biosynthetic pathways
(a) Phosphatidylglycerol
(b) Diphosphatidylglycerol
(c) Eis(monoacylg1ycero)phosphate
8. Phosphatidylglycerol in pulmonary surfactant and amniotic fluid
9. Lipid storage diseases and bis(monoacylg1ycero)phosphate metabolism
(a) Congenital conditions
(b) Acquired lipidoses
(c) Possible mechanism of bis(monoacylg1ycero)phosphate storage
10. Concluding remarks

References

215
216
216
217
217
218
218
219
220
22 1
22 1
226
228
228
23 1
232
235
238
238
238
240
24 1
24 1
244
246
247
249
250

25 1
252
253
255

Chapter 7. Inositol phospholipids, by J.N. Hawthorne

263

1. Discovery
2. Chemistry
(a) Phosphatidylinositol and its phosphates
(b) Phosphatidylinositol mannosides
(c) Sphingolipids containing inositol
3. Distribution in tissues and fatty acid composition
(a) Distribution
(b) Fatty acid composition

263
263
263
265
266
267
267
268


4.

Biosynthesis
(a) Phosphatidylinositol
(b) Phosphatidylinositol phosphates
(c) Phosphatidylinositol mannosides
(d) Sphingolipids containing inositol
5. Catabolic pathways
(a) Hydrolysis of phosphatidylinositol
(b) Hydrolysis of polyphosphoinositides
(c) Hydrolysis of other inositol lipids
6. Subcellular localization of metabolic pathways
7. Phosphoinositide metabolism and receptor activation
(a) Phosphatidylinositol
(b) The calcium-gating hypothesis
(c) The role of polyphosphoinositides
8. Inositol lipids and diabetic neuropathy
9. Conclusions
References

268
268
269
270
270
270
270
27 I
27 1
27 1
272
272

273
214
276
276
276

Chapter 8. Phospholipid transfer proteins, by J.-C. Kader, D. Douady and P. Muzliak

2 79

I. Discovery
2. Methods for the determination of transfer activities
(a) Transfer between natural membranes
(b) Transfer between artificial and natural membranes
(c) Transfer between liposomes
3. Distribution in living cells
(a) Animal cells
(i) Beef tissues
(ii) Rat tissues
(iii) Human plasma
(b) Plants and microorganisms
4. Biochemical properties
(a) Isoelectric point, M,-value and amino acid composition
(b) Molecular specificity
(c) Specificity for membranes
(d) Immunological properties
5. Mode of action
(a) Phospholipid transfer proteins as carriers
(i) Phospholipid monolayers
(ii) Binding experiments

(b) Interactions between phospholipids and phospholipid transfer proteins
(c) Net transfer
(i) Transfer proteins are able to insert PI or PC into membranes deficient in these
phospholipids
(ii) Transfer proteins are able to leave the membrane devoid of any lipid, after the
transfer process
(iii) Transfer proteins are able to catalyze a net mass transfer
(d) Control of phospholipid transfer activity by membrane properties
(e) Different steps of the exchange process
(i) Binding of phospholipid to the protein
(ii) Formation of a collision complex between the proteins and the membrane
(iii) Release of phospholipid

219
280
280
282
282
283
284
284
285
286
286
287
287
29 1
292
292
292

292
292
293
294
296
296
296
297
297
299
299
299
300


(iv) Detachment of phospholipid from the membrane
(v) Detachment of the protein with or without bound phospholipid
6. Phospholipid transfer proteins as tools for membrane research
(a) Asymmetric distribution and transbilayer movement of lipids
(i) Liposomes
(ii) Erythrocytes
(iii) Mitochondria
(iv) Microsomes
(v) Microorganisms
(b) Manipulation of the phospholipid composition
7. Physiological role
8. Conclusions
References

300

300
300
30 I
301
30 1
302
303
303
304
304
307
307

Chapter 9. Phospholipases, by H . van den Bosrh

313

1. Introduction
2. Phospholipases A ,
(a) Occurrence and assay
(b) Purified enzymes and properties
3. Phospholipases A ,
(a) Occurrence and assay
(b) Purified enzymes and properties
(c) Regulatory aspects
(i) Regulation of phospholipase A, activity by zymogen-active enzyme conversion
(ii) Regulation of phospholipase A , activity by availability of Ca2+ ions
(iii) Regulation of phospholipase A activity by interaction with regulatory proteins
4. Lysophospholipases
(a) Occurrence and assay

(b) Purified enzymes and properties
5. Functions of phospholipases A and lysophospholipases
(a) Phospholipid turnover
(b) Release of prostaglandin precursors
6. Phospholipases C
(a) Occurrence and assay
(b) Purified enzymes and properties
7. Phospholipases D
(a) Occurrence and assay
(b) Purified enzymes and properties
8. Concluding remarks
References

313
314
3 I4
316
320
320
32 I
323
324
324
325
327
327
33 1
334
334
335

337
337
340
344
344
348
3 50
35 1

Chapter 10. On the mechanism ofphospholipase A , . by A . J . Slotboom, H . M . Verheij and G.H. de
Haas

359

1.
2.
3.
4.

Introduction
Purification and assays
Structural aspects
Kinetic data
(a) Monomeric substrates
(b) Micellar substrates
(i) Micelles of short-chain lecithins

359
360
363

368
369
371
37 1


(ii) Mixed micelles of phospholipids with detergents
(c) Monomolecular surface films of medium-chain phospholipids
(d) Phospholipids present in bilayer structures
(e) Reversible inhibition of phospholipase A,
(f) Monomeric or dimeric enzymes or higher aggregates?
5. Chemically modified enzymes
(a) Specific amino acids
(i) Sulphydryl groups and serine
(ii) Histidine
(iii) Tryptophan
(iv) Methionine
(v) Lysine
(vi) Carboxylate groups
(vii) Arginine
(viii)a-Amino group
(ix) Tyrosine
(b) Miscellaneous
(i) Modifications of PLA with ethoxyformic acid anhydride
(ii) Cross-linking of PLA
(iii) Photoaffinity labelling
(iv) Semisynthesis of pancreatic phospholipase A
6. Ligand binding
(a) Binding of Ca2+
(i) Pancreatic phospholipases A

(ii) Venom phospholipases A,
(b) Binding of monomeric zwitterionic substrate analogues
(c) Binding to aggregated lipids
(i) Pancreatic PLA
(ii) Snake venom PLA
7. Immunology
8. The 3-dimensional structure
9. Catalytic mechanism
10. Prospects
References

374
377
379
387
387
389
389
390
390
392
393
394
395
396
397
398
399
399
40 1

‘401
40 1
404
404
404
405
407
409
409
413
414
415
419
424
426

Chapter I ! . Genetic control of phospholipid bilayer assembly, by C.R.H . Raetz

435

1. Introduction
2. Approaches to the isolation of Escherichia coli mutants defective in phospholipid metabolism
(a) Isolation of auxotrophs and supplementation of phospholipids by fusion
(b) Analogs or inhibitors of metabolism
(c) Radiation suicide
(d) ‘Brute force’
(e) Enzymatic colony sorting on filter paper
3. Genetic approaches to phospholipid metabolism in yeasts and fungi
4. Genetic approaches to phospholipid metabolism in higher mammalian cells
(a) Transfer of animal cell colonies to filter paper and its application to somatic cell genetics

5. General properties of E. coli phospholipid mutants
6. E. coli mutants in phosphat;.dic acid synthesis
(a) Glycerol-3-phosphate acyltransferase K, mutants ( plsB)
(b) Mutants in the biosynthetic glycerol-3-phosphate dehydrogenase (gps-4)
(c) Mutants in diacylglycerol kinase ( d g k )

435
436
436
437
437
438
438
441
442
442
445
447
447
45 1
45 1


7. E. coli mutants in CDP-diacylglycerol synthesis

(b) Cytidine auxotrophs ( p y r G )
(c) CDP-diacylglycerol hydrolase (cdh )
8 . E. coli mutants in phosphatidylethanolamine synthesis
(a) Phosphatidylserine synthase ( p s s )
(b) Phosphatidylserine decarboxylase ( p s d )

9. E. coli mutants in polyglycerophosphatide synthesis
(a) Phosphatidylglycerophosphate synthase ( pgsA and pgsB)
(b) Cardiolipin synthase ( c l s )
10. E. coli mutants in membrane lipid turnover and catabolic enzymes
(a) Mutants unable to generate membrane-derived oligosaccharides
(b) Mutants in catabolic enzymes ( pldA)
1 1. Molecular cloning of E. coli genes coding for the lipid enzymes
12. Further genetic approaches to the control of E. coli phospholipid gene expression
13. Choline and inositol auxotrophs of fungi and yeasts
(a) Neurospora crassa
(b) Saccharomyces cereoisiae and other yeasts: inositol auxotrophs
(c) Choline auxotrophs of S. cereoisiae
14. Genetic modification of membrane phospholipid synthesis in mammalian cells
(a) Characterisation of inositol auxotrophs of CHO cells
(b) Autoradiographic detection of CHO mutants defective in phosphatidylcholine synthesis
(c) Other in situ assays for detection of lipid enzymes in CHO colonies
15. Summary
References

452
452
454
454
455
455
456
456
456
458
458

458
459
459
462
464
464
465
466
468
468
468
412
412
474

Subject Index

419

(a) CDP-diacylglycerol synthase (cds)


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1
CHAPTER 1

Phosphatidylserine, phosphatidylethanolamine
and phosphatidylcholine

G.B. ANSELL and S. SPANNER
Department of Pharmacology, The Medical School, Birmingham B15 2TJ, U.K.

I . Introduction
This chapter deals with the metabolism of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in mammalian cells.
Although basic mechanisms for the synthesis and catabolism of these major cell
components have been known for some years there have been many recent investigations on their metabolism and possible function. Therefore this account, while
summarising well-established facts, covers some of the more recent advances in some
detail. Although original sources are usually cited, references to reviews rather than a
series of papers are sometimes given.

2. Discovery and chemistry
(a) Phosphatidylcholine and bsophosphatidylcholine
Between 1846 and 1847 Gobley isolated from egg-yolk and brain a lipid which he
called ‘‘lecithin’’ (Gk. lekithos, egg-yolk) 111 and from which he could obtain
glycerophosphoric acid and fatty acids. Diakanow [2,3] and Strecker [4] showed that
this lipid contained the base choline, originally isolated from hog bile by Strecker [5]
(Gk. chol2, bile) and the two workers were able to deduce a provisional structure for
lecithin. The subsequent hlstory of lecithin was documented by MacLean and
MacLean [6], Wittcoff [7] and Ansell and Hawthorne [8]. It was not until 1950 that
Baer and Kates [9] by chemical synthesis showed that lecithin was based on
L-a-glycerophosphate (L-3-glycerophosphate or D- 1-glycerophosphate, deriving from
D-glyceraldehyde) like all other naturally occurring glycerophospholipids. Other
methods of synthesis are given by Strickland [lo]. The nomenclature of phospholipids has undergone numerous modifications in the last two decades [8,10,11] and
account has been taken of the fact that glycerol does not possess rotational
symmetry. The latest recommendations are those of the IUPAC-IUB Commission
on Biochemical Nomenclature [ 1 11 and the stereospecific numbering system is now
used for all phospholipids. Thus lecithin is 1,2-diacyl-sn-glycero-3-phosphocholine
or
Hawthorne/AnseN (eds.) Phospholipids

0 Elsevier Biomedical Press, I982


TABLE 1
h)

Phosphatidylcholine, phosphatidylethanolamine. phosphatidylserine and lysophosphatidylcholineconcentrations in various tissues
Tissue

Total
phospholipid
( B mol/g)

Phosphatidylcholine
(% TPL)

Phosphatidylethanolamine
(4% TPL)

Phosphatidylserine
(4% TPL)

I2
40 (incl.
plasmal)
34
1
21
24
20

19
24
25
22
26
21
18
30
26

8
13

-

16
21
I
1
6 (with PI)
8
8
8
4
3
4
4
3
3

-

Brain.
grey matter,

rat
man

60.2
50.9

25
39

white matter,
myelin
Kidney

82.8
man
man
rat
36.6
man
22.2
rat
man
rat
17.5
man

24.7
rat
11.3
man
16.9
ox
28.1
guinea pig 30.6
rat
15.2
man
21.5
rat
1.5
man
2.9
rat
4.2
man
3.9
man
436

31
24
34
33
54
53
42

41
51
48
53
50
36
40
64
70
42
29
40

3
23
28
28

37.9
41.3
4.3
14.4

48
44
90
68

24
28

4
12

Lung
Spleen
Skeletal muscle
Pancreas
Heart
Plasma
Erythrocytes
Platelets
(nmol/ 109 ,
Liver
Bile
Amniotic fluid
(pmo1/100 ml)

rat
man
rat
man

1

-

trace
11

14

9
3
3
1

8

Lysophosphatidylcholine
(4% TPL)

Ref.

Unpublished results

-

29

-

1

3
-

3
1
2
3
trace

-

1

4
23
1
4
2
1
1
1
(1

34

46
34
33
313
33
38
36
35. 315
6. 7
3 14

n
3

34

t,

41
12

$
9
3

3


TABLE 2

-_

Major molecular species of phosphatidylcholine in the rat as I& of the total
Fatty acids

Brain

Liver

Lung

Kidney

R.b.c.

Plasma

Gastric

Intestine

Bile

2
B

mucosa
16:0/16:0
18 :0/16 :0

16.0
4.0

4.0

16:0/16: 1
16:0/18: 1

-

-

30.0

4.0

-

-

-

-

-

-

-

6.5

-

9

As

-

-

-

31.0
7.0

-

-

14.5

5.5

12.0, 8.0
10.0. 16.6

-

-

-

-

-

-

-

-

-

-

18:0/18: 1

12.0

-

-

-

-

-

14.0

-

-

16 :O / 18 : 2
I8 :0/18 : 2

10.0

12.4
5.6

19.4
16.0

-

-

-

24.6
15.8

53.7
9.9

18: 1/16:0

10.0

-

12.0

7.0

-

-

-

-

-

I6 :0/18: 3

-

-

-

-

-

-

6.0

16:0/20:4
18:0/20:4

8.0
10.0

15.0

22.0
25.0

16:0/22:6
18 :0/22 : 6

-

18:0/16: 1

-

15.7, 20.0
10.0, 9.0, 9.0

18.9, 22.0
25.0, 20.9
4.8
5.6

10.0, 6.4. 8.0
5.6

-

8.0

5.4

7.5

-

-

-

-

-

-

5.8
13.3
6.9
4.8

3

-

-

8.0

a

-

20.3

25.0, 41.4
4

3

9

-

-

14.7

-

14.0
8.6

-

-

-

-

-

-

-

3

0
3
(D

-

Values taken from 149-54).

w


4

G.B. Ansell and S. Spanner

3-sn-phosphatidylcholine. In most naturally occurring lecithins the l-position is
esterified with a saturated fatty acid and the 2-position with an unsaturated one but
there are notable exceptions (see Table 2).
Lysolecithin ( 1-, or 2-lysophosphatidylcholine, or 1- or 2-acyl-sn-glycero-3-phosphocholine) also occurs naturally in tissues though at very much lower levels than
lecithin (Table 1) [12-141. It is, of course, a significant component of blood plasma
(p. 32) as was found conclusively by Gjone et al. [ls]. It is likely that all naturally
occurring lysolecithins have the 1-acyl structure (note that 1-acyl-sn-glycero-3-phosphocholine is 2-lysophosphatidylcholine) and that the fatty acid is saturated as first
suggested by Liidecke in 1905 [16]. The high levels of lysolecithin reported in the

older literature (see 171) arise as a result of phospholipase activity post mortem.

(b) Phosphatidylethanolamine (3-sn-phosphatidylethanolamine)
Thudichum [ 171 separated a nitrogen- and phosphorus-containing lipid fraction from
brain tissue which he distinguished from lecithin by its relative insolubility in warm
ethanol. He called it “kephalin” and obtained ethanolamine from it as a hydrolysis
product (though he thought this base was a breakdown product of choline and not
naturally occurring). “Kephalin” or “cephalin” is now known to be a mixture of
ethanolamine-, serine-, and inositol-containing phospholipids but it was not until
1913 that one of the contributing bases was shown to be ethanolamine [ 18,191. Rudy
and Page [20] isolated an ethanolamine glycerophospholipid from the cephalin
fraction of brain tissue which they reasonably assumed to be phosphatidylethanolamine. However, it is not easy to separate phosphatidylethanolamine from
tissues on a preparative scale, especially if they contain ethanolamine plasmalogen as
do brain, cardiac muscle and skeletal muscle (see [10,21]) and it is probable that
Rudy and Page’s preparation contained plasmalogen. It is likely in retrospect,
therefore, that the first pure preparations of phosphatidylethanolamine from natural
sources were those of Lea et al. [22] from egg-yolk and Klenk and Dohmen [23] from
liver. The phospholipid was first synthesised by Baer et al. [24]; other methods of
synthesis are given by Strickland [lo]. The fatty acid in the 1-position is usually
saturated and that in the 2-position unsaturated.
(c) Phosphatidylserine (3-sn-phosphatidylserine)

Although MacArthur [25] had demonstrated the presence of a-amino nitrogen in
Thudichum’s “kephalin” it was not until 1941 that Folch and Schneider [26] showed
that an a-amino-P-hydroxylic acid was present. In the same year Folch [27]
identified the amino acid as L-serine and showed that phosphatidylserine was a
component of the kephalin fraction. In 1948 Folch proposed a structure [28] and
Baer and Maurukas [28a] showed that this phospholipid, when reduced, was
which they synthesised.
identical to 1,2-distearoyl-sn-glycero-3-phospho-~-serine

The structures of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine are given in Fig. 1.


Phosphatidylserine, -ethanolamine, -choline

5

c H ~ O -CO-R'
R~O-O-C-H

C H ~ O - PO(OH ) - O C H ~ C H ~ N * ( C
H ~ )

or

(I )

-0 C H p C H 2 NH z

(11)

N HZ

I

or -OCH2CH-COOH

(111)

Fig. 1. 1,2-Diacyl-sn-glycero-3-phosphocholine

(phosphatidylcholine) (i) where R'- and R ' - are the fatty
acyl substituents. In phosphatidylethanolamine (ii) and phosphatidylserine (iii) the choline is replaced by
ethanolamine and serine respectively.

3. Determination and distribution in animal tissues
The phospholipid content of mammalian organs varies from organ to organ and
from species to species. Table 1 gives, where possible, the total phospholipid content
in pmol/g wet weight of tissue and the phosphatidylcholine [ 1 I], phosphatidylethanolamine [ 121, phosphatidylserine [ 131 and lysophosphatidylcholine content as a
percentage of the total phospholipid in two species, rat and man. For a more
comprehensive, but unfortunately now outdated survey the reader is referred to the
chapter by White [29] and to journals relevant to the organs concerned.
Although much work has been carried out on the fatty acid content of phospholipids, the values for the molecular species are more difficult to find. This in part
is due to the rather complex methodology involved. In Table2 values are given for
the molecular species of phosphatidylcholine in various tissues of the rat and in
Tables 3 and 4 values for phosphatidylethanolamine and phosphatidylserine in
muscle and kidney of various animals.
TABLE 3
Molecular species of phosphatidylethanolaminein various tissues of different animals
Skeletal muscle

Kidney

Rat

Dog

Human

Pig

Sarcoplasmic
reticulum of skeletal
muscle
Rabbit

l6:0/18: 1
l8:0/18: I
I6 : 0/18 : 2
16 : 0/20: 4
18 :0/20: 4
18 : 0/22 : 6
18: 1/18: 1
18: l / 2 0 : 4

-

12.1

-

13.1
11.6
13.9
37.3

14.0
21.0
28.0
9.3

10.8
27.3
25.9

-

Values taken from [ 5 5 ] and [56].

10.3
35.6

14.9
17.9
11.9
14.9
23.9

-

-

-

11.2

6.9
18.1

-

6.7
-


G.B. Ansell and S. Spanner

6
TABLE 4

Molecular species of phosphatidylserine as 56 of total in skeletal muscle of the rabbit

16:0/18:1

18:0/18: 1
18 :0/20 :3
18: 0/22 : 3
18 : 0/20 :4
18 :0/22 : 5
18:0/22 :6
a

Sarcoplasmic reticulum a

Sarcotubular vesicle

11.0
8.0
17.0
5 .O
50.0

4.0
-

14.0
8.0
11.0
20.0
8.0
25.0

-

Includes phosphatidylinositol. Values taken from [55) and 157).

The methods for the isolation of phospholipids and their subsequent separation
into classes dependent upon the nature of the base are now well established.
Phospholipids containing choline, ethanolamine and serine are readily extracted into
chloroform-methanol (2 : 1, v/v) from mammalian tissues [30]and this is the
method adopted by most workers. Early methods of isolation of individual lipids by
column chromatography or paper chromatography following the removal of the
fatty acids have, on the whole, given way to two-dimensional thin-layer chromatography. The lipids can then be quantified by the determination of the phosphorus
content or in experiments involving radiolabelled compounds, be assayed for radioactivity. The individual lipids may also be separated on silica gel and assayed for
their fatty acid content by gas-liquid chromatography. For details of these methods
the reader is referred to the reviews by Spanner [21] and Nelson [31].
The determination of the molecular species of the lipid is more complex. For one
method and for relevant references the reader is referred to the paper by Kawamoto
et al. [32].

4. Biosyn thesis
In section 2 the phospholipids were described in the order in which they were

discovered but it seems more logical to reverse this order when discussing the
biosynthetic pathways because it is known that phosphatidylserine can give rise to
phosphatidylethanolamine which in turn can be converted to phosphatidylcholine
though these are not the only pathways.

(a) Phosphatidylserine
There are two established pathways for the biosynthesis of phosphatidylserine, a
phospholipid which accounts for 5- 10%of the total phospholipid in eukaryotic cells.
One is a Ca2+-mediated exchange reaction of L-serine with another phospholipid,


Phosphatidylserine, -ethanolamine, -choline

7

probably phosphatidylcholine, and until very recently, was thought to be the sole
method by which phosphatidylserine is synthesised in animal tissues. The other
pathway is the reaction between CDP-diacyl glycerol and L-serine, confined apparently to bacteria and plants.
(i) Base-exchange reaction
In 1959 Hubscher et al. [58] noted that L-serine could be incorporated into
phosphatidylserine in a liver mitochondria1 preparation by a reaction likely to be
independent of energy supply but dependent on Ca*+ ions. Subsequently, the
energy-independent reaction was shown largely to be confined to the microsomal
fraction (endoplasmic reticulum) [59], with a K,, for serine of about 0.5 mM and
optimum pH of 8.3 with 25 mM Ca2'. In Ehrlich ascites cells, however, the
exchange reaction is found in mitochondria, possibly due to malignant transformation [60]. The reaction has been extensively investigated, particularly in brain tissue,
by Porcellati and his co-workers [61-631. The K,, for serine seems to depend on the
Ca2+ concentration [63]. Base exchange also occurs with choline (p. 16) and
ethanolamine (p. 12) and the question arises whether a single enzyme is responsible
and what the preferred lipid acceptor is. The work of Kanfer [64] and Gaiti et al. [63]

indicated that more than one enzyme is involved and the partial purification of the
L-serine base-exchange enzyme has been reported [65]. A microsomal fraction from
brain tissue was solubilised with a mixture of detergents and the protein precipitate
obtained by 35-60% saturation with ammonium sulphate fractionated on Sephadex
B and DEAE-cellulose columns. Ethanolamine plasmalogen and phosphatidylethanolamine were good acceptor lipids ( K , , serine 0.4 mM); other phospholipids
could not serve as acceptors and the incorporation of serine was not inhibited by
ethanolamine or choline. In cultured brain cells there is a preferential incorporation
of serine into 1-alkyl-2-acyl-glycerophosphoserine as well as diacylglycerophosphoserine [66]. The purified enzyme of Taki and Kanfer [65] had no
phospholipase D activity though earlier studies [67] had suggested that this phospholipase was identical with the base-exchange enzyme. The molecular mechanism
for the exchange is unknown. For an extensive account of the base-exchange
reaction for serine and the other phospholipid bases the reader is referred to a recent
review by Kanfer [68] in which the relationship of the responsible enzymes to
phospholipase D is also discussed.
(ii) Other reactions
In bacteria it is well established that phosphatidylserine is formed by the reaction of
L-serine with CDP-diacylglycerol catalysed by phosphatidylserine synthase (CDP-diacylglycero1:t-serine 0-phosphatidyltransferase, EC 2.7.8.8)

L-serine + CDP-diacylglycerol

-

phosphatidylserine + CMP

and first observed in a cell-free system from E. coli [69,70]. It also occurs in plants
and protozoa [71] but as far as is known does not occur in animal tissues.
Over 20 years ago Hubscher et al. [58] noted an incorporation of L-serine into the


8

G.B. Ansell and S. Spanner

phosphatidylserine of mitochondria isolated from liver which was dependent on
MgZf and ATP and stimulated by CMP. This was confirmed by Bygrave and
Biicher [72] but the finding was puzzling since other work had more or less
eliminated mitochondria as organelles capable of synthesising phospholipids containing nitrogen bases de novo [73,73a] except for the formation of phosphatidylethanolamine by the decarboxylation of phosphatidylserine (p. 9). More recent work
has, however, demonstrated an ATP- and CMP-stimulated incorporation of L-serine
into the phosphatidylserine of mitochondria of Ehrlich ascites cells [60]. Although
the incorporation was also stimulated by the presence of phosphatidic acid, which
suggested the involvement of CDP-diacylglycerol, the addition of the latter had no
effect. Furthermore, serine did not stimulate the incorporation of “P from
[ 32 Plphosphatidic acid into phosphatidylserine in the presence of CTP or the release
of [ I4C]CMP from [ ‘‘C]CDP-diacylglycerol and so the involvement of CDP-diacylglycerol had to be rejected. Kiss [74] had noted earlier that stimulation of
phosphatidylserine formation in heart slices by phosphatidic acid was unaffected by
CTP and thought that it might be caused by a reversal of the action of a
phospholipase D.
The ATP-stimulated incorporation of serine into mitochondria1 phosphatidylserine therefore has been difficult to explain. It is possible that there are two
base-exchange mechanisms, one dependent on, and one independent of, ATP, a view
originally put forward by Bygrave and Biicher [72] and supported by the experiments of Yavin and Zeigler [66] with cultured brain cells. Recently a new explanation of the ATP-stimulated synthesis has been put forward though it was studied in
brain microsomes and not liver mitochondria [75] but is interesting in that it will
explain phosphatidic acid-stimulation as well. In the presence of Ni2+ ions, ATP
promotes the conversion of phosphatidic acid to pyrophosphatidic acid (P,P‘bis( 1,2-diacyl-sn-glycero-3-pyrophosphate).
This then appears to react directly with
L-serine to give phosphatidylserine. The reaction is inhibited by -SH inhibitors such
as p-hydroxymercuribenzoate which do not affect the Ca2+-dependent pathway.
Presumably the final fatty acid composition of phosphatidylserine is determined
by the acyltransferase reactions which operate for phosphatidylethanolamine and
-choline (pp. 12 and 17) but only one study appears to have been carried out [76].
(b) Phosphatidylethunolumine

There are four established pathways for the biosynthesis of this phospholipid.
(i) The decarboxylation of phosphatidylserine
phosphatidylserine

-.

phosphatidylethanolamine

+ C02

(ii) The transfer of ethanolamine by its phosphorylation and transfer to a
diacylglycerol acceptor from CDP-ethanolamine
ethanolamine

+ ATP

-+

phosphoethanolamine

+ ADP