81

Biochimica et Biophysics Acfa, 441 (1976) 81-92 0 Elsevier Scientific Publishing Company, Amsterdam

-Printed

in The Netherkids

BBA56811 PHOSPHOL~~ BIOSYNT~SIS IN SARCOPLASMIC RE~CUL~ MEMBRANE DURING DEVELOPME~

M.

GABRIELASARZALAand MARIAPILARSKA

Department of Biochemistry of Nervous System and Muscle, Nencki Institute of Expe~me~ta~ Biology, 3 Pasteur Str., 02493 Warsaw ~Polaffd~

(ReceivedMarch 3rd, 1976)

summary Biosynthesis of phosphatidic acid, phosphatidylcholine and phosphatidylethanolamine in the sarcoplasmic reticulum membnme has been investigated. The results show that sarcoplasmic reticulum, in addition to its main function, i.e. transport and accumulation of Ca2+, is able to synthetize phospholipids by the same pathways as endoplasmic reticulum of other tissues. The changes of activity of enzymes involved in phospholipid biosynthesis during muscle development have been analysed. The extent of sn-glycero-3-phosphate and lysophosphatidylcholine acylation by acyl-CoA or free fatty acids in the presence of ATP and CoA is the same at every stage of development. The specific activity of glycerolphospha~ acyl~nsfe~e(s) increases progressively during development up to about the 10th day of postnatal life and then decreases to the adult level. Linoleate esterifies sn-glycero-3-phosphate to a higher extent than palmitate, especially during postnatal period. The main product of sn-glycero-3-phosphate acylation is phosphatidic acid. The specific activity of Iysolecithin acyltransferase increases from the low embryonic value to a rn~~urn at about the 3rd day of postnatal life, followed by a decrease to the adult value. The activity of cholinephosphotransferase decreases from a high value observed during the earliest embryonic period studied until the 3rd day before birth, and then begins to increase again from about the 5th day of postnatal life. The activity of eth~ol~~ephosphotr~sfe~e decreases continuously with age. The main product of phosphatidylethanolamine methylation is phosphatidylmonomethylethanolamine. The specific activity of phosphatidylethanolamine Abbreviations: HEPES. N-2hydroxycthvlpiperrtlne-N’~~~~fo~c b~(2-~~~~yl)-N,~’ tetraecetie acid.

acid: EGTA, ethylene&ycol-

82 methyltransferase increases from the embryonic period to a maximum between the 4th and the 9th day of.postnatal life followed by a decrease to the adult value.

Introduction Sarcoplasmic reticulum of skeletal muscle represents a form of endoplasmic reticulum which is highly specialized in active calcium transport. Its main function is regulation of the Ca” concentration in the muscle cell during the contraction-relaxation cycle. Sarcoplasmic reticulum membrane, which is fragmented during the homogenization of muscle, is isolated with a high degree of purity in the form of microsomal vesicles. These vesicles are able to accumulate and release Ca*+ [ 1,2]. It has been well established that the presence of phospholipids is essential for the ATPase activity and ability for Ca’+-uptake of sarcoplasmic reticulum vesicles [3-51. Recent studies suggest that these properties also depend on the fatty acid composition of phospholipids [6]. The lipid pattern of the sarcoplasmic reticulum membrane from adult muscle has been reported by many investigators [5,7-121, but the changes in lipid content and composition during development of the sarcoplasmic reticulum membrane have been shown only recently [ 13,141. The metabolism of lipids in the sarcoplasmic reticulum membrane was SQ far studied in a fragmentary way and only in the microsomal fraction isolated from muscle of adult animals. These observations have indicated [ 15-181 that the sarcoplasmic reticulum membrane is able to synthesize phospholipids, although the activities of enzymes involved in phospholipid metabolism are lower than those in liver endoplasmic reticulum. The aim of the present study was to investigate in detail the properties of enzymes involved in phospholipid biosynthesis in the sarcoplasmic reticulum membrane with emphasis on the changes in their activity during ontogenesis. Materials and Methods Preparation of the microsomal fraction of skeletal muscle Skeletal muscle microsomes, i.e. vesicles of fragmented sarcoplasmic reticulum, were prepared from rabbit leg and back skeletal muscle at various stages of development, from 24day-old. embryos until 21 days of postnatal development, and from adult animals. Muscle mince was homogenized in 5 ~01s. of 0.1 M KC1 and 20 mM histidine, pH 7.2. The fractions between 8000 X g and 50 000 X g were collected, suspended in 0.25 M sucrose and purified on a continuous sucrose density gradient. For details of the procedure, see ref. 19. Preparation of substrates was synthesized enzymatically and purified sn-[ l- 14C]Glycero-3-p hosphate by paper chromatography according to Scherphof and Van Deenen [ 201. CDP[32P]ethanolamine was prepared according to Chojnacki and Metcalfe [211.

83

l-Acyl-sn-glycerophosphocholine (lysophosphatidylcholine) was prepared by degradation of egg yolk phosphatidylcholine with phospholipase A2 from Crotulus adarmrnteus (EC 3.1.1.4) according to Lands and Hart [ 221. Lysophosphatidylcholine was suspended to a final concentration of 1 mM in 0.1 M KCl, 20 mM histidine, pH 7.2, by means of ultrasonication using a MSE, London sonicator. 1,2-Diacyl-sn-glycerols were prepared according to Zwaal et al. [ 231, by degradation of phosphatidylcholine isolated from muscle microsomes with phospholipase C from Clostridium welchii, in a two-phase system containing 5 mM CaCl,, 100 mM KCl, 20 mM histidine, pH 7.2, and diethyl ether. Freshly prepared solutions of 1,2diacylsnglycerols were evaporated to dryness under Nz atmosphere. Residues were suspended in 20 mM histidine, pH 7.2, containing 0.03% Tween 20 by sonication for 1 min using a MSE sonicator.

Assays for enzymatic ac tiuity The activity of acyl-CoA:sn-glycerol-3-phosphate

O-acyltransferase(s) (EC 2.3.1 .15), glycerolphosphate acyltransferase( s)) was measured in an incubation medium containing: 200 nmol of sn-glycero-3-phosphate, 60 nmol of fatty acids (sodium salts), 100 I.cmol HEPES, pH 7.2, 10 pmol dithiothreitol, 20 pmol MgC12, 100 lmol ATP, 200 nmol CoAand microsomes (1 mg protein) in a final volume of 2 ml. Incubation was carried out for 30 min. The activity of acyl-CoA:acylglycero-3-phosphocholine 0-acyltransferase (EC 2.3.1.23, lysolecithin acyltransferase) was measured in an incubation medium containing: 100 nmol of sonicated emulsion of lysophosphatidylcholine, 60 nmol labelled fatty acids (sodium salts), 100 pmol HEPES, pH 7.2, 100 pmol ATP, 200 nmol CoA, 10 pmol dithiothreitol, 20 pmol MgC12, and microsomes (200 pg protein) in a final volume of 2 ml. Incubation was carried out for 15 min. The activity of CDPcholine: 1,2diacylglycerol cholinephosphotransferase (EC 2.7 8.2)) cholinephosphotransferase) and CDPethanolamine : 1,2diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1, ethanolaminephosphotransferase) was measured in an incubation medium containing: 50 nmol of CDP[Me-“C]choline or 50 nmol of CDP[32P]ethanolamine, 0.4 mg 1,2diacylsn-glycerol, 100 pmol HEPES, pH 7.6, 20 pmol MgC12, 10 E.cmol dithiothreitol, 200 nmol EGTA and muscle microsomes (1 mg protein) in a final volume of 2 ml. Incubation was carried out for 30 min. The activity of S-adenosyl-L-methionine:phosphatidylethanolamine Nmethyltransferase (EC 2.1 .1.17), phosphatidylethanolamine methyltransferase) and S-adenosyl-L -methionine : phosphatidylmonomethylethanolamine Nmethyltransferase (phosphatidylmonomethylethanolamine methyltransferase) was measured in an incubation medium containing 60 nmol S-adenosyl-L[Me-14C]methionine, 100 pmol HEPES, pH 7.4, 10 I.tmol dithiothreitol, 10 pmol MgC12 and muscle microsomes (3 mg protein) in a final volume of 2 ml. Incubation was carried out for 30 min. All incubations were carried out under N2 atmosphere in a shaking waterbath at 37°C. The reactions were stopped by addition of 8 ml of a chloroform/ methanol (2 : 1, v/v) mixture and lipids were extracted from the incubation medium according to Bligh and Dyer [ 241.

84

Determinations of all enzymatic activities were performed on at least seven microsomal preparations, each isolated from separate litters. The data given on the graphs are the mean values from all experiments. The range was always less than +lO% of mean values. Analytical procedures Individual phospholipids were separated via silica thin-layer chromatography using chloroform/methanol/water (65 : 25 : 4, v/v) as a solvent. Phosphatidic acid was separated on silica gel plates impregnated with 0.25 N oxalic acid, using light petroleum/acetone/formic acid (75 : 25 : 0.25, v/v) as a solvent. To separate monoacyl-sn-glycero-3-phosphate, phosphatidic acid and fatty acids, oxalic acid-impregated plates were developed with chloroform/methanol/HCl (87 : 13 : 0.5, v/v). When labelled phosphatidylcholine was isolated from incubation mixtures containing labelled fatty acids, the plates were first developed in chloroform/light petroleum/formic acid (65 : 33 : 2, v/v) and then run in a solvent for phospholipid separation. Various lipid components were detected by exposure to iodine vapour and identified by co-chromatography with lipid standards. Phosphatidic acid was eluted from silica gel with chloroform/methanol (1 : 1, v/v) and phosphatidylcholine, phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine, with chloroform/ methanol (2 : 8, v/v) mixtures. After evaporation to dryness under Nz, the residues were dissolved in a chloroform/methanol (1 : 1, v/v) mixture and aliquots were taken for radioactive determination by means of liquid scintillation counter. Protein was determined by the method of Lowry et al. [25] using bovine serum albumin as standard. Chemicals Palmitic and linoleic acids, ATP, CoA, CDPcholine, S-adenosyl-L-methionine and phospholipase C from Clostridium welchii were purchased from Sigma Chemical Company, U.S.A. Creatine phosphate and creatine phosphokinase were obtained from Calbiochem. Palmitoyl-CoA was supplied from Serva and phospholipase A from Crotalus adamanteus snake venom from Koch-Light Laboratories Ltd. CDP[Me-‘4C]choline, [ 14C]linoleic and [ 3H]palmitic acid, S-adenosyl-L-[Me-‘4C]methionine were supplied by the Radiochemical Centre Amersham, England. All solvents were redistilled before use. Results Glycerolphosphate acyltransferase(s) activity The extent of phosphatidic acid synthesis catalyzed by muscle microsomes was measured in the presence of sn-glycero-3-phosphate, free fatty acids, ATP and CoA. Glycerolphosphate acyltransferase(s) from muscle microsomes displayed a broad optimum pH, ranging between 6.7 and 8.0. Dithiothreitol caused an increase of incorporation of fatty acids in accordance with previous findings with liver microsomes [ 261. Preliminary experiments performed on microsomes isolated at various stages of muscle development showed that the extent of phosphatidic acid formation

85

was virtually the same when palmitoyl-CoA was used as acyl donor, as in the case when pahnitate was used in the presence of ATP and CoA. These results indicate that the activation of free fatty acids by acid : CoA ligase (AMP) is not a rate-limiting step of acylation of sn-glycero-3-phosphate and that the long-chain fatty acid activation system is already present in the sarcoplasmic reticulum membrane in the prenatal period. Since, however, the muscle microsomal fraction contains a highly active Mg’+- and Ca”-stimulated ATPase, a high concentration of ATP or creatine phosphate and creatine phosphokinase as an ATP regenerating system were necessary in order to maintain a sufficient level of ATP. The incorporation of sn-[ 1-14C] glycero-3-phosphate into phosphatidic acid in the presence of MgATP and CoA and in the absence of added fatty acids, was in the early stages of development as high as about ‘70% of that observed in the presence of palmitoyl-CoA; it was only 25%, when microsomes from mature muscles were used. This may be the result of a higher content of endogenous fatty acids in the microsomes from embryonic muscles. For instance we have found 50-70 nmol of free fatty acids per mg protein in the muscle microsomes of newborn animals in comparison to 20-25 nmol per mg protein in the adult stage. Under standard incubation conditions, 85-909~ of the acylation products of exogenous sn-glycero-3-phosphate were 1,2diacyl-sn-3-phosphate and lo15% monoacyl-sn-3-phosphate. This observation indicates that under the conditions of the experiment, the first acylation step is rate-limiting. Incorporation of labelled fatty acids into neutral lipids was negligible. The extent of phosphatidic acid synthesis by muscle microsomes from various stages of development, in the presence or absence of sn-glycero-3-phosphate, was measured in the presence of [14C]linoleate and [3H)palmitate. Fig. 1 shows that the specific activity of glycerolphosphate acyltransferase(s) increased progressively with development and reached maximum activity around the 10th day of life, followed by a decrease to the adult value. Up to the 5th day of postnatal development, both fatty acids were incorporated to a similar extent; in the later stages; linoleic acid was more effective as an acyl donor. Some incorporation of fatty acids into phosphatidic acid was observed also in the absence of exogenous sn-glycero-3-phosphate. It may be due to the presence of endogenous sn-glycero-3-phosphate in the muscle microsomes, or due to the exchange of fatty acids by a deacylation-reacylation cycle involving phosphatidic acid. Lysolecithin acy&ransferase actiuity Incorporation of labelled fatty acids into phosphatidylcholine catalyzed by muscle microsomes was measured in the presence or absence of l-acyl-snglycero-3-phosphocholine. Similarly as for acylation of sn-glycero-3-phosphate the extent of lysophosphatidylcholine acylation was the same when acyl-CoA or free fatty acids in the presence of ATP and CoA were used as acyl donor. For the same reason as in the case of measurement of glycerolphosphate acyltransferase(s) activity, a high concentration of ATP was used. The specific activity of lysolecithin acyltransferase of the microsomes isolat-

86

I

I

I

30

I

t

10

5

‘ 15

t

2Of

Age (days) Fig. 1. Glycerolphosphate acyltransferase(s) activity of microsomal fraction during muscle development. Samples of mlcrosomal fraction (containing 1 mg protein) isolated at various stages of development were incubated with 30 nmol [3HJpabnitate and 30 nmol (‘4CJlinoleate in the presence or absence of 200 run01 of sn-glyeero-3-phosphate. For detafls of the assay, see Material and Methods. (a, :x) run01 of pslmitate and (0. o) run01 of linoleate incorporated in the presence (full symbols) and in the absence (empty symbols) of exogenous sn-glycero-3-phosphat.e. Birth

H

Age

(days)

Fig. 2. Lysolecithin acyltransferase activity of microsomal fraction durfng muscle development. Samples of microsomal fraction (containing 0.2 mg protein) isolated from muscles at various stages of develonment were incubated with SO nmol [‘H]pahnitate and 30 nmol [14CJlinoleate in the Presence or absence of 100 nmol of sonicated emulsion of l-acyl-sn-glycero-3qhosphooholine. For details Of the assay, see Materials and Methods. (A, A) nmol of pabnitate and (a, of nmol of linoleate incorporated in the Presence (full symbols) or absence (empty symbols) of exogenous I-acytsn-glyeero-3-phosphocholine.

87

ed from muscle at various stages of development is presented in Fig. 2. The enzyme activity increased from a low embryonic value to a maximum between the 2nd and 10th day after birth, then continuously decreased to the adult value. When palmitate and linoleate were added together as acyl donors for the acylation of l-acyl-sn-glycero-3-phosphocholine, linoleic acid was incorporated to a much higher extent than palmitate during the whole assayed period of development. The difference was especially high in the early postnatal period, indicating a high specificity for acylation of the 2-position. In the absence of exogenous Iysophosphatidylcholine, the incorporation of linoleic acid was still relatively high, particularly in the early postnatal period. Choline- and ethanolaminephosphotransferases

activity

Activities of choline- and ethanoloaminephosphotransferases were determined in the presence of microsomes, 1,2diacyLsnglycerols and radioactive CDPcholine or CDPethanolamine . The stimulation of phosphotransferase activity by exogenous, 1,2diacylsn-glycerols depend on their origin. The most effective were 1,2diacyLsnglycerols obtained from phosphatidylcholine isolated from skeletal muscle microsomes, which stimulated the incorporation of phosphocholine to a much higher extent than those obtained from phosphatidylcholine derived from other sources, such as egg yolk or liver. The pHdependence curve was very similar for both transferases, with the optimum between pH 7.0 and 8.0. The optimum concentration of Mg2+ was

H

I 25

30

I

I

I

5

10

/

Age(days)

Fig. 3. Cholinephosphotreasferw ectivity of mlcroeomal fraction duzlng muecle development. Semples of mtcrosomel fraction (containing 1 mg protein) lsoleted from muscles at vulous stegee of development were incubeted with 50 nmol CDPIMe-“Clcholine In the prewnce (0) or ebsence (0) of 0.4 mg 1.2 diacyl+nQycerols. For deteils of the away, see Matedal end Methods.

88

found to be 10 mM; however higher concentrations of this cation caused considerable inhibition. The activity of both enzymes was higher in the presence of di~io~reitol. Fhosphatidyl~hol~e and phosphatidyleth~ol~~e synthesis was higher in the presence of EGTA; Ca2’ inhibited phosphotransferase activity. The activity of cholinephosphotransferase in the microsomal fraction isolated at various stages of development, was determined with and without the addition of 1,2diacyl-sn-glycerols. Fig. 3 shows that the activity of the enzyme at about the 24th day of prenatal life was the same as in the adult stage. It decreased sharply during embryonic development until the 3rd day before birth, then remained at about the same level until about the 5th day of postnatal development and finally increased to the adult value. The addition of 1,2-diacylsn-glycerols obtained from phosphatidylcholine of adult muscle microsomes, stimulated phosphatidylcholine formation to roughly the same extent (about 3 times) during the whole assayed period. The extent of phosphoeth~ol~ine incorporation (not shown in the figure) was the highest at the end of prenatal and beginning of postnatal life (13.2 nmol per mg microsomal protein). From the 7th day after birth, it decreased to the adult value of 3.7 nmol of phosphoethanolamine incorporated per mg protein. FhosphutidyiethQno~amine methy~trunsfe~ses activity The activities of methyltransferases were determined in the presence of phos-

Age (days 1

Fig. 4. Phosphatidylethanolammine metbyltransfersse activity of micxosomal fraction during muscle development. Samples of microsomal fraction (containing 3 mg protein) isolated from muscles at various stages of development were incubated with 60 nmol S-adenosyl-L-[Me-14CImetbionine. For details of the assay, see Materials and Methods. Ordinate: nequiv. of -CH3 incorporated into phosphatidylmonometbylethanolamine per mg microsomal protein.

89

phatidylethanolamine present in muscle microsomes and S-adenosyl-L-[Me“C]methionine. During the whole assayed period of development, about 8090% of total radioactivity incorporated into phospholipids was found in phosPhosphatidyldimethylethanolamine and phatidylmonomethylethanolamine. phosphatidylcholine were only minor products of methylation. The addition of dithiothreitol caused an increase of incorporation of methyl groups; Ca” had no effect on the methylation of phosphatidylethanolamine. The extent of the formation of phosphatidylmonomethylethanolamine in the presence of microsomes isolated from muscles at various stages of development is shown in Fig. 4. The amount of phosphatidylmonomethylethanolamine formed per mg protein was the same in the embryonic period as in the adult stage. The activity of phosphatidylethanolamine methyltransferase increased from embryonic value to a maximum between the 4th and 9th day after birth and then decreased to the adult value. Discussion In spite of numerous studies concerning structure, composition and function of sarcoplasmic reticulum (for review, see refs. 1, 2) the phospholipid metabolism of this membrane has been studied fragmentarily and in adult muscle only. Waku and Lands [ 151 found the presence of lysolecithin acyltransferase and Pennington and Worsfold [16], the activity of cholinephosphotransferase in rabbit sarcoplasmic reticulum. Previous studies from this laboratory have also indicated that the microsomes isolated from mature muscle are able to synthesize phospholipids [ 17,18,27] ; however, specific activity of these enzymes has been found to be much lower than that of microsomes from liver or intestinal mucosa [ 28-301. In this work, the activity of the enzymes involved in the synthesis of phosphatidic acid, phosphatidylcholine and phosphatidylethanolaine via various pathways, as the synthesis de novo, methylation of phosphatidylethanolamine and acylation of lysophosphatidylcholine have been investigated. All the results obtained indicate that the biosynthesis of phospholipids in skeletal muscle cell proceeds in the same way as in the liver, and that the sarcoplasmic reticulum plays, in this respect, a role analogous to that of endoplasmic reticulum. We have shown recently [14,31], that considerable changes in the amount and composition of the lipid constituents of the sarcoplasmic reticulum membrane occur during muscle development. The most significant are: a decrease of the phospholipid to protein ratio, a continuous decrease of the content of phosphatidylethanolamine and an increase of the content of phosphatidylcholine (when expressed as the relative percentage of the total phospholipid content), and changes in the pattern of the molecular species of phosphatidylcholine. In the present study, these observations have been extended to the investigations of the changes in the activity of enzymes responsible for the synthesis of phosphatidic acid, phosphatidylcholine and phosphatidylethanolamine in the sarcoplasmic reticulum during development. In Fig. 5, the changes in the activity of all investigated enzymes during de-

90

Birth

% 500,

1 450.

400

350.

300.

250.

200

150*

I

. 25

30

5 Age

10

15

20

kiOy5)

Fig. 5. Changes in the activities of enzymes involved in phospholipid synthesis in sarcoplasmic reticulum during development. The activity of each enzyme from Figs. 14 is expressed as the percentage of the activity found in sarcoplasmic reticulum isolated from muscles of adult aalmals. (0) 1ysolecitMn acyltransferase. (0) dycerolphwhate acyltransferase(s), (a) cholinephosphotransferase. (0) ethanolaminephosphotransferase. (A) phosphatldylethanolamine methyltransferase.

velopment are presented, expressed as the percentage of activity in the mature sarcoplasmic reticulum. The highest activity of glycerolphosphate acyltransferase(s) is observed in early postnatal development. Thus,the most active synthesis of phosphatidic acid, which is an intermediate in the synthesis of both neutral lipids and phospholipids, takes place at the time when an intensive process of muscle differentiation and a rapid increase of muscle mass occur. The activity of lysolecithin adyltransferase is also the highest during the first days after birth. The pattern of the changes of lysolecithin acyltransferase activity during development coincides rather closely with the changes of the composition of fatty acids in phosphatidylcholine. The significant increase of the amount of unsaturated fatty acids is observed during this period of postnatal development [ 13,311. Contrary to the pattern of acyltransferases, the activity of cholinephosphotransferase, being the highest at about the 5th day before birth, decreased continuously until about the 2nd day of postnatal life. Only from this period does the increase in the amount of phosphatidylcholine in the sarcoplasmic reticulum seem to be correlated directly with the increase of cholinephosphotransferase activity, i.e. with the synthesis de novo of phosphatidylcholine. Contrary to changes in the activity of cholinephosphotransferase in muscle microsomes during development, the activity of this enzyme in liver micro-

91

somes [32] is low in early embryonic life and increases significantly from 2 to 3 days before birth to the maximum value observed at about the 8th day of postnatal life. The highest activity of ethanolaminephosphotransferase has been found in muscle microsomes from embryonic and newborn rabbits, during the period when the amount of phosphatidylethanolamine already decreases. Comparison of the amounts of both phosphatidylcholine and phosphatidylethanolamine, on one hand, and the activities of corresponding phosphotransferases on the other, suggests the conversion of phosphatidylethanolamine to phosphatidylcholine by the methylation pathway. Indeed, the highest activity of methyltransferases is observed between the 3rd and the 10th day of postnatal life. However, the results of this work suggest that the three-step methylation of phosphatidylethanolamine does not contribute to a significant extent to the synthesis of phosphatidylcholine in the sarcoplasmic reticulum membrane during the whole period of development. Acknowledgements The authors wish to thank Professor T. Chojnacki, for kindly supplying of CDP[32P]ethanolamine and Dr J. Zborowski for that of sn-[1-i4C]glycero-3phosphate. The skilful technical assistance of Mr. S. Stachowski is gratefully acknowledged. References 1 Hasselbach. W. (1964) Prog. Biophys. Mol. Biol. 14.167-222 2 Martonosi. A. (1971) in Biomembranes (Mauson. L.A.. ed.) Vol. 1. pp. 191-269. York 3 Martonod. A., Donley. J. and Halpin, R.A. (1968) J. Biol. Chem. 243.61-70 4 Fiehn. W. and Hasselbach. W. (1970) Eur. J. Biochem. 13.510-518 5 Meissner. G. and Fleischer. S. (1972) Biochim. Biophys. Acta 255.19-33

Plenum Press, New

6 Warren. G.B.. Toon, P.A.. Birdsall. N.J.M.. Lee. A.G. and Metcalfe. J.C. (1974) Biochemistry, 13, 5501-5507 7 Drabikowski. W.. Dominas. H. and Dabrowska. M. (1966) Acts Biochbn. Polon 13.12-24 8 Waku. K.. Uda. Y. and Nakazawa. Y. (1971) J. Biochem. (Tokyo) 69.483-491 9 MacLennan, D.H.. Seeman. P., Bes. g.H. and Yip. C.C. (1971) J. Biol. Chem. 246, 2702-2710 10 Fiehn. W.. Peter. J.B.. Mead. J.F. and Gan-Elephano. M. (1971) J. Biol. Chem. 246, 5617--5620 11 Marai. L. and Kuksls. A. (1973) Can. J. Biochem. 51.1248-1261 12 Mar& L. and Kuksis. A. (1973) Can. J. Biochem. 51.1365-1379 13 Boland. R., Martonosi. A. and Tillack, T.W. (1974) J. Biol. Chem., 249.612-623 14 Sarzafa, M.G., Pilarska. M., Zubnycka, E. and Michalak, M. (1975) Eur. J. Biochem. 57.25-34 15 Waku, K. and Lands, W.E.M. (1968) J. Biol. Chem. 243.2654-2659 16 Pennington, R.J. and Worsfold. W. (1969) Biochim. Biophys. Acta 176.774-782 17 Sanafa. M.G. and Pilamka. M. (1972) 16th International Conference on the Biochemistry of Lipids. the Hague. Abstract No. C38 18 Pfiarska. M. and Sarzafa. M.G. (1974) 9th FEBS Meeting. Budapest, Abstracts No. s6a 38 19 Drabikowski. W.. Sarzafa, M.G., Wroniszewska, A., Lagwidska. E. and Dnewiecka. B. (1972) Biochim. Biophys. Acta 274.158-170 20 Schewzhof. G.L. and Van Deenen. L.L.M. (1966) Biochim. biophys. Acta 113.417-420 21 Chojnacki. T. and Metcalfe. R.F. (1966) Nature Lond. 210.947-948 22 Lands, W.E.M. and Hart, P. (1964) J. Lipid. Rer. 5. 81-87. 23 Zwaal. R.F.A., Roclofsan. B.. Comfurius. P. and Van Deenen. L.L.M. (1971) Biochim. Biophys. Acta 233.474479 24 BliI. E.G. and DYU, W.J. (1969) Can. J. Blocham. Physiol. 37.911-917 25 Lowry. P.H.. Rosebrough, N.J., Farr. A.L. and Randall, R.J. (1951) J. Biol. Chem. 193. 265-275 26 Monroy, G.. Rola. F.H. and Puhnan. M.E. (1972) J. Biol. Chem. 247.68844894

92 27 SarzaIa. M.G. 28 29 30 31 32

(1974)

17th International Conference

on the Biochemistry

of Lipids, Milan, Abstr.

No B15 Webster, G.R. (1965) Biochbn. Biophys. Acta 98, 512-519 Subbaiah. P.V., Sastry, P.S., Ganguly, J. (1970) Biochem. J. 118,241-246 Daae, L.N.W. (1973) Biochim. Biophys. Acta 306, 186-193 Pilarska. M. and Samala, M.G. (1975) 10th FEBS Meeting, Paris, Abstract No 1038 Weinhold. P.A., Skinner. R.S. and Sanders, R.D. (1973) Biochim. Biophys. Acta 326.43-51