Phosphatidylserine biosynthesis in mitochondria from ehrlich ascites tumor cells

258

Biochimica et Biophysics Acta, 619 (1980) 268-266 @ E~vier/North-Ho~and Biomedical Press

BBA 57614

PHOSPHATIDYLSERINE BIOSYNTHESIS IN M~OCHONDR~ EHRLICH ASCITES TUMOR CELLS

JOLANTA

BARAfiSKA

Department of Cellular Biochemistry, 02-093 Warszawa (Poland~ (Received

FROM

Nencki Institute of Experimental

Biology, Pasteura 3,

January 17th, 1980)

Key words: Pkosphatidylserine biosynthesis; Phosphatidylcholine; Phosphatidyletkanolamine; (Ehrlich ascites mitochondria)

Phosphatidic acid;

Summary 1. Isolated mitochondria of Ehrlich ascites tumor cells incorporated [‘*Clserine into phosphatidylserine and partly into its decarboxylation product, phosphatidyleth~ol~ine, while phosphatidylser~e was the sole product with microsomes. 2. The incorporation of [‘4C]serine into mitochondrial phospholipids was stimulated by Ca2+ which indicated the operation of the Ca2+dependent baseexchange mechanism, virtually absent in mammalian tissue mitochondria. The finding cannot be attributed to microsomal contamination. 3. The inco~oration of [14C]serine into mitochond~~ phospholipids was also stimulated by ATP, both in the presence and in the absence of calciumcomplexing agents. The stimulation by ATP was insensitive to penicillin and streptomycin, thus pointing that this process was not of bacterial origin. 4. The latter process was further stimulated by phosphatidic acid and phosphatidic acid precursors, but not by CDP diacylglycerol. 32P from neither ]y-32P]ATP nor [32P]phosphatidic acid was incorporated into phosphatidylserine in the presence of CTP and L-serine. The release of [14CJCMP from [14C]CDP diacylglycerol was not stimulated by L-serine. 5. It is concluded that [ 14C]serine incorporation into mitochondrial phospholipids by ATP-dependent process does not fit to any of the pathways of phospholipid biosynthesis described so far.

Abbreviation: EGTA, ethyleneglyeol-bis(P-aminoethyl

ether)-Ns?l’-tetraacetic

acid.

259

Introduction It is generally assumed that nitrogen-containing phospholipids cannot be synthesized de novo in mitochondria (cf. Ref. 1). Therefore, the striking feature observed previously in this laboratory [2] was that, in Ehrlich ascites tumor cells incubated with [32P]phosphate at or below 2O”C, the specific labeling of phospholipids was higher in mitochondria than in microsomes. It was even more intriguing that the main labeled phospholipid formed in mitochondria under these conditions appeared to be phosphatidylethanolamine whose specific labeling exceeded the specific labeling of this phospholipid in microsomes. After incubation at 37”C, the labeling was higher in microsomes than in mitochondria and phosphatidylcholine was the main labeled phospholipid in both kinds of particles. One of the explanations for this predominant labeling of mitochondrial phosphdlipids might be the assumption that mitochondria are able to synthesize phosphatidylethanolamine. However, this process might be normally masked by the prevailing synthesis proceeding in the endoplasmic reticulum and becomes only evident when the latter is decreased by lowering the temperature. Since phosphatidylethanolamine can be formed in mitochondria by decarboxylation of phosphatidylserine [ 3,4], the question appears whether Ehrlich ascites mitochondria are perhaps able to synthesize de novo the latter phospholipid. In animal tissues, phosphatidylserine is formed by Ca2’-stimulated baseexchange reaction which has been mainly found in the microsomal fraction [3--51. A de novo synthesis via CDP diacylglycerol has been described so far for bacteria [6] and plants [ 71 and is generally believed not to occur in animal tissues. Nevertheless, an ATP-dependent and CMP-stimulated incorporation of serine into phosphatidylserine in isolated liver mitochondria has been observed by Hiibscher et al. [8,9] and Bygrave and Biicher [lo]. The mechanism of this synthesis was, however, obscure. The present study provides evidence that phosphatidylserine can be formed in Ehrlich ascites mitochondria in the Ca”-stimulated base-exchange reaction and an ATP-dependent process. Several properties of this latter process are described and it is concluded that it does not fit to any of the known pathways of phosphatidylserine biosynthesis. A preliminary report on this study has already been presented [ 111. Materials and Methods Ehrlich ascites tumor cells, grown in the peritoneal cavity of white mice, strains R III and CFW, were harvested 7-9 days after transplantation. The isolation and disruption of the cells by osmotic shock and isolation of mitochondria were performed according to Borst [ 121. The microsomal fraction was obtained by centrifugation of the post-mitochondrial supernatant at 100 000 X g for 1 h. Our previous study [2] showed that NADPH-cytochrome c reductase (EC 1.6.2.4) and glucose-6-phosphatase (EC 3.1.3.9), enzymes usually considered to be characteristic for microsomes, were also present in Ehrlich ascites mitochondria and by that unsuitable as indicators of microsomal contamination. Therefore, the purity of the fractions was checked as described [2]

by using cholesterol esterase (EC 3.1.1.13) as a marker of microsomes and cytochrome oxidase (EC 1.9.3.1) as a marker of mitochondria. Determination of marker enzymes in the present study showed about 10% cross-contamination of microsomal and mitochondrial fractions. All incubations were carried out under constant shaking at 30°C with air as the gas phase. Incorporation of [ 14C]serine into phospholipids was studied in the following incubation media: A, 60 mM KCl, 10 mM phosphate buffer (pH 7.4), 6 mM MgC&, 0.6 mM sn-glycerol 3-phosphate, 0.3 mM sodium paimitate, 0.2 mM CoA, oligomycin 5 ,ug per mg protein or 2 mM NaN3, and 50 PM L-[3-14C]serine (specific activity 48 Ci/mol); B, 60 mM imidazole buffer (pH 7.4), 3 mM CaC& and 44.5 E.IML-[3-‘4C]serine (specific activity 56 Ci/mol). The amount of subcellular fractions corresponded to 2 mg protein. The final volume was 1.0 ml. Incorporation of [ “Plphosphatidic acid into phospholipids was examined in the medium containing 60 mM KCl, 10 mM phosphate buffer (pH 7.4), 10 mM MgC&, 10 mM 2-mercaptoethanol, 1.5 mM CTP, 2 mM EGTA, [32P]phosphatidic acid (1.5 - lo6 cpm) and sonicated mitochondria (3 mg protein) in a final volume of 1.0 ml. In experiments where incorporation of [ 14C]serine was examined, the incubation was terminated by the addition of trichloroacetic acid to the final concentration of 10%. The precipitate was washed three times with .5% trichloroacetic acid containing low concentration of unlabeled serine. Lipids were extracted from the precipitate with CH30H/CHC13 (1 : 2, v/v). In other experiments incubations were terminated by addition of CH30H/CHC13 (2 : 1, v/v) and lipids were extracted according to Bligh and Dyer [ 131. After extraction of lipids, phospholipids were separated and identified by thin-layer chromatography on silica gels G and H (E. Merck AG, Darmstad, F.R.G.) with the developing systems described in the text. Protein was determined by the biuret method [14] with serum albumin as standard. Radioactivity was measured with a scintillation spectrometer making use of the Cerenkov effect for 32P [ 151 or using liquid scintillation cocktails for 14C. L-[3-14C]Serine and [U-14C]CTP were from the Radiochemical Centre (Amersham, England). [32P]Phosphatidic acid was synthesized by incubating rat liver mitochondria with glycerol 3-[32P]phosphate as described by Zborowski and Wojtczak [ 161 and subsequently separating on a silicic acid column. rat-Glycerol 3-[32P]phosphate for this synthesis was prepared by heating inorganic [32P]phosphate with glycerol [ 171. Phosphatidic acid (sodium salt, from egg lecithin) and CDP diacylglycerol were from Koch-Light (Colnbrook, England). CoA was from Boehringer (Mannheim, F.R.G.) and was reduced before use by a 2-fold excess of reduced glutathione. Results Table I shows that the incorporation of [ 14C]serine into phospholipids of Ehrlich ascites mitochondria was stimulated by either ATP plus CTP or Ca*+.

261 TABLE

I

INCORPORATION Ehrlich ods). EGTA

ascites

OF [‘4C]SERlNE

mitochondria

Other additions

INTO MITOCHONDRIAL

were incubated

PHOSPHOLIPIDS

for 60 min in 1.0 ml of medium

A (see Materials and Meth-

were as indicated.

Additions

(mM)

Serine incorporation (pmol/mg protein) Expt.

1

Expt. 2

0 0 0

none ATP 7 mM + CTP 1.5 mM CaCl2 3 mM

10 32 115

33 52 123

1 1 1 1

none ATP 7 mM CTP 1.5 mM ATP 7 mM + CTP 1.5 mM

3 33 10 43

5 42 6 51

The effect of the two nucleotides was not due to a possible calcium contamination, as it was also maintained’in the presence of EGTA. The stimulation was produced mainly by ATP. CTP alone was essentially without effect, but together with ATP it had an additional slightly stimulatory effect which was, however, not statistically significant (six experiments). The addition of penicillin and streptomycin (60 ,ug each per ml of the incubation medium and all solutions used for the preparation of cells and mitochondria) had no effect on serine incorporation. The effect of Ca” in mitochondria cannot be explained by microsomal contamination, since under the same conditions, the Ca2+-stimulated incorporation of serine in microsomes was not much higher and amounted to 130-180 pmol/mg protein per h. Contrary to this, in rat liver under the same conditions we obtained the values of 855 and 55 pmol/mg protein per h for the microsomal and mitochondrial fractions, respectively. In imidazole buffer which is more suitable for the base-exchange reaction [ 51, the Ca’+-stimulated serine incorporation in subcellular fractions from the Ehrlich ascites tumor was also similar in mitochondria and microsomes and amounted to 1.0 and 1.3 nmol/mg protein per h, respectively. Under similar conditions the incorporation into rat liver subcellular fractions was 2,4 and 0.3 nmol/mg protein per h in microsomes and mitochondria, respectively. Phospholipids formed during incubation of [ 14C]serine with subcellular particles of Ehrlich ascites tumor cells were analyzed by thin-layer chromatography. Most of the incorporated radioactivity was found in phosphatidylserine (Table II). However, in mitochondria, a substantial percentage was also found in phosphatidylethanolamine, both in the presence of EGTA plus ATP plus CTP as well as in the presence of CaCl,. This was apparently due to the action of phosphatidylserine decarboxylase (EC 4.1.1.65) located exclusively in mitochondria [3,4]. In microsomes about 90% of the label was present in phosphatidylserine. Table III shows that the stimulation of [ 14C]serine incorporation by ATP is further potentiated by precursors and cofactors of phosphatidic acid synthesis, i.e. fatty acids, glycerol 3-phosphate and CoA. CTP has an additional slight

262 TABLE

II

INCORPORATION

OF [14ClSERINE

INTO INDIVIDUAL

PHOSPHOLIPIDS

Mitochondrial and mlcrosomal phospholipids formed during incubation with [14Clserlne, as shown in Table I. were separated by thin-layer chromatography [181. The radioactivity of spots corresponding to phosphatidylserine and phosphatidylethanolamine is expressed as percentage of total radioactivity incorporated. Numbers in parentheses represent numbers of experiments. Phospholipids

Phosphatidylserine Phosphatidylethanolamine

Mitochondria

Microsomes +CaCl2

+EGTA+ATP+CTP

+CaC12

(3)

(2)

61-64 21-23

73-75 13-18

(2)

80-9 3-

1 5

stimulatory effect. Addition of phosphatidic acid instead of its precursors and cofactor8 has a much lower effect which could be, however, increased by CTP and CMP. CDP diacylglycerol in concentrations of 10 ,uM and 1 mM was without effect on [ “C]8erine incorporation in both the absence and the presence of mercaptoethanol and at pH 7.4 and 8.0 (not shown). To check that the sample of CDP diacylglycerol used in this experiment was not decomposed, an experiment with rat liver microsomes and [3H]inositol was performed. In this case CDP diacylglycerol increased the incorporation of inositol into phospholipids. It might be, however, assumed that externally added CDP diacylglycerol had no acces8 to sites where the reaction with [ 14C]8erine was located. Therefore, in the next experiment (Fig. 1) sonicated mitochondria of Ehrlich ascites cells were incubated with [32P]phosphatidic acid, CTP and either L-serine, sn-glycerol &phosphate, or myo-inositol. In the sample in which only [32P]phos-

TABLE

III

EFFECT OF COFACTORS AND PRECURSORS ON THE INCORPORATION MITOCHONDRIAL PHOSPHOLIPIDS IN THE PRESENCE OF EGTA

OF [14C]SERINE

INTO

The incubation medium contained 60 mM KCl, 1 mM EGTA, 10 mM phosphate buffer (PH 7.4), 6 mM MgCI2. 10 ng oligomycin and mitochondria (2 mg protein) in 1.0 ml. The additions were as indicated: 7 mM ATP. 1.5 mM CTP, 2 mM CMP, 0.2 mM CoA. 0.6 mM snglycerol 3-phosphate, 0.3 mM sodium palmitate. 0.5 mM phosphatidic acid (sodium salt) emulsified before use by sonlfication. 1.5 mM mercaptoethanol was added to all samples, except of the samples with CoA where 0.4 mM reduced glutathione was present instead. Incubation time was 60 min. Additions

Serine incorporation (pmollmg protein) Expt.

None ATP ATP+CoA+glycerol 3phosphate+palmitate ATP+CoA+glycerol 3-phosphate+palmitate+CTP Phosphatidic acid Phosphatidic acid+CTP Phosphatidic acid+CMP

2.8 8.6 17.0 18.5 8.2 13.0 15.6

1

Expt. 2 1.8 10.5 20.0 28.4 6.0 24.2 18.8

263

Solvent 4)-

front

CL COP-DC

PG

PS PA

M---Of 1

2

3

4

5

igin

6

Fig. 1. Incorporation of [32Plphosphatidic acid into mitochondrial phospholipids. Sonicated Ehrlich ascites mitochondria were incubated for 60 min in the medium described under Materials and Methods. After incubation. lipids were extracted and phosphollpids separated on silica gel G in CHC13/CH30H/ 23% NHdOH/HzO (65 : 35 : 2.5 : 2.5; v/v). The spots were visualized by autoradiogxaphy. Indications: 1, no additions, zero time control; 2. no additions; 3, myo-inositol added; 4, L-serine added; 5. sn-glycerol 3-phosphate added; 6. standard of [14Clphosphatidylserine. Spots were identified by comparing their positions with those of known standards: CL. cardiolipin; CDP-DG. CDP diacylglycerol; PG. phosphatdylglycerol; PI, phosphatidylinositol; PS. phosphatidylserine; PA, phosphatidic acid.

phatidic acid and CTP were present (sample 2 in Fig. 1) new radioactive spots appeared and were identified as [32P] CDP diacylglycerol and [ 32P]phosphatidylglycerol. Addition of inositol (sample 3) resulted in the appearance of [ 32P]phosphatidylinositol, probably due to microsomal contamination. Addition of glycerol 3-phosphate (sample 5) gave rise to a drastic increase of the radioactivity of the phosphatidylglycerol spot and the appearance of a new spot as cardiolipin. Contrary to this, addition of L-serine (sample 4) did not originate in any new radioactive spot. These results were confirmed by the separation of phospholipids in other systems of thin-layer chromatography [18,19]. In order to further examine the possibility of phosphatidylserine synthesis by the CDP diacylglycerol pathway, the following experiment was performed. Ehrlich ascites mitochondria were first preincubated with sn-glycerol 3-phosphate, palmitate, CoA and ATP, i.e. under conditions for the synthesis of phosphatidic acid. Thereafter, [ 14C]CTP was added to promote the accumulation of [14C]CDP diacylglycerol. At this step AMP was also present to prevent the hydrolysis of CDP diacylglycerol [20]. Finally, such pretreated mitochondria were incubated with either sn-glycerol3-phosphate or L-serine and checked for the formation of [ 14C]CMP. It appeared that glycerol 3-phosphate promoted a release of [ 14C]CMP, whereas L-serine was without effect (Fig. 2). Finally, a possibility was checked for a phosphorylation of serine to phosphorylserine and its further incorporation into phosphatidylserine. Ehrlich

264

Time

(min)

Fig, 2. Release of [14CICMP from [‘4ClCDP diacylglycerol in Ehrhch ascites mitochondria. Mitochondria (30 mg protein) were incubated for 30 min in 10 ml medium containing 60 mM KCl, 10 mM phosphate buffer (PH 7.4), 6 mM MgCl2, 7 mM ATP, 0.3 mM sodium palmitate, 0.2 mM CoA. 2 mM NaNa, 1 mM EGTA and 1 mM sn-glycerol 3-phosphate. After sedimenting by centrifugation at 100 000 X g for 30 min. mitochondria were resuspended and incubated in 8 ml of the medium containing 60 mM KCl, 10 mM phosphate buffer (PH 7.4), 10 mM MgCl2, 2 mM EGTA, 10 mM 2-mercaptoethanol, 3 mM AMP, and 8 yCi [U-14ClCTP (specific activity 477 Ci/mol). After 30 min mitochondria were sedimented again, resuspended and incubated in the same medium but without CTP in portions containing 4 mg mitochondrial protein (corresponding to about 50 000 cpm [14ClCDP diacylglycerol) in 2 ml medium. without or with either 1 mM L-serine or 1 mM sn-glycerol 3phosphate. Aliquots of 0.25 ml were withdrawn at various time intervals, extracted with CH2OH/CHC13 (2 : 1, v/v) [133, and the upper watermethanol phase was counted for [‘4CICMP. o-o , no additions; l -b, L-serine added; A-, sn-glycerol3phosphate added.

ascites mitochondria were aerobically incubated with highly labeled inorganic [ 32P]phosphate and L-serine under conditions promoting formation of [32P]ATP by oxidative phosphorylation (in the presence of succinate and 2-0x0glutarate). After extraction and separation of phospholipids no labeled phosphatidylser~e could be detected. Discussion It is well established that Ca2+dependent base-exchange (pathway 1, Fig. 3) occurs in mammalian liver in the microsomal but not in the mitochondrial fraction [ 3-5 ] . However, the present investigation shows that the Ca’+stimulated process occurs in Ehrhch ascites mitochondria at a rate similar to that in microsomes. A contamination of the mitochondrial fraction by microsomes not exceeding lo%, as estimated on the basis of cholesterol esterase activity, indicates that the base exchange can be intrinsic to these mitochondria. The occurrence of this enzymic process in mitochondria was first reported by Dennis and Kennedy 1211 for ~e~~~~~~e~~ p~r~fo~~is and has recently been demonstrated in mitochondria from Morris hepatoma 7777 [22]. The latter authors suggest that the malignant transformation rather than the rapid growth may be responsible for unsual intracellular localization of this process. Thus, Ehrlich ascites tumor cells can be the next example leading to the unusual intracellular localization of the Ca’*-stimulated base-exchange reaction.

265

0

Phosphatidylethanolamine (phosphatidylsarjne, phosphat~dylcholine)

ethanolamine

serine @ Phosphatidic acid + CTP -f

CDPdiacylglycero~ -)-+

ATP

phosphatidylserine

+--se&e + ?

CMP

Ppi

CMP CTP

@ Serine + ATP -$ ADP

phosphorylserine

+ PPi

/

diacylglycerol

CDPserine

Fig. 3. Possible and speculative pathways for the incorporation

of serine into phosphatidylserine.

The present study shows that mitochondria of Ehrlich ascites cells also incorporate serine into p~osphatidylseri~e by an ATPdependent mechanism. Similar observations have been made a long time ago with liver mitochondria by Hiibscher et al. [8,9] and Bygrave and Biicher [lo]. This ATP-dependent incorporation was not affected by including penicillin and streptomycin in the isolation and incubation media, thus indicating that it was, most likely, not of bacterial origin. The mechanism of this process remains unclear. It was shown in the present study that phosphatidic acid and precursors and cofactors of its synthesis were also stimulatory. Quite recently, Haldar et al. [23] have presented evidence for the synthesis of phosphatidic acid in Ehrlich ascites microsomes but have not been able to demonstrate the presence of glycerolphosphate acyltransferase in mitochondria. This is in disagreement with our previous observations [ 2] that phosphatidic acid is synthesized in Ehrlich ascites mito~hon~a in situ. No matter whether the synthesis of phosphatidic acid was intrinsic to mitochondria or was due to mi~ro~mal contamination, it is highly likely that it occurred in mitochon~i~ prep~tions used in the present investigation, The stimulation by phosph~tidic acid might suggest that the biosynthetic pathway described for bacteria [24] and plants [i’], with CDP diacylglycerol as intermediate (see pathway 2 in Fig. 3), is also operating in animal mitochondria. The stimulatory effect of CMP reported by Hiibscher et al. [8,9] and Bygrave and Biicher [lo] and observed in the present study might also suggest the participitation of the CDP diacylglycerol pathway due to the reversible reaction: CDP dia~ylglycerol + L-serine * CMP f phosphatidylserine. This seems to be subs~tiated by observations of Kiss [25] that in pulsechase expe~en~ on rat heart slices the incorpomtion of inorganic [32P]phosphate into phosphatidylserine immediately followed the incorporation

266

into phosphatidic acid. The CDP diacylglycerol-mediated mechanism for liver microsomes, though operating at a very slow rate, has been recently proposed by Jefsema and Morre f26]. However, we were unable to demonstrate any stimulation of serine incorporation by added CDP diacylglycerol. Neither did serine stimulate the incorporation of 32P from [32P]phosphatidic acid into phosphatidylserine in the presence of CTP, and the release of [ *‘%]CMP from [ 14C]CDP diacylglycerol. Therefore, pathway 2 (Fig. 3) seems unlikely. A speculative possibility that serine is in~orpo~ted into phosphatidylserine in a way similar to the incorporation of choline, that is via the formation of CDPserine (pathway 3, Fig. 3), was also ruled out on the basis that no radioactivity from [fl, T-~~P]ATP was recovered in phosphatidylserine. Therefore, a hitherto unknown mechanism must be responsible for the ATPdependent incorporation of serine into phosphatidylserine in Ehrlich ascites mitochondria. One of the possibilities to be considered may be the stimulation by ATP of the base-exchange reaction. Acknowledgements I wish to thank Professor Lech Wojtczak for his constant interest and helpful discussions during this study and for valuable comments on the manuscript, and Jadwiga Klonowska and Maria Bednarek for skilful technical assistance. References 1 MeMurray, W.C. (1973) in Form and Function of Phospho~pi~ (An&, G.B., Hawthorne, J.N. and Dawson. R.M.C.. eds.), BBA Library, Vol. 3, pp. 205-261, EIsevier, Amsterdam 2 Barafiska. J. and Banskabeva, V.B. (19’76) FEBS Lett. 66.24-29 3 Dennis, E.A. and Kennedy, E.P. (1972) J. Lipid Res. 13.263-267 4 Van Golde. L.M.G.. Raben, J.. Battenburg. J.J., Fleischer, B.. Zambrano. F. and Fleischer. S. (1974) Biochim. Biophys. Acta 360. x79-192 5 Bjerve, K.S. (1973) Biocbim. Biophys. Acta 296.549662 6 Lennarz, W.J. (1970) in Lipid Me~bo~ @Yak& S-J., ea.), pp. 156-184, Academic Press, New York 7 Marsha& M-0. and Kates, M. (1974) Can. J. Biochem. 62.469-482 8 Hfibscher, G.. DiIs. R.R. and Paver. W.F.R. (1968) Nature 182.1806-1807 9 Hiibscher. G.. DiIs. R.R. and Paver. W.F.R. (1959) Biochim. Biophys. Acta 36, 518-528 10 Bygrave, F.L. and Bficher, Th. (1968) Eur. J. Biochem. 6.256-263 11 Barairska. J. and Wojtczak, L. (1974) 17th International Conference on the Biochemistry of Lipids, Milan, Abstracts, p. 27 12 Borst, P. (1960) J. Biophys. Biochem. Cytol. 7,381-383 13 B&h, E.G. and Dyer, W.J. (1959) Can. 3. Bioehem. Physiol. 37.911-917 14 GomalI, A.G.. BardawilI, C.J. and David, M.M. (1949) J. Biol. Chem. 177.751-766 16 Gould, J.M., Cather. R. and Winget, G.D. (1972) Anal. Biochem. 50.540-548 16 Zborowski, J. and Wojtcsak, L. (1969) Biochim. Biophys. Acta 187, 73-84 17 Kennedy, E.P. (1953) J. Biol. Chem. 201. 399412 18 Neskovic, N.M. and Kostic, D.M. (1968) J. Chromatogr. 35.297-300 19 Skipski, V.P., Peterson, R.F. and Barclay. M. (1964) Biochem. J. 90, 374-377 20 Van Heusden. G.P.H. and van den Bosch, II. (1978) Eur. J. Bioehem. 84.405412 21 Dennis, E.A. and Kennedy, K.P. (1970) J. Lipid Res. 11,394403 22 Hostetler, K.Y., Zenner, B.D. and Morris. H.P. (1979) J. Lipid Res. 20,607-613 23 HaIdar, D.. Tso. W.W. and PuBman. ME. (1979) J. Biol. Chem. 264,45024509 24 Kanfer, J. and Kennedy, E.P. (1964) J. Biol. Chem. 239. 1720-1726 25 Kiss, 2. (1976) Eur. J. Biochem. 67. 557-661 26 Jelsema, C.L. and Morre, D.J. (1978) J. Biol. Chem. 253,7960-‘7971