A comparison of lysophosphatidylcholine acyltransferase activities in neuronal nuclei and microsomes isolated from immature rabbit cerebral cortex

223

Biochirnica et Biophysics Acta, 666 (1981) 223-229 Else~er~North-Hound Biomedical Press BBA 57918

A COMPARISONOF LYSOPHOSPHATIDY~HOLINEACYLTRANSFERASEACTIVITIESIN NEURONAL NUCLEIAND MICROSOMESISOLATEDFROM IMMATURERABBIT CEREBRALCORTEX R.R. BAKER and H.-Y. CHANG Neurology Program, Department of Medicine, 6368 Medical Sciences Bldg., Universityof Toronto, Toronto M5S IA8 (Canuda) (Received April Sth, 1981)

Key words: Ly~pho~~utidy~choline acyl~a~sferuse; N~cieus; ~~cro~me~ (rabbit cerebral cortex)

Using neuronal nuclei (N1) and microsomes (pa) isolated from cerebralcortices of lSday-old rabbits, the activity of lysophosphatidylcholine acyltmsferase (acyl CoA: 1-acyl-m-glycerol-3-phosphorylcholineacyltransferase)was studied u&g palmitoyl-, oleoyl- and arachidonoyl-CoA and a pool of lysophosphatidylcholine labelled with [ 3H]paImitate, [ 3H]stearate or [ 3H]oleate. GeneraUy,in the acylation of the three radioactivelysophosphatidylchohes with arachidonoyl-CoA, the N,-specific acylation activities we= two to seven times those of P3, For oleoyl-CoA smaller N1 : P, specific activity ratios were found, differing significantly from unity for only the l-palmitoyl and l-stearoyl lysophosphatidylcholines. The N1 : P3 ratios for the two unsaturated acyl-CoA thioesters were usually found to increase as the lysophosphatidylcholine concentration was lowered from 100 to 25 @i. Thus, nuclear acylation rates, p~cu~y with ~~hidonoyl~oA, wefe less affected by lowering the acceptor concent~tion than were microsomal activities. At the high lysophosphatidylcholine con~n~tion (100 &i), ~chidonoyl~oA was a superior substrate to oleoyl-CoA in the nuclear acylations of the l-palmitoyl or l-stearoyl acceptors. Such a preference was never seen for the microsomal fraction. Using oleoyl- and arachidonoyl_CoA,the nuclear enzymes also showed a greater preference for the l-palmitoyl homologue over the l-oleoyl homologue than did the microsomal enzymes. These results support the existence of neuronal nuclear lysophosphatidylchole acyltransferases with different substrate preferencesthan shown by the microsomal fraction.

Introduction Following intrathecal injections of 13H]palmitate, 13H]oleate or f3H] arac~donate and [“4C]glycerol into IS-day-old rabbits, we isolated several subfractions, including microsomes, from radioactively labelled nerve cell bodies of cerebral cortex. Of these subfractions the purified nuclear fraction had phospholipids with the highest ‘H specific radioactivities and the highest 3H : 14C double label ratios [l]. Thus, the existence of neuronal nuclear lysophospholipid acyltransferases was suggested. Using in vitro assays with subfractions isolated from cerebral cortex (1 5-day-old rabbit) we recently demonstrated 2-3fold higher specific activities with oleoyl-CoA for a neuronal nuclear fraction over the microsomal frac0005~2760/8~/0000-0000/$02.50

0 1981 ElsevierfNorth-Holland

tion in the acylation of I-acyl-sn-glycerol-3-phosphorylcholine (labelled with [3H]palmitate) 121. We further isolated nuclear envelopes from neuronal nuclei and demonstrated a 6-fold en~chment in this lysophosphatidylcho~ne acyltransferase activity over the parent nuclei [2]. It was the aim of this study to investigate the characteristics of this nuclear acylation activity using different acyl-CoA thioesters and l-acyl-sn-glycerol-3.phosphorylcholines labelled with different radioactive fatty acids, and to compare these features with those of the microsomal fraction. Materialsand Methods Materials. Coenzyme A (free acid), p~~toyl- and oleoyK!oA were purchased from P-L Biochemicals Biomedical Press

224

Inc. (Milwaukee, WI). Nu Chek Prep Inc. (Elysian, MN) supplied arachidonoyl and oleoyl chlorides and Monsanto Ltd. (Nitro, WV) provided the Santoquin (ethoxyquin). Phosphatidylcholine (carrier) was obtained from Serdary Research Laboratories (London, Ontario). New England Nuclear Corp. (Lachine, P.Q.) provided the [9,10-3H]oleic acid (10.0 Ci/mmol) and [9,10-3H] palmitic acid (16.8 Ci/mmol). Male, New Zealand white rabbits (15 days of age) were purchased from Riemens Fur Ranches (St. Agatha, Ontario). Preparation of subcellular fractions. The neuronal nuclear fraction Ni was prepared from cerebral cortices following the method of Thompson [3] as described by Baker and Chang [4]. In this procedure nuclei were sedimented from homogenates prepared in 2.0 M sucrose/ 1 mM MgCis , further purified by discontinuous gradient centrifugation and then washed and sedimented three times using low-speed differential centrifugation. The microsomal fraction Pa was prepared by sedimenting a post-~tochondrial supernatant at 120 000 X g for 60 min [2]. Both fractions Nr and Ps were finally suspended in 10 mM Tris-WC1 (pH 7.4) containing 0.32 M sucrose and were used as fresh preparations or stored in small aliquots on dry ice for up to 10 days. Preparation of radioactive I-acyl-sn-glycerol-3phospho~lcholines. Approximately 1 mCi of either [3H]palmitate, [3H]oleate or [3H]stearate was prepared as a complex with bovine serum albumin and injected intrathecally into 15-day-old rabbits as described by Baker and Chang [ 11. The ]3H]stearate used was prepared by hydrogenation of the [3HJoleate methyl ester [5], followed by isolation of [3H]stearate methyl ester by argentation thin-layer chromatography [I] and hydrolysis of the ester in ethanolic KOH [6]. The rabbits were killed 20 min post-injection, the cerebral cortices were removed and [3H]phosphatidylcholine isolated by column and thin-layer chromatography 121. l- ]“H] Acyl-SE-glycerol-3-phosphorylcholine was produced by phospholipase Az (Crotalus adamanteus) hydrolysis of the t3H] phosphatidylcholine and purified by silicic acid column chromatography 121. Preparation of acyi-CoA thioesters. Arachidonoyiand oleoyl-CoA thioesters were prepared by the method of Reitz et al. [7] as described by Baker and Thompson [8] using commercially supplied acyl

chlorides. Santoquin was dissolved in benzene and purified using alumina chromatography (Brockntan activity I) with benzene as eluant (loading: 50 ~1 Santoquin/S g alumina). The yellow-orange Santoquin was dried under nitrogen and added only to media containing tetrahydrofuran (freshly distilled), Post-synthesis Santoquin was removed from the thioester acid precipitates by ethyl ether extraction. Solutions of acyl-CoA thioester products were prepared in aqueous 10 mg% hydroquinone and stored at -7O’C. Lysophosphatidylcholine acyltransferase assay. The conversion of lysophosphatidylcholine into phosphatidylcholine by acylation with acyl-CoA thioesters (acyl-CoA: 1-acyl-srz-glycerol-3-phosphorylcholine was essentially as acyltransferase) monitored described by Holub et al. [9]. The assay mixture contained 50 mM Tris-HCl (pH 7.4) 20-100 FM acylCoA, 25-200 I.IM lysophosphatidylcholine (concentration of total pool of lysophosphatidylcholine molecular species, with one species labelled as the [l-3HJpalmitoyl, [l-3H]stearoyl or [f-3H]01eoyl derivative) and 0. I-O.3 mg protein in a final volume of 0.5 ml. Incubations were usually for 3 min at 37°C in a shaking water bath and were stopped on ice by the addition of 10 ml of c~oroform~nlethanol(2 : 1, v/v). Unlabelled carrier phosphatidylcholine was added (10 /lg ofphosphorus), lipids were extracted [2] and radioactive phosphatidylcholine isolated by thinlayer chromatography [9] and eluted from the gel ’ [2]. Radioactivity measurements were carried out as described by Baker and Chang [2]. Matching assays in the absence of acyl-CoA thioesters or in the presence of boiled enzyme were run as blanks. The synthesis of radioactive phosphatidylcholines was determined using the specific radioactivities of individual fatty acids in the lysophosphatidylcholine substrates. Protein, marker enzymes and nucleates. Protein was determined by the method of Lowry et al. [lo] using bovine serum albumin as standard. Assays for DNA and RNA 141, cholinesterase, c~tochronle oxidase and N-acetylglucosaminidase [I] and NADPHcytochrome c reductase [ 1 l] have been reported previously.

225 Results and Discussion Protein, nucleates and marker enzymes in subfractions As shown in Table I, of the two subfractions studied, the neuronal nuclei (N,) had the higher DNA content and an RNA : DNA ratio of 0.53 in close agreement with earlier results [4]. The microsomal fraction (Ps) had a specific RNA content (100 pg/mg protein) which was very close to values reported for microsomal fractions from rat brain [ 121. Fraction P3 had the higher specific activity of cholinesterase, a marker for endoplasmic reticulum and plasma membranes of cholinergic cells, and of NADPH-cytochrome c reductase, a marker for endoplasmic reticulum [ 131. However, nuclear envelopes isolated from rat liver have been shown to have relatively high specific activities of many enzymes (including NADPH-cytochrome c reductase and glucosed-phosphatase) considered to be markers for other membranes [ 141. Thus, the activity of cholinesterase and NADPH-cytochrome c reductase in N1 need not solely indicate microsomal contamination. The specific activity of lactate dehydrogenase in fraction N1 assayed in the presence of Triton X-100 [4], was less than 4% of that found in the homogenate. This is indicative of a lack of cellular contamination in N1. We have shown previously substantial differences between N1 and P3 in their relative contents of phosphatidylinositol, phosphatidylserine and sphingo-

myelin [2]. The phospholipid profile in N1 was also quite different in this regard when compared with other cellular and subcellular fractions [ 151. It has been proposed that the use of heavy sucrose gradients in the isolation of nuclei effe’ctively removes microsomal contamination [ 161. There was also little mitochondrial or lysosomal contamination in either N1 or Ps as judged by cytochrome oxidase and N-acetylglucosaminidase, respectively. Substrates for lysophosphatidylcholine acyltransferase The composition of the fatty acids of the l-acylsn-glycerol-3-phosphorylcholine in mol% was as follows: 16 : 0, 58.8 * 0.9;, 16 : 1, 3.1 f 0.3; 18 : 0, 15.3kO.7; 18:1, 18.7kO.7; 18:2, 4.1kO.6; means f S.D. (n = 12). The molar ratio of fatty acid : phosphate in this lysophosphatidylcholine was 0.99 f 0.04, mean + S.D. (n = 11). The lysophosphatidylcholine substrate was labelled by one of three radioactive fatty acids and argentation thin-layer chromatography of derived fatty acid methyl esters revealed that 95% of the radioactivity was associated with saturated fatty acids in the cases of [3H]palmitate and [‘Hlstearate and 95% of the [3H]oleate radioactivity was recovered in the monoene fraction. The specific radioactivities for the three labelled lysophosphatidylcholines were: (2.36 + 0.14) - 10’ dpm/ nmol palmitate, mean f S.D. (n = 5); (8.45 + 0.54) . lo3 dpmlnmol oleate, mean + S.D. (n = 4) and

TABLE I MARKER ENZYMES, PROTEIN AND NUCLEATE CONCENTRATIONS CEREBRAL CORTEX

IN SUBCELLULAR

FRACTIONS OF RABBIT

Units of enzyme specific activity are nmol/min per mg protein. The concentrations of protein and nucleates are given as mg/g wet wt. cerebral cortex and rg/mg protein, respectively. Values are means -+ S.D. of three or more individual determinations. Homogenate

Protein DNA RNA Cholinesterase NADPH-cytochrome c reductase Cytochrome oxidase N-Acetylglucosaminidase

75.9 k 3.1 15.9 2 0.8 37.8 _+ 2.7 81.6 + 7.4 1.23+ 0.3 68.5 f 13.9 22.2 _+ 0.5

Nl (neuronal nuclei)

p3

1.51 240 128 19.1 1.9 4.4 2.9

12.9 + 4.7 + 99.6 f 161.8 + 5.02 1.7 + 15.2 +

f 0.19 k4I + 16 + 2.9 _+ 0.4 t 1.0 + 0.2

(microsomes) 1.8 1.7 6.6 14.7 0.6 0.2 0.7

226

(3.52 f 0.37) * lo3 dpm/nmol stearate, average f range (n = 2). The commercial palmitoyl-CoA and oleoyl-CoA had molar ratios of phosphate : palmitate or : oleate of 2.7 and 2.6, respectively, (3.0, theoretical) and showed ratios of absorbance at 232/260 nm, 250/260 nm and 280/260 nm similar to previous results given for oleoyl-CoA [2]. The oleoyl-CoA and arachidonoyl-CoA synthesized in our laboratory had molar ratios of phosphate : fatty acid of 3.5 and 2.8, respectively. Oleoyl-CoA preparations from both sources were equally good substrates. The acyl-CoA thioesters were dissolved in 10 mg% hydroquinone as this antioxidant did not inhibit the acylation reactions but preserved the unsaturated acyl-CoA thioesters as viable substrates for at least 6 weeks at -70°C. Santoquin, at very low concentrations in the incubation medium, brought about a substantial inhibition (up to 60%) of the acylation of lysophosphatidylcholine. Lysophosphatidylcholine acyltransferase activities in subfractions In our assays we have used as acceptor l-acyl-snglycerol-3-phosphorylcholine derived from phosphatidylcholine of cerebral cortex of rabbit brain. This lysophosphatidylcholine substrate is a mixture of three principal molecular species, e.g. in 100 E.IMlysophosphatidylcholine would be found 1-palmitoyl, I-stearoyl and I-oleoyl homologues at 59, 15 and 19 PM concentrations, respectively. We have labelled each of the homologues with the appropriate radioactive fatty acid and have used three separately labelled lysophosphatidylcholine substrates. Thus, we have measured individually the formation of l-palmitoyl, 1stearoyl or 1-oleoyl phosphatidylcholine during a competition with the remaining, unlabelled lysophosphatidylcholine molecular species. This is a more effective way of assessing selectivities based on the fatty acid in the acceptor [9] as little preference has been shown by rat liver microsomes in the use of pure, synthetic molecular species of lysophosphatidylcholines [ 171. As well, a mixture of lysophosphatidylcholines derived from endogenous phosphatidylcholine may be a better approximation of the lysophosphatidylcholines available in vivo for acylation. In the lysophosphatidylcholine acyltransferase assays there was no significant production of phosphatidylcholine in the presence of boiled enzyme (or

in the absence of enzyme). However, in the absence of acyl-CoA thioesters, there was a small but significant production of phosphatidylcholine as a result of the activity of lysophosphatidylcholine transacylase. These transacylase specific activities in fraction Nr and P3 were approximately lo-20% of the corresponding lysolecithin acyltransferase values. Thus, transacylase blanks were run for each new lysolecithin acyltransferase assay condition tested. The lysophosphatidylcholine acyltransferase rates varied in a linear manner with time for at least 3 or 4 min and with enzyme concentration up to 0.3 or 0.4 mg protein per 0.5 ml incubation volume. The effect of concentration of acyl-CoA thioesters demonstrated that a plateau in the lysophosphatidylcholine acyltransferase rates was maintained over a range of 20 to 100 I.IM concentration. For the lysophosphatidylcholine acceptor, maximum acylation rates were established at 100 PM and these were maintained or declined slightly at 150 and 200 PM. We have tested acceptor concentrations of 100, 50 and 25 I.IM as selectivities for arachidonoyl-CoA over oleoyl-CoA in 1-acyl-snglycerol-3-phosphorylcholine acylation were more pronounced at lower lysophosphatidylcholine concentrations using rat liver micr,osomes [ 181. The acylation rates in the formation of l-palmitoy1 phosphatidylcholine are given in Table II. The specific activities for fraction Ni using the two unsaturated acyl-CoA thioesters ranged from two to seven times the corresponding values for P3, as seen by the Nr : P3 activity ratios. The Nr : P3 ratios for arachidonoyl-CoA were higher at each concentration of lysophosphatidylcholine than the corresponding values for oleoyl-CoA. These differentials increased with decreasing lysophosphatidylcholine concentration for both unsaturated acyl-CoA thioesters. This indicated that the nuclear acylation activities were less affected by decreasing acceptor concentrations. The size of the N 1 : P3 ratios demonstrated that the lysophosphatidylcholine acyl transferase activity in fraction Nr is nuclear in origin and cannot be ascribed to microsomal contamination. Recently, we have shown that nuclear envelopes prepared from neuronal nuclei have 6-fold higher specific lysophosphatidylcholine acyltransferase activities than the parent nuclei [2], indicating a concentration of this acylation activity in the nuclear membranes. It is likely that the location of other enzymes in the nuclear

227 TABLE II THE FORMATION OF l-PALMITOYL SPECIES OF PHOSPHATIDYLCHOLINE DURING ACYLATION OF MIXED l-ACYLGLYCERYLPHOSPHORYLCHOLINES WITH ACYLCoA THIOESTERS BY SUBFRACTIONS OF CEREBRAL CORTEX Standard incubation conditions were employed using SO-75 I.~M~achidonoyi~oA (20 : 4 CoA) or 100 PM oleoyl-CoA (18 : I CoA) or paImitoyl-CoA (16 : 0 CoA), 0.1-0.2 mg protein and 25, 50 or 100 PM l-acyl-~~-~ycerol-3-phosphoryicbo~e Iabelled with [ 3H]palmitate. All values given are means + S.D. of three or more separate experiments (except for one single value for P3 and palmitoyl-CoA at 25 PM). The subfractions used were Nr (neuronal nuclei) and Pa (microsomes). Specific activity ratios Nr : P3 and 20 : 4/18 : 1 CoA are also presented. Those ratios which differ significantly from unity (P< 0.01, based on Student’s t-test) are followed by superscript a. Concentration (rM)

100

Subfraction

Nr p3

Nr :P3 50

Nt p3

Nr :Ps 25

Nr p3

Nr :P3

l-Palmitoylphosphatid~l~ho~ne

formed (nmol/lO mm per mg protein)

16: OCoA

18: 1CoA

20:4CoA

20 : 4/18 : 1 CoA

8.1 f 1.5 6.4 _+0.5 1.3

21.5 1: 3.2 11.7 t 1.5 1.8 a

32.3 -c 4.1 12.2 -c 2.9 2.7 a

1.5 a 1.0

5.3 f 0.3 4.2 _+0.3 1.3

19.4 F 3.1 8.7 k 1.3 2.2 a

24.1 t 2.5 5.7 + 1.0 4.2 a

1.2 0.7 a

3.6 f 0.1 3.6 1.0

16.1 t 1.7 4.9 rt 0.3 3.3 a

14.4 z!z2.9 2.1 + 0.4 6.9 a

0.9 0.4 a

envelope may be obscured by the relatively large dilution of such activities by chromatin proteins in intact nuclei. This effect has been demonstrated by Van Golde et al. [ 191 who found si~i~cant lysophosphatidylcholine acyltransferase, 1,2-diacylglycerol acyltransferase and cholinephosphotransferase activities in nuclear fractions from bovine liver when such activities were expressed in terms of membrane phospholipid instead of total nuclear protein. Fraction Ps is a ~crosom~ fraction derived from whole cerebral cortex of Sday-old rabbits and is not a purely neuronal microsomal fraction. From homogenates of nerve cell bodies prepared from this tissue we have isolated a second microsomal fraction with a very high specific RNA content (469 ggfmg protein). This second microsomal fraction showed very similar specific lysophosphatidylcholine acyltransferase activities to fraction P3 in the acylation of lysophosphatidylcholine labelled with [‘HI palmitate. The selectivity shown for the two unsaturated acyl-C!oA thioesters is given by the 20 : 4f 18 : 1 CoA ratios in Table II. The specific activities shown by fraction Nr for arachidonoyl-CoA were significantly higher only at the 100 /.&I lysophosphatidylcholine concentration, while at the 25 I.IMconcentratron the

acyl-CoA thioesters were used equally well. Even more striking was the situation for fraction P3 which showed no preference for arachidonoyl-CoA at the high lysophosphatidylcho~e con~ntration and a definite preference for oleoyl-CoA at the 50 and 25 FM acceptor concentrations. This demonstrated an important qualitative difference in the acylation of lysophosphatidylcholine by the two subcellular fractions. This decline in acylation with the polyunsaturated acyl-CoA thioester at lower acceptor concentrations is the reverse of the situation reported for rat liver microsomes [ 181. The acylation rates for 1-oleoyl-sn-glycerol-3.phosphorylcholine are given in Table III. The specific activities for this unsaturated molecular species of lysophosphatidylcho~e were generally less than 50% of the corresponding values for the I-palmitoyl homologue (Table II). This can be attributed, in part, to the 4.fold higher concentration of the 1-palmitoyl acceptor in the lysophosphatidylcholine substrate. However, in certain cases (e.g. for fraction Ni at the 100 MM acceptor concentration) the rates for the I-palmitoyl homologue were close to six times the values for the 1-oleoyl homologue, suggesting a possible preference for this saturated lysophosphatidylchotwo

228 TABLE III THE FORMATION OF l-OLEOYL SPECIES OF PHOSPHATIDYLCHOLINE DURING ACYLATION OF MIXED l-ACYLGLYCERYLPHOSPHORYLCHOLINES WITH ACYL-CoA THIOESTERS BY SUBFRACTIONS OF CEREBRAL CORTEX The incubation conditions were as noted in Table II but with 1-acyl-sn-glycerol-3-phosphorylcholine labelled with [3H]oleate. All values are means + S.D. of three or more separate experiments. Subfractions and specific activity ratios are as designated in Table II. Those ratios which differ significantty from unity (P < 0.01, based on Student’s t-test) are followed by the superscript a. Concentration (@M)

100

Subfraction

Nr p3

N, :P3 50

Nr p3

Nr :P3 25

Nl p3

N1 :P3

I-Oleoyl phosphatidylcholine

formed (nmol/lO min per mg protein) ____--

16:OCoA

18:lCoA

20 : 4 CoA

20:4/18:1CoA

1.4 f 0.2 1.7 kO.2 0.8

4.8 It 0.3 3.9 t 0.6 1.2

5.9 + 0.7 3.4 t 0.5 1.7a

1.2 0.9

1.2 + 0.2 1.3 i- 0.2 0.9

4.2 + 0.4 3.2 f 0.4 1.3

4.8 + 0.4 2.1 f 0.3 2.3 a

1.1 0.7 a

0.5 _+0.1 0.9 f 0.1 0.6

3.5 f 1.0 2.3 k 0.2 1.5

3.7 t 0.2 1.1 + 0.1 3.4 a

1.1 0.5 a

line. Absolute proof of this would require the use of an acceptor population with equal concentrations of the two molecular species. The Nr : Ps specific activity ratios shown in Table III for oleoyl-CoA and arachidonoyl-CoA were approximately 50% of the corresponding ratios seen in Table II. Only for arachidonoyl-CoA were the specific nuclear acylation rates significantly higher than the corresponding microsomal activities. Thus, the nuclear fraction, in the acylation of 1-oleoyl lysophosphatidylcholine, did not show the same superiority over the microsomal fraction as was evident with the 1-palmitoyl acceptor. This disproportionate use of the two lysophosphatidylcholine substrates is another qualitative difference seen in the acylation by the nuclear and microsomal enzymes. With the 1-oleoyl lysophosphatidylcholine there was no significant preference shown by the nuclear fraction for the arachidonoyl-CoA over the oleoyl-CoA, as the 20 : 4/18 : 1 CoA specific activity ratios were quite close to unity. This contrasted with the preference shown to arachidonoyl-CoA by fraction Nr using the 1-palmitoyl lysophosphatidylcholine (Table II, at 100 PM lysophosphatidylcholine). On the other hand, the microsomes showed a decline in 20 : 4118 : 1 CoA specific activity ratios with decreasing lysophosphatidylcholine acceptor concentrations which was simi-

lar for both 1-oleoyl and 1-palmitoyl homologues. The specific activities for the conversion of 1-stearoyl lysophosphatidylcholine into phosphatidylcholine are given in Table IV. The concentrations of 1stearoyl and 1-oleoyl acceptors in the lysophosphatidylcholine population were similar (15 and 19 ,uM, respectively), yet the 1stearoyl homologue had acylation rates which were generally less than 60% of those for the 1-oleoyl lysophosphatidylcholine (Table III). Thus, a preference for the unsaturated 18 carbon homologue may be suggested. In the acylation of the I-stearoyl homologue the Nr : P3 ratios for arachidonoyl-CoA were similar to the corresponding values seen for the I-palmitoyl acceptor (Table II) and a preference for arachidonoyl-CoA over oleoyl-CoA was shown by the nuclear fraction at 100 PM lysophosphatidylcholine. Again the microsomal fraction showed no preference for this polyunsaturated acylCoA over oleoyl-CoA. Based on Tables II, III and IV a preference for arachidonoyl-CoA could be demonstrated for the nuclear fractions when 100 PM concentrations of 1-saturated lysophosphatidylcholines were used as acceptors. The highest Nr : P3 ratios were also seen for these two lysophosphatidylcholine homologues with this polyunsaturated acyl-CoA. The summation of the nuclear acylation rates for the three 1-acyl lyso-

229

TABLE IV THE FORMATION OF l-STEAROYL SPECIES OF PHOSPHATIDYLCHOLINE DURING ACYLATION OF MIXED l-ACYLGLYCERYLPHOSPHORYLCHOLINES WITH ACYL-CoA THIOESTERS BY SUBFRACTIONS OF CEREBRAL CORTEX The ~cubation conditions were as noted in Table II but with l-acyl-so-glycerol-3-phospho~lcholine labelled with [3H]oIeate, All values are means f S.D. of three or more separate experiments. Subfractions and specific activity ratiosare asdesignated in Table II. The ratios which differ significantly from unity (P < 0.05, based on Student’s t-test) are followed by the superscript a. Concentration (PM)

100

Subfraction

Nl p3

N1 :P3 50

Nl p3

N1 :P3

I-Stearoyl phosphatidylcholine

formed (nmol/lO min per mg protein)

16:OCoA

18: 1CoA

20:4CoA

20 : 4/l 8 : 1 CoA

0.6 -+0.1 0.4 t 0.1 1.5

2.1 t 0.3 1.6 + 0.4 1.3

3.8 rt 0.9 1.1 2 0.3 3.5 a

1.8 a 0.7

0.5 + 0.1 0.4 t 0.1 1.3

1.9 + 0.2 1.0 + 0.2 1.9a

2.1 it 0.7 0.5 f 0.1 5.4 a

1.4 0.5

phosphatidylcholines (at 100 $l total lysophosphatidylcholine concentration) were: 20 : 4 CoA, 42.0; 18 : 1 CoA, 28.4; 16 : 0 CoA, 10.1 nmolfl0 min per mg protein. In preliminary studies of lysophosphatidylcholine acyltransferases in subfractions derived from cerebral cortices of 9.week-old rabbits we have shown that neuronal nuclei did have higher specific activities than microsomes for the l-saturated lysophosphatidylchol~e acceptors with arac~donoyland oleoyl-CoA. Acknowledgements This work was supported by a grant from the Medical Research Council of Canada. R.R.B. is a Scholar of the Medical Research Council. References 1 Baker, R.R. and Chang, H.-Y. (1980) Biochem. J. 188, 153-161 2 Baker, R.R. and Chang, H.-Y. (1981) Can. J. Biochem., in the press 3 Thompson, R.J. (1973) J. Neurochem. 21,19-40 4 Baker, R.R. and Chang, H.-Y. (1980) Can. J. Biochem. 58,620-628

5 Eybel, C.E. and Simon, R.G. (1970) Lipids 5, 590-596 6 Kates, M. (1972) in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T.S. and Work, E., eds.), 1st edn., Vol. 3, p. 363, Else~~/No~h-Hound, Amsterdam 7 Reitz, R.C., Lands, W&M., Christie, W.W. and Holman, R.T. (1968) J. Biol. Chem. 243,2241-2246 8 Balier, R.R. and Thompson, W. (1973) J. Biol. Chem, 248,7060-7065 9 Holub, B-J., MacNaughton, J.A. and Piekarski, J. (1979) Biochim. Biophys. Acta 572,413-422 10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and RandalI. R.J. (1951) J. Biol. Chem. 193,265-275 11 Baker, R.R. (1979) Brain Res. 169,65-81 12 Morgan, I.G., Wolfe, L.S., Mandel, P. and Combos, G. (1971) Biochim. Biophys. Acta 241,737-751 13 Miller, E.K. and Dawson, R.M.C. (1972) Biochem. J. 126, 805-821 14 Franke, W.W. (1974) Phil. Trans. R. Sot. (Lond.) B 268, 67-93 15 White, D.A. (1973) in Form.and Function of Phospho. lipids (An& G.B., Hawthorne, J.N. and Dawson, R.M.C., eds.) 2nd edn., p. 441, Elsevier Scientific Publishing Co., Amsterdam 16 Thompson, R.J. (1975) J. Neurochem. 25,811-823 17 Brand& A.E. and Lands, W.E.M. (1967) Biochim. Biophys. Acta 144,605-612 18 Okuyama, H., Yamada, K. and Ikezawa, H. (1975) J. Biol. Chem. 250,1710-1713 19 Van Golde, L.M.G., Fleischer, B. and Fleischer, S. (1971) Biochim. Biophys. Acta 249,318-330