Abstract
Treatment of Ehrlich ascites cells with 2 mM oleic acid causes a greater than 10-fold increase in the formation of platelet-activating factor (PAF; 1-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine) from the de novo precursor of PAF, 1-[3H]alkyl-2-acetyl-sn-glycerol. Under these conditions, CTP:phosphocholine cytidylyltransferase activity, which is known to catalyze the rate-limiting step in phosphatidylcholine biosynthesis, was stimulated 32% (p less than 0.001) over control cells. Surprisingly, the dithiothreitol-insensitive choline-phosphotransferase activity, which catalyzes the final step in PAF biosynthesis, was reduced approximately 95% in membranes isolated from cells that were pre-treated with 2 mM oleic acid. However, calculations of product formation at this reduced cholinephosphotransferase activity revealed that it was still sufficient to accommodate the increased synthesis of PAF observed in the intact oleic acid-treated cells. Kinetic studies and experiments done with cells treated with phenylmethylsulfonyl fluoride (an acetylhydrolase inhibitor) indicate the various metabolic products formed are derived through the following sequence of reactions: 1-alkyl-2-acetyl-sn-glycerol----1-alkyl-2-acetyl-sn-glycero-3- phosphocholine----1-alkyl-2-lyso-sn-glycero-3-phosphocholine----1-alkyl- 2(long-chain) acyl-sn-glycero-3-phosphocholine. These results indicate PAF is the source of alkylacylglycerophosphocholine through the action of an acetylhydrolase and a transacylase as shown in other cell systems. The relative amounts of PAF, lyso-PAF, and alkylacylglycerophosphocholine produced after treatment of the cells with oleic acid in the absence of the phenylmethylsulfonyl fluoride inhibitor indicate that the acylation rate for lyso-PAF is considerably slower (i.e. rate-limiting) than the deacetylation of PAF by acetylhydrolase. We further conclude that the final step in the de novo pathway for PAF biosynthesis is under the direct control of CTP:phosphocholine cytidylyltransferase, which emphasizes the importance of this regulatory (rate-limiting) step in the biosynthesis of both phosphatidylcholine and PAF.
Highlights
Treatment of Ehrlich ascites cells with 2 mM oleic glycero-3-phosphocholine or alkylacetyl-GroPCho)’ is a poacid causes a >lO-fold increase in the formation of tent biologically active phospholipid with diverse physiologiplatelet-activating factor
Because of the importance of cytidylyltransferase in the regulation of the biosynthesis of phosphatidylcholine [16], we investigated whether cells with minimal in chloroform:methanol:glacial acetic acidwater (50:30:8:5, v/v), whereas neutral lipids were separated on boric acid (4%)-impregnated Silica Gel G chromatoplates developed in ch1oroform:methanol (97:3, v/v)
We incubated cell suspensions for 10 min a t 37 "C, and after the cells were separated from the media by centrifugation (500 X g for 10 min) at Stimulation of P A F Biosynthesis and Its Metabolites Obleyic 4 "C, the totallipids were extracted by the method of Bligh and Dyer Acid and Sodium Oleate i n EAC-Biosynthesis of total cho
Summary
Preparation of PHIAlkylacetyl-Gro(I-[9,10-3H]Hexadecyl-2-acetylsn-gly~erolJ-[~H]Alkylacetyl-Growas prepared by hydrolysis of 1[9,10-3H]hexadecyl-2-acetyl-sn-glycero-3-phosph~ho(l[i3nHe]alkylacetyl-GroPCho, 56 pCi/nmol, a generous gift from Dr Steven Wyrick, University of North Carolina, Chapel Hill, NC) with phospholipase C (Bacilluscereus, type V from Sigma), as described by Mavis et al [23]. Measurement of CytidylyltransferaseActivity-EAC (20 X lo' cells) were incubated in a total volume of 20 ml of Hepes-buffered DMEM at 37 "C for 20 min in the presence of either 1%BSA (controls) or 1%BSA plus 2 mM oleic acid. Estimation of EndogenousFree FattyAcid Levels-EAC from four These cell suspensions were either used directly to examine the micewere collected, washed as already described, counted, and the metabolism of [3H]alkylacetyl-Gro,or the cells were first recovered lipids extracted using 2% glacial acetic acid in the methanol portion by centrifugation (500 X g for 10 min) and homogenized for use in of the Bligh and Dyer method [26].
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