J. Biol. Chem., Vol. 263, Issue 12, 5656-5661, 04, 1988
Stimulation of the de novo pathway for the biosynthesis of platelet- activating factor (PAF) via cytidylyltransferase activation in cells with minimal endogenous PAF production
ML Blank, YJ Lee, EA Cress and F Snyder
Medical and Health Sciences Division, Oak Ridge Associated Universities, Tennessee 37831.
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.
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