Blank M L, Lee Y J, Cress E A, Snyder F
Medical and Health Sciences Division, Oak Ridge Associated Universities, Tennessee 37831.
J Biol Chem. 1988 Apr 25;263(12):5656-61.
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.
用2 mM油酸处理艾氏腹水细胞，可使血小板活化因子（PAF；1-[3H]烷基-2-乙酰基-sn-甘油-3-磷酸胆碱）从其从头合成前体1-[3H]烷基-2-乙酰基-sn-甘油的生成量增加10倍以上。在这些条件下，已知催化磷脂酰胆碱生物合成限速步骤的CTP：磷酸胆碱胞苷转移酶活性，比对照细胞提高了32%（p<0.001）。令人惊讶的是，催化PAF生物合成最后一步的二硫苏糖醇不敏感胆碱磷酸转移酶活性，在用2 mM油酸预处理的细胞分离的膜中降低了约95%。然而，根据这种降低的胆碱磷酸转移酶活性计算产物生成量表明，它仍足以满足完整的油酸处理细胞中观察到的PAF合成增加。动力学研究以及用苯甲基磺酰氟（一种乙酰水解酶抑制剂）处理细胞的实验表明，形成的各种代谢产物是通过以下反应序列产生的：1-烷基-2-乙酰基-sn-甘油----1-烷基-2-乙酰基-sn-甘油-3-磷酸胆碱----1-烷基-2-溶血-sn-甘油-3-磷酸胆碱----1-烷基-2（长链）酰基-sn-甘油-3-磷酸胆碱。这些结果表明，如在其他细胞系统中所示，PAF是通过乙酰水解酶和转酰基酶的作用产生烷基酰基甘油磷酸胆碱的来源。在用油酸处理细胞且不存在苯甲基磺酰氟抑制剂的情况下，产生的PAF、溶血PAF和烷基酰基甘油磷酸胆碱的相对量表明，溶血PAF的酰化速率比PAF被乙酰水解酶脱乙酰化的速率慢得多（即限速）。我们进一步得出结论，PAF生物合成从头途径的最后一步受CTP：磷酸胆碱胞苷转移酶的直接控制，这强调了该调节（限速）步骤在磷脂酰胆碱和PAF生物合成中的重要性。
