Winkler J D, Sung C M, Hubbard W C, Chilton F H
Division of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Biochem Pharmacol. 1992 Nov 17;44(10):2055-66. doi: 10.1016/0006-2952(92)90109-v.
Recent studies suggest that the first step in platelet-activating factor (PAF) biosynthesis, 1-alkyl-2-lyso-GPC (lyso PAF) formation, may be initiated by the selective transfer of arachidonate from 1-alkyl-2-arachidonoyl-GPC to an acceptor lyso phospholipid by a CoA-independent transacylase activity (CoA-IT). The present study was designed to determine whether the formation of 1-alkyl-2-lyso-GPC and the release of arachidonic acid can occur by different mechanisms. These experiments examined both the formation of 1-[3H]alkyl-2-lyso-GPC from 1-[3H]alkyl-2-acyl-GPC and the release of arachidonic acid from membrane phospholipids as determined by GC/MS in neutrophil homogenates under various conditions. The addition of unlabelled lyso phospholipids to neutrophil homogenates stimulated the time-dependent formation of 1-[3H]alkyl-2-lyso-GPC from 1-[3H]alkyl-2-acyl-GPC. Without exogenous lyso phospholipids, little 1-[3H]alkyl-2-lyso-GPC was formed in this reaction. The activity which catalyzed the formation of 1-[3H]alkyl-2-lyso-GPC had characteristics identical to CoA-IT as indicated by the fact that both reactions were: independent of Ca2+, Mg2+, CoA and CoA fatty acids, located in microsomal fractions, and stable in 10 mM dithiothreitol. In sharp contrast to the aforementioned reaction, addition of lyso phospholipids did not affect the quantity of arachidonic acid released from membrane phospholipids. Furthermore, there was a Ca(2+)-independent release of arachidonic acid from membrane phospholipid that was increased 4 to 5-fold after the addition of 5 mM Ca2+. Finally, Ca(2+)-dependent arachidonic acid release was inhibited by putative phospholipase A2 inhibitors, aristolochic acid and scalaradial, at concentrations where neither the production of 1-[3H]alkyl-2-lyso-GPC nor Ca(2+)-independent arachidonic acid release was altered. Together these data imply that there may be different mechanisms involved in the formation of 1-alkyl-2-lyso-GPC and arachidonic acid from membrane phospholipids.
近期研究表明，血小板活化因子（PAF）生物合成的第一步，即1-烷基-2-溶血甘油磷脂酰胆碱（溶血PAF）的形成，可能是由一种不依赖辅酶A的转酰基酶活性（CoA-IT）将花生四烯酸从1-烷基-2-花生四烯酰甘油磷脂酰胆碱选择性转移至受体溶血磷脂而启动的。本研究旨在确定1-烷基-2-溶血甘油磷脂酰胆碱的形成和花生四烯酸的释放是否通过不同机制发生。这些实验检测了在各种条件下，中性粒细胞匀浆中由1-[3H]烷基-2-酰基甘油磷脂酰胆碱形成1-[3H]烷基-2-溶血甘油磷脂酰胆碱以及膜磷脂中花生四烯酸的释放情况（通过气相色谱/质谱法测定）。向中性粒细胞匀浆中添加未标记的溶血磷脂可刺激1-[3H]烷基-2-酰基甘油磷脂酰胆碱随时间形成1-[3H]烷基-2-溶血甘油磷脂酰胆碱。若无外源性溶血磷脂，此反应中几乎不形成1-[3H]烷基-2-溶血甘油磷脂酰胆碱。催化1-[3H]烷基-2-溶血甘油磷脂酰胆碱形成的活性具有与CoA-IT相同的特征，这体现在两个反应均：不依赖Ca2+、Mg2+、辅酶A和辅酶A脂肪酸，定位于微粒体部分，且在10 mM二硫苏糖醇中稳定。与上述反应形成鲜明对比的是，添加溶血磷脂并不影响膜磷脂中释放的花生四烯酸的量。此外，存在一种不依赖Ca2+的膜磷脂中花生四烯酸的释放，添加5 mM Ca2+后其增加4至5倍。最后，推测的磷脂酶A2抑制剂马兜铃酸和辐花内酯在不改变1-[3H]烷基-2-溶血甘油磷脂酰胆碱生成量和不依赖Ca2+的花生四烯酸释放量的浓度下，抑制了依赖Ca2+的花生四烯酸释放。这些数据共同表明，从膜磷脂形成1-烷基-2-溶血甘油磷脂酰胆碱和花生四烯酸可能涉及不同机制。
