Kramer R M, Jakubowski J A, Deykin D
Boston University School of Medicine, MA.
Biochim Biophys Acta. 1988 Apr 15;959(3):269-79. doi: 10.1016/0005-2760(88)90200-7.
The metabolism of platelet-activating factor (PAF) and arachidonic acid is linked through the common intermediate 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (alkylarachidonoyl-GPC). Hydrolysis of alkylarachidonoyl-GPC by phospholipase A2 may initiate the biosynthesis of both PAF and eicosanoids, since alkyllyso-GPC is formed for acetylation to PAF and arachidonic acid is liberated for conversion to biologically active metabolites. In order to elucidate the regulation and functional role of human platelet phospholipase A2 in the pathway leading to the formation of both classes of lipid mediators, we have characterized its action upon alkylarachidonoyl-GPC. Human platelet phospholipase A2 was solubilized and then partially purified in the presence of n-octyl-beta-D-glucopyranoside (octyl glucoside). Hexadecylarachidonoyl-GPC was prepared biosynthetically using platelet sonicates, purified by two-step high-performance liquid chromatography (HPLC) and suspended in buffer by sonication. Our results indicate that deacylation of alkylarachidonoyl-GPC by platelet phospholipase A2 has an absolute requirement for Ca2+. It occurs at submicromolar concentrations of free Ca2+ and exhibits a biphasic Ca2+-dependence with activity plateaus at 10 microM and 2 mM. Phospholipase A2-mediated hydrolysis of alkylarachidonoyl-GPC is increased 2-fold by albumin and is enhanced 5-fold if 1,2-dioleoylglycerol is incorporated into the substrate dispersion. The substrate dependence and specificity of platelet phospholipase A2 for 1-alkyl- vs. 1-acyl-linked subclasses of arachidonic acid containing phosphatidylcholine was examined with 1-O-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (hexadecylarachidonoyl-GPC) and 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (palmitoylarachidonoyl-GPC). We found that the substrates were deacylated equivalently. We conclude that, in stimulated platelets, in the presence of increased levels of cytoplasmic free Ca2+ and newly generated diacylglycerol, alkylarachidonoyl-GPC may be rapidly hydrolyzed by phospholipase A2 and may serve as a precursor of both PAF and eicosanoids.
血小板活化因子（PAF）和花生四烯酸的代谢通过共同中间体1-烷基-2-花生四烯酰基-sn-甘油-3-磷酸胆碱（烷基花生四烯酰基-GPC）相联系。磷脂酶A2对烷基花生四烯酰基-GPC的水解可能启动PAF和类二十烷酸的生物合成，因为烷基溶血-GPC形成后可乙酰化为PAF，而花生四烯酸被释放出来用于转化为生物活性代谢物。为了阐明人血小板磷脂酶A2在导致这两类脂质介质形成的途径中的调节作用和功能，我们对其作用于烷基花生四烯酰基-GPC的情况进行了表征。人血小板磷脂酶A2被溶解，然后在正辛基-β-D-吡喃葡萄糖苷（辛基葡萄糖苷）存在下进行部分纯化。十六烷基花生四烯酰基-GPC通过血小板超声裂解物进行生物合成制备，经两步高效液相色谱（HPLC）纯化，并通过超声处理悬浮于缓冲液中。我们的结果表明，血小板磷脂酶A2对烷基花生四烯酰基-GPC的脱酰作用对Ca2+有绝对需求。它在游离Ca2+的亚微摩尔浓度下发生，并且表现出双相Ca2+依赖性，在10 microM和2 mM时活性达到平稳。白蛋白可使磷脂酶A2介导的烷基花生四烯酰基-GPC水解增加2倍，如果将1,2-二油酰基甘油掺入底物分散液中则增强5倍。用1-O-十六烷基-2-花生四烯酰基-sn-甘油-3-磷酸胆碱（十六烷基花生四烯酰基-GPC）和1-棕榈酰基-2-花生四烯酰基-sn-甘油-3-磷酸胆碱（棕榈酰花生四烯酰基-GPC）研究了血小板磷脂酶A2对含花生四烯酸的磷脂酰胆碱的1-烷基与1-酰基连接亚类的底物依赖性和特异性。我们发现这些底物的脱酰作用相同。我们得出结论，在受刺激血小板中，在细胞质游离Ca2+水平升高和新生成的二酰基甘油存在的情况下，烷基花生四烯酰基-GPC可能被磷脂酶A2迅速水解，并可能作为PAF和类二十烷酸的前体。
