Venable M E, Nieto M L, Schmitt J D, Wykle R L
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.
J Biol Chem. 1991 Oct 5;266(28):18691-8.
The first step in the synthesis of platelet-activating factor (PAF) in stimulated neutrophils is generally accepted to be hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (1-O-alkyl-2-acyl-GPC), with 1-O-alkyl-2-arachidonoyl-GPC being the preferred precursor. Characterization of the enzymatic activity responsible for the hydrolysis of 1-O-alkyl-2-arachidonoyl-GPC has been hampered by lack of an active and reliable cell-free system for study. In the present studies, membrane preparations containing 1-O-[3H]alkyl-2-arachidonoyl-GPC were prepared from intact human neutrophils that had been labeled using 1-O-[3H]hexadecyl-2-lyso-GPC. When the labeled membrane preparations were incubated in the presence of unlabeled 1-O-alkyl-2-lyso-GPC (5 microM), rapid deacylation (up to 25% of the label in 10 min) of the 1-O-[3H]alkyl-2-arachidonoyl-GPC to 1-O-[3H]alkyl-2-lyso-GPC (lyso-PAF) was observed. The deacylation activity appeared to be the same in preparations from resting or stimulated cells. No requirement for Ca2+, various nucleotides, or protein kinase activation could be demonstrated. A number of observations indicated that [3H]lyso-PAF is formed in the system by the action of the CoA-independent transacylase present in the cells rather than by phospholipase A2. Both 1-O-alkyl-2-lyso-GPC and 1-acyl-2-lyso-GPC elicited deacylation of 1-O-[3H]alkyl-2-arachidonoyl-GPC, whereas neither 3-O-alkyl-2-lyso-GPC nor 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphorylcholine, which should act as detergents but are not transacylase substrates, effected deacylation. The deacylation activity and CoA-independent transacylase activities were blocked in parallel by a number of inhibitors and by heat inactivation. In preparations containing 1-O-alkyl-2-[3H]arachidonoyl-GPC, no release of free [3H]arachidonic acid was observed. However, a shift of the [3H]arachidonate into exogenous 1-O-tetradecyl-2-lyso-GPC was observed in the system. These findings are consistent with the generation of [3H]lyso-PAF by the CoA-independent transacylase activity.
在受刺激的中性粒细胞中，血小板活化因子（PAF）合成的第一步通常被认为是1-O-烷基-2-酰基-sn-甘油-3-磷酸胆碱（1-O-烷基-2-酰基-GPC）的水解，其中1-O-烷基-2-花生四烯酰基-GPC是首选前体。由于缺乏用于研究的活性且可靠的无细胞系统，负责1-O-烷基-2-花生四烯酰基-GPC水解的酶活性的表征受到了阻碍。在本研究中，使用1-O-[3H]十六烷基-2-溶血-GPC标记完整的人中性粒细胞，制备含有1-O-[3H]烷基-2-花生四烯酰基-GPC的膜制剂。当在未标记的1-O-烷基-2-溶血-GPC（5 microM）存在下孵育标记的膜制剂时，观察到1-O-[3H]烷基-2-花生四烯酰基-GPC快速脱酰基化为1-O-[3H]烷基-2-溶血-GPC（溶血-PAF）（10分钟内高达标记的25%）。脱酰基活性在静息或受刺激细胞的制剂中似乎相同。未证明对Ca2+、各种核苷酸或蛋白激酶激活有需求。一些观察结果表明，[3H]溶血-PAF是由细胞中存在的不依赖CoA的转酰基酶作用在该系统中形成的，而不是由磷脂酶A2形成的。1-O-烷基-2-溶血-GPC和1-酰基-2-溶血-GPC均引发1-O-[3H]烷基-2-花生四烯酰基-GPC的脱酰基作用，而3-O-烷基-2-溶血-GPC和1-O-烷基-2-O-甲基-rac-甘油-3-磷酸胆碱（它们应起去污剂作用但不是转酰基酶底物）均未影响脱酰基作用。脱酰基活性和不依赖CoA的转酰基酶活性被多种抑制剂和热失活同时阻断。在含有1-O-烷基-2-[3H]花生四烯酰基-GPC的制剂中，未观察到游离[3H]花生四烯酸的释放。然而，在该系统中观察到[3H]花生四烯酸盐向外源1-O-十四烷基-2-溶血-GPC的转移。这些发现与不依赖CoA的转酰基酶活性产生[3H]溶血-PAF一致。
