Venable M E, Olson S C, Nieto M L, Wykle R L
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157.
J Biol Chem. 1993 Apr 15;268(11):7965-75.
Resting human neutrophils acylate 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (1-O-alkyl-2-lyso-GPC; lyso-PAF) specifically with arachidonate (AA); upon stimulation, however, the specificity is lost and other fatty acid residues are added. The major goals of this study were to compare the various acylation reactions present in the cells and to determine the cause of the specificity loss upon stimulation. The CoA-independent transacylase was active in neutrophil homogenates and was found to be both highly specific for AA and stereospecific, requiring 1-O-alkyl-2-lyso-GPC for activity. Homogenates also contained acyl-CoA:1-radyl-2-lyso-sn-glycero-3-phosphocholine acyltransferase activity, which transferred acyl chains from oleoyl-, linoleoyl-, or linolenoyl-CoA to both 1-alkyl and 1-acyl acceptors, but preferred the 1-acyl acceptor when arachidonoyl-CoA was used. The CoA-dependent and -independent activities co-sedimented on a discontinuous Percoll gradient in a single band containing plasma membrane and possibly other membranes. CoA alone promoted nonspecific acylation in the homogenates. The AA-specific acylation was attenuated up to 80% in sonicates of ionophore-stimulated cells, whereas the CoA-dependent acyltransferase remained unchanged. Potential phospholipid AA donors for the transacylase were substantially depleted in the stimulated cells but could not account for the large decrease in acylation. An accumulation of 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine (alkenyl-2-lyso-GPE), which acts as a competing substrate, appeared to be the major cause of the reduced AA-specific acylation of lyso-PAF observed in the stimulated preparations. Removal of the alkenyl-2-lyso-GPE restored the activity, whereas the addition of alkenyl-2-lyso-GPE (2 microM) to resting membrane preparations resulted in a marked decrease in transacylation of lyso-PAF.
静息状态下的人中性粒细胞可将花生四烯酸（AA）特异性地酰化到1-O-烷基-2-溶血-sn-甘油-3-磷酸胆碱（1-O-烷基-2-溶血-GPC；溶血血小板活化因子）上；然而，在受到刺激时，这种特异性会丧失，其他脂肪酸残基会被添加进来。本研究的主要目的是比较细胞中存在的各种酰化反应，并确定刺激后特异性丧失的原因。不依赖辅酶A的转酰基酶在中性粒细胞匀浆中具有活性，并且发现它对AA具有高度特异性且具有立体特异性，其活性需要1-O-烷基-2-溶血-GPC。匀浆中还含有酰基辅酶A：1-基-2-溶血-sn-甘油-3-磷酸胆碱酰基转移酶活性，该活性可将来自油酰基、亚油酰基或亚麻酸酰基辅酶A的酰基链转移到1-烷基和1-酰基受体上，但当使用花生四烯酰基辅酶A时，它更倾向于1-酰基受体。依赖辅酶A和不依赖辅酶A的活性在不连续的Percoll梯度上共同沉降在一个包含质膜以及可能其他膜的单一条带中。单独的辅酶A会促进匀浆中的非特异性酰化。在离子载体刺激的细胞的超声破碎物中，AA特异性酰化最多可减弱80%，而依赖辅酶A的酰基转移酶则保持不变。转酰基酶潜在的磷脂AA供体在受刺激的细胞中大量减少，但无法解释酰化作用的大幅下降。作为竞争底物的1-O-烯丙基-1'-烯基-2-溶血-sn-甘油-3-磷酸乙醇胺（烯基-2-溶血-GPE）的积累似乎是在受刺激的制剂中观察到的溶血血小板活化因子的AA特异性酰化降低的主要原因。去除烯基-2-溶血-GPE可恢复活性，而向静息膜制剂中添加烯基-2-溶血-GPE（2 microM）会导致溶血血小板活化因子的转酰化作用显著降低。
