Enzymatic studies of lyso platelet-activating factor acylation in human neutrophils and changes upon stimulation.

Abstract

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.