Receptor-mediated activation of a phospholipase A2 in rabbit neutrophil plasma membrane.

Abstract

Using the exogenous substrate [1-14C]oleate-labeled autoclaved Escherichia coli, the chemotactic factors fMet-Leu-Phe, complement C5a, and leukotriene B4 [(5S,12R)-dihydroxy-6-cis,8-trans,11-trans,14-cis-icosatetraenoic acid] were shown to stimulate a phospholipase A2 of isolated plasma membranes of rabbit peritoneal neutrophils. Each of the chemotactic factors shows a biphasic concentration dependence with the optimal concentrations occurring at 1, 10 and 0.1 nM, respectively. The specific antagonists of fMet-Leu-Phe binding, carbobenzoxy-Phe-Met and tert-butoxycarbonyl-Phe-Leu-Phe, effectively block the stimulation by fMet-Leu-Phe, indicating that the activation is receptor mediated. .DELTA.6-trans-leukotriene [(5S,12R)-dihydroxy-all-trans-6,8,10,14-icosatetraenoic acid], a biologically inactive stereoisomer of leukotriene B4, does not stimulate phospholipase activity, suggesting that the enhancement by leukotriene B4 is also receptor mediated. The unstimulated and activated phospholipase exhibit a broad range of maximal activity between pH 7.0 and pH 8.5, both with an optimal pH of 8.5. The activation of the phospholipase by fMet-Leu-Phe is completely calcium dependent; no increase in activity is demonstrable if fMet-Leu-Phe is added in the absence of exogenous Ca or in the presence of EGTA [ethyleneglycol-bis [.beta.-aminoethylether]-N,N''-tetraacetic acid]. In contrast, the unstimulated plasma membrane activity of the phospholipase, as well as the activity arising after stimulation, is relatively insensitive to the concentration of Ca, being inhibited by < 50% in the presence of 10 mM EGTA. The phospholipase hydrolyzes 1-[1-14C]palmitoyl-2-acyl-sn-glycerophosphoethanolamine to form only radioactive lysophosphatidylethanolamine as the product, indicating that the enzyme has an A2 specificity.