Receptor-mediated activation of a phospholipase A2 in rabbit neutrophil plasma membrane.
Using the exogenous substrate [1-14C]oleate-labeled autoclaved Escherichia coli, we have demonstrated that the chemotactic factors fMet-Leu-Phe, complement component C5a, and leukotriene B4 [(5S,12R)-dihydroxy-6-cis,8-trans,11-trans,14-cis-icosatetraenoic acid] 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 t-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 calcium or in the presence of EGTA. 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 calcium, being inhibited by less than 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.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002628:Chemoreceptor Cells / Q000502:physiology*; D002630:Chemotactic Factors / Q000494:pharmacology*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D007700:Kinetics; D009504:Neutrophils / Q000201:enzymology*; D010740:Phospholipases / Q000097:blood*; D011817:Rabbits; D013329:Structure-Activity Relationship
D002630:Chemotactic Factors; D004533:Egtazic Acid; D010740:Phospholipases; D002118:Calcium