A Calcium-dependent Mechanism for Associating a Soluble Arachidonoyl-hydrolyzing Phospholipase A2 with Membrane in the Macrophage Cell Line

Arachidonoyl-hydrolyzing phospholipase AZ plays a central role in providing substrate for the synthesis of the potent lipid mediators of inflammation, the eicosanoids, and platelet-activating factor. Although Ca2+ is required for arachidonic acid release in vivo and most phospholipase A, enzymes require Ca2’ for activity in vitro, the role of Ca” in phospholipase A2 activation is not understood. We have found that an arachidonoyl-hydrolyzing phospholipase AZ from the macrophage-like cell line, RAW 264.7, exhibits Ca2+dependent association with membrane. The intracellular distribution of the enzyme was studied as a function of the Ca2+ concentration present in homogenization buffer. The enzyme was found almost completely in the 100,000 x g soluble fraction when cells were homogenized in the presence of Ca2+ chelators and there was a slight decrease in soluble fraction activity when cells were homogenized at the level of Ca2+ in an unstimulated cell (80 nM). When cells were homogenized at Ca2+ concentrations expected in stimulated cells (230-450 nM), 60-70% of the phospholipase A2 activity was lost from the soluble fraction and became associated with the particulate fraction in a manner that was partly reversible with EGTA. Membraneassociated phospholipase A2 activity was demonstrated by [3H]arachidonic acid release both from exogenous liposomes and from radiolabeled membranes. With radiolabeled particulate fraction as substrate, this enzyme hydrolyzed arachidonic acid but not oleic acid from membrane phospholipid, and [3H]arachidonic acid was derived from phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinosiCol/phosphatidylserine. We suggest a mechanism in which the activity of phospholipase AZ is regulated by Ca2+: in an unstimulated cell phospholipase A2 is found in the cytosol; upon receptor ligation the cytosolic Ca2+ concentration increases, and the enzyme becomes membraneassociated which facilitates arachidonic acid hydrolysis.