title: A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7.
abstract: Arachidonoyl-hydrolyzing phospholipase A2 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 A2 enzymes require Ca2+ for activity in vitro, the role of Ca2+ in phospholipase A2 activation is not understood. We have found that an arachidonoyl-hydrolyzing phospholipase A2 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. Membrane-associated 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 phosphatidylinositol/phosphatidylserine. We suggest a mechanism in which the activity of phospholipase A2 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 membrane-associated which facilitates arachidonic acid hydrolysis.
authors.affiliation: Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.
authors.forename: J Y
authors.initials: JY
authors.lastname: Channon
authors.forename: C C
authors.initials: CC
authors.lastname: Leslie
mesh_terms: D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D008081:Liposomes / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2
chemical_list: D001095:Arachidonic Acids; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D016718:Arachidonic Acid; D004533:Egtazic Acid; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium