REGULATION OF LYSOPHOSPHOLIPASE ACTIVITY OF THE 85-KDA PHOSPHOLIPASE A(2) AND ACTIVATION IN MOUSE PERITONEAL-MACROPHAGES

The regulation of the lysophospholipase activity of the 85-kDa cytosol ic phospholipase A(2) (PLA(2)) was studied in vitro and in stimulated macrophages. Bovine serum albumin was found to inhibit lysophospholipa se activity of the recombinant 85-kDa PLA(2) when assayed at a relativ ely low substrate concentration. Inhibition could be reversed if the s ubstrate concentration was increased or if Ca2+ was present in the ass ay. Incubation of recombinant enzyme with macrophage membranes and lip id extracts from macrophage membranes resulted in the release of arach idonic acid, as well as, stearic acid, which is enriched at the sn-1 p osition of macrophage phospholipids. This suggests that with a bilayer substrate the PLA(2) can sequentially deacylate the sn-2 then sn-1 ac yl groups. This was verified by demonstrating that the phospholipids, phosphatidylcholine and phosphatidylinositol, were hydrolyzed to glyce rophosphocholine and glycerophosphoinositol by incubation with recombi nant 85-kDa PLA(2). The 85-kDa enzyme was identified as the main lysop hospholipase activity in mouse peritoneal macrophage cytosols. Additio n of Ca2+ to the assay enhanced activity, but this effect decreased as the substrate concentration was increased. Incubation of macrophages with zymosan increased the lysophospholipase activity of the 85-kDa PL A(2) in cytosols. Phosphorylation of recombinant PLA(2) with mitogen-a ctivated protein kinase resulted in an increase in lysophospholipase, as well as, PLA(2) activity. In macrophages stimulated with zymosan re lease of stearic acid (18:0) and palmitic acid (16:0) was observed in addition to arachidonic acid (20:4). These results are consistent with a role of the 85-kDa PLA(2) in regulating lysophospholipid levels in macrophages during zymosan stimulation.