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 cytosolic phospholipase A(2) (PLA(2)) was studied in vitro and in stimulated macrophages. Bovine serum albumin was found to inhibit lysophospholipase activity of the recombinant 85-kDa PLA(2) when assayed at a relatively low substrate concentration. Inhibition could be reversed if the substrate concentration was increased or if Ca2+ was present in the assay. Incubation of recombinant enzyme with macrophage membranes and lipid extracts from macrophage membranes resulted in the release of arachidonic acid, as well as, stearic acid, which is enriched at the sn-1 position of macrophage phospholipids. This suggests that with a bilayer substrate the PLA(2) can sequentially deacylate the sn-2 then sn-1 acyl groups. This was verified by demonstrating that the phospholipids, phosphatidylcholine and phosphatidylinositol, were hydrolyzed to glycerophosphocholine and glycerophosphoinositol by incubation with recombinant 85-kDa PLA(2). The 85-kDa enzyme was identified as the main lysophospholipase activity in mouse peritoneal macrophage cytosols. Addition 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 PLA(2) in cytosols. Phosphorylation of recombinant PLA(2) with mitogen-activated protein kinase resulted in an increase in lysophospholipase, as well as, PLA(2) activity. In macrophages stimulated with zymosan release 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.