Abstract
The regulation of the lysophospholipase activity of the 85-kDa cytosolic phospholipase A2 (PLA2) was studied in vitro and in stimulated macrophages. Bovine serum albumin was found to inhibit lysophospholipase activity of the recombinant 85-kDa PLA2 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 PLA2 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 PLA2. 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 PLA2 in cytosols. Phosphorylation of recombinant PLA2 with mitogen-activated protein kinase resulted in an increase in lysophospholipase, as well as, PLA2 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 PLA2 in regulating lysophospholipid levels in macrophages during zymosan stimulation.
Highlights
Metabolized to a number of potent proinflammatory eicosanoids
In this study we have examined whether the lysophospholipase activity of the 85-kDa PLA2 is regulated in zymosan-stimulated macrophages and whether it functions as a lysophospholipase under physiological conditions
Inhibition of Lysophospholipase Activity by Albumin-Prior to our studies to investigate lysophospholipase activity in stimulated macrophages, experiments were carried out to optimize the lysophospholipase assay of the 85-kDa PLA2
Summary
Materials-Pathogen-free female ICR mice (8 weeks old) were obtained from Harlan Sprague-Dawley. For measuring PLA 2 activity, assays contained 1-hexadecyl-2-[3HJarachidonoyl-PC (30 J.LM) (100,000 dpm/assay) cosonicated with 9 J.LM dioleoylglycerol, 1 mg/ml fatty acid-free BSA, 150 mM NaCl, and 50 mM Tris, pH 7.4, for macrophage cytosols or in 50 mM Tris, pH 7.6, for recombinant PIA". Membranes (20 J.Lg of protein) or sonicated lipids extracted from 10 J.Lg of membrane protein (0.7 J.Lg of lipid phosphorus) were incubated with purified PIA" (50 ng) in 50 mM Tris, pH 7.6, containing 1 mM CaCI 2, 100 J.Lg/ml of fatty acid-free BSA, 150 mM NaCl at 37°C with shaking in a total volume of 50 J.Ll. The reaction was stopped by the addition of 100 J.Ll of ice-cold methanol, to which was added 50 ng of [13C4Jpalmitic acid, 50 ng [2H3Jstearic acid, 10 ng of [2H2Joleic acid, and 10 ng of [2H8Jarachidonic acid. For analysis of the stoichiometry of phosphorylation the 32P-labeled PLA 2 was separated by SDS-polyacrylamide gel electrophoresis, the band excised and counted by Cerenkov counting to determine the moles phosphate incorporated per mol of PLA, (16)
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