Catalytic mechanism and function of phosphatidate-specific phospholipases A2

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A phospholipase A2 (PLA2) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca^<2+> ions for activity, and exhibited both phosphatidic acid-preferring PLA2 and monoacylglycerol lipase (MGL) activities with a modest specificity toward unsaturated acyl chains, but not lysophospholipase, and di- and triacylglycerol lipase activities. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidino)-phenylmethanesulfonyl fluoride and methylarachidonyl fluorophosphonate inhibited both activities to a similar extent, indicating a single active site is involved in PLA2 and MGL catalysis. We sequenced about 10 tryptic peptides of PA-PLA2 by capillary reverse phase HPLC/electrospray ionization (ESI) ion trap mass spectrometry, and found that it belonged to a new family of PLA2 enzymes. The optimal pH for PLA_2 activity (around 5.5) differed from that for MGL activity (around 8.0), but both activities broadly extended over a physiologically relevant pH range from pH 5.5 to 7.5. At pH 5.5 the enzyme also hydrolyzed bis (monoacylglycerol) phosphate (lysobisphosphatidic acid, LBPA) that has been hitherto known as a PLA2-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from crude liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA2, and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal-phase HPLC/ESI ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic acid, lysobisphosphatidic acid, and monoacylglycerol in the endosome/lysosome system with mildly acidic milieu.