Purification and Properties of a Phospholipase A2/Lipase Preferring Phosphatidic Acid, Bis(monoacylglycerol) Phosphate, and Monoacylglycerol from Rat Testis

• Published in: The Journal of biological chemistry Vol. 277; no. 46; pp. 43674 - 43681
• Main Authors: Ito, Masafumi, Tchoua, Urbain, Okamoto, Mitsuhiro, Tojo, Hiromasa
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 15.11.2002
• Subjects: Acyltransferases - metabolism; Animals; Catalysis; Chloroquine - pharmacology; Chromatography, High Pressure Liquid; Esterases - metabolism; Glycerides - chemistry; Glycerides - isolation & purification; Hydrogen-Ion Concentration; Hydrolysis; Lipid Metabolism; Liver - metabolism; Lysophospholipids - chemistry; Lysophospholipids - isolation & purification; Male; Mass Spectrometry; Monoglycerides; Pancreas - enzymology; Phospholipases A - metabolism; Phospholipases A2; Protein Structure, Tertiary; Rats; Rats, Sprague-Dawley; Substrate Specificity; Testis - enzymology; Testis - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M202817200

Phospholipase A 2 (PLA 2 ) 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 PLA 2 and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, ( p -amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA 2 and lipase activities. The sequence of NH 2 -terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA 2 activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA 2 -resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA 2 , and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments.