Purification and properties of a phospholipase A2/lipase preferring phosphatidic acid, bis(monoacylglycerol) phosphate, and monoacylglycerol from rat testis.
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.
Ito
Masafumi
M
Department of Molecular Physiological Chemistry (H-1), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Japan.
Tchoua
Urbain
U
Okamoto
Mitsuhiro
M
Tojo
Hiromasa
H
D000217:Acyltransferases / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002738:Chloroquine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004950:Esterases / Q000378:metabolism; D005989:Glycerides / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D050356:Lipid Metabolism; D008099:Liver / Q000378:metabolism; D008246:Lysophospholipids / Q000737:chemistry* / Q000302:isolation & purification*; D008297:Male; D013058:Mass Spectrometry; D050178:Monoglycerides; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology / Q000378:metabolism*
D005989:Glycerides; D008246:Lysophospholipids; D050178:Monoglycerides; C012786:bis(monoacylglyceryl)phosphate; D002738:Chloroquine; D000217:Acyltransferases; D004950:Esterases; D010741:Phospholipases A; D054467:Phospholipases A2