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J. Biol. Chem., Vol. 277, Issue 46, 43674-43681, November 15, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/46/43674    most recent M202817200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ito, M. Articles by Tojo, H. Search for Related Content PubMed PubMed Citation Articles by Ito, M. Articles by Tojo, H. Social Bookmarking                      What's this?

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

Masafumi Ito, Urbain Tchoua, Mitsuhiro Okamoto, and Hiromasa Tojo

From the Department of Molecular Physiological Chemistry (H-1), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

Phospholipase A₂ (PLA₂) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca²⁺ ions for activity and exhibited both phosphatidic acid-preferring PLA₂ 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 PLA₂ and lipase activities. The sequence of NH₂-terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA₂ 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 PLA₂-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 PLA₂, 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.

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