2000 Fiscal Year Final Research Report Summary
Catalytic mechanism and function of phosphatidate-specific phospholipases A2
| Project/Area Number |
11670120
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General medical chemistry
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| Research Institution | Osaka University |
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Principal Investigator |
TOJO Hiromasa Osaka University Graduate School of Medicine, Associate Professor, 医学系研究科, 助教授 (90135707)
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| Project Period (FY) |
1999 – 2000
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| Keywords | phospholipase A2 / phosphatidic acid / lysobisphosphatidic acid / monoacylglycerollipase / phospholipids / testis / lysophosphatidic acid |
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| Research Abstract |
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.
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[Publications] Lu, T., Ito, M., Tchoua, U., Takemori, H., Okamoto, M., and Tojo, H.: "Identification of Essential Residues for Catalysis of Rat Intestinal Phospholipase B/Lipase"Biochemistry. (In press). (2001)
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「研究成果報告書概要(欧文)」より
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[Publications] Koyama, M., Ito, S., Nakajima, A., Shimoya, K., Azuma, C., Suehara, N., Murata, Y., and Tojo, H.: "Elevation of group II phospholipase A2 in serum and amniotic fluid in preterm labor"Am.J.Obstet.Gynecol.. 183. 1537-1543 (2000)
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「研究成果報告書概要(欧文)」より
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[Publications] Sawaguchi, A., Ide, S., Kawano, J., Nagaike, R., Oinuma, T., Tojo, H., Okamoto, M., and Suganuma, T.: "Reappraisal of potassium permanganate oxidation applied to lowicryl K4M embedded tissues processed by high pressure freezing/freeze substitution, with special reference to differential staining of the zymogen granules of rat gastric chief cells"Arch.Histol.Cytol.. 62. 447-458 (1999)
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「研究成果報告書概要(欧文)」より
