| Project/Area Number |
63480490
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
INOUE Keizo Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (30072937)
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| Co-Investigator(Kenkyū-buntansha) |
ARAI Hiroyuki Faculty of Pharmaceutical Sciences, Instructor, 薬学部, 助手 (40167987)
UMEDA Masato Faculty of Pharmaceutical Sciences, Instructor, 薬学部, 助手 (10185069)
KUDO Ichiro Faculty of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (30134612)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1990: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1989: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1988: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Keywords | phospholipase A2 / platelets / arachidonate metabolism / calcium ion / degranuration / purification / subcelluar localization / nitrogen cavitation |
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| Research Abstract |
(1) When a rabbit platelet soluble fraction was prepared by sonication and subjected to heparin--Sepharose column chromatography, phospholipase A2 activity was detected in both heparin-binding and non-binding fractions. The activity detected in the heparin-binding fraction appeared to belong to the secretory 14-KDa group II phospholipase A2, because it bound to anti- human 14-KDa group II phospholipase A2 monoclonal antibody. The activity found in the heparin- non-binding fraction did not appreciably react with the same antibody. When platelets were gently disrupted by the nitrogen cavitation method, the heparin-non-binding phospholipase A2 was mainly recovered in the platelet cytosolic fraction. The heparin-non-binding fraction hydrolyzed a phospholipid bearing an arachidonoyl residue at the sn-2 position more effectively than one with a linoleoyl residue. Thus, rabbit platelets contain two kinds of phospholipase A2 ; one is the secretory 14--kDa group II phospholipase A2 in the granu
… More
le fraction and the other is a cytoslic phospholipase A2 with a preference for arachidonoyl residue.
(2) The heparin-non-binding phospholipase A2 activities were purified to near homogeneity from rabbit and human platelets. Their molecular weights were about 88 kDa and 90 kDa, respectively. The purified enzymes exhibited a fatty acid preference ; it hydrolyzed phospholipid bearing an arachidonoyl residue at the sn-2 position more effectively than that with a linoleoyl residue. The catalytic activity of the purified enzyme with phosphatidylcholine or phosphatidylethanolamine increased sharply in the presence of between 10^<-7> and 10^<-6> calcium ion. These characteristics differ from those. Therefore, these high-molecular weight enzymes are classified to a novel phospholipase A2 and may participate in the stimulus-dependent release of arachidonoyl residues in rabbit platelets.
(3) Monoclonal antibodies were raised against rabbit platelet 88-kDa phospholipase A2. These antibodies reacted with phospholipase A2 activities detected in rabbit lung, liver and brain, indicating the exisetence of enzymes with similar characteristics. Less
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