| Project/Area Number |
60440031
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Kyoto University |
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Principal Investigator |
MURACHI Takashi Faculty of Medicine, Kyoto University Professor, 医学部, 教授 (10089104)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Harutaka Institute for Virus Research, Kyoto University Professor, ウイルス研究所, 教授 (10027310)
KANNAGI Reiji Faculty of Medicine, Kyoto University Lecturer, 医学部, 講師 (80161389)
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| Project Period (FY) |
1985 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥22,700,000 (Direct Cost: ¥22,700,000)
Fiscal Year 1987: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1986: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1985: ¥16,000,000 (Direct Cost: ¥16,000,000)
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| Keywords | CALPASTATIN / INHIBITORY DOMAIN / CALPAIN / プロテオリシス / 細胞内プロティナーゼ / 繰返しドメイン構造 / 脳下垂体前葉 / ACTH産生細胞 / 細胞内プロテイナーゼ / カルモデュリン様蛋白質 |
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| Research Abstract |
Calpastatin is an endogenous inhibitor protein acting specifically on calpain (Ca^<2+>-dependent cysteine proteinases). With calpain, calpastatin constitutes an intracellular regulatory system as related to Ca^<2+>-induced proteolysis. The present three-year project for 1985-1987 aimed at the elucidation of the mechanism of inhibition of calpain by calpastatin, particularly that occurring inside a cell. The followings are the major results obtained.
(1) Very wide, but somewhat uneven, distribution of calpastatin in various mammalian tissues has been demonstrated by immunohistochemical methods.
(2) Two different molecular species of calpastatin, showing 68- and 107-kDa mobilities on SDS-Polyacrylamide gel electrophoresis, were isolated and characterized.
(3) Molecular cloning of pig calpastatin has led to the elucidation of the primary structure of 713-amino acid residues, which contained a four-repetitive-domain structure.
(4) Expression in E. coli of cDNAs for the four domains has revealed that each domain, having approximately 140 amino acid residues, possesses inhibitory activity against calpain. Using techniques for site-directed mutagenesis, several different fragments were created from a calpastatin unit domain, and they were compared with respect to inhibitory potency. It was thus concluded that a central portion of a unit domain, composed of some 50 amino acid residues, is essential for the inhibition of calpain activity.
The present study has provided fundamental information as to the in vitro mechanism of calpastatin action on calpain, and it has also given some clue how to elucidate the mechanism of its action in vivo.
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