1987 Fiscal Year Final Research Report Summary
Regulation of intracellular proteolysis by calpastatin
| 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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| Keywords | CALPASTATIN / INHIBITORY DOMAIN / CALPAIN / プロテオリシス |
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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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