| Project/Area Number |
62300011
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| Research Category |
Grant-in-Aid for Co-operative Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
広領域
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| Research Institution | Kyoto University |
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Principal Investigator |
HIROMI Keitaro Kyoto University, Faculty of Agric., Prof. Emeritus., 農学部, 名誉教授 (50025425)
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| Co-Investigator(Kenkyū-buntansha) |
AKASAKA Kazuyuki Kyoto University, Faculty of Science, Assoc. Prof., 理学部, 助教授 (50025368)
MITSUI Yukio Nagaoka University of Technology, Faculty of Engin., Prof., 工学部, 教授 (40012637)
OHTA Takahisa University of Tokyo, Faculty of Agric., Prof., 農学部, 教授 (30011844)
MIURA Kin-ichiro University of Tokyo, Faculty of Engin., Prof., 工学部, 教授 (30000227)
ISHII Shin-ichi Hokkaido University, Faculty of Pharm. Sci., Prof., 薬学部, 教授 (90001031)
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| Project Period (FY) |
1987 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥9,800,000 (Direct Cost: ¥9,800,000)
Fiscal Year 1989: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1988: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1987: ¥4,900,000 (Direct Cost: ¥4,900,000)
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| Keywords | Protein Engineering / Site-directed Mutagenesis / Proteinase Inhibitor / Subtilisin Inhibitor / Metallo-proteinase Inhibitor / NMR of Proteins / X-ray Crystallography of Protein / Interaction of Enzyme and its inhibitor / プロテアーゼインヒビター / ズブチリシンインヒビター / 金属プロテアーゼインヒビター / 酵素-インヒビター相互作用 / 酵素ーインヒビター相互作用 |
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| Research Abstract |
SSI denotes here Streptomyces subtilisin inhibitor and SMPI, Streptomyces metallo-proteinase inhibitor. SSI was found to inhibit also Streptomyces metallo-proteinases, SGMPA & SGMPB, and Ki was determined with a novel fluorogenic substrate; SSI gene was cloned, site-directed mutagenesis was applied on it, and characters of the mutant SSI's and of SSI-expression system were elucidated; Clones producing mondclonal antibodies, IgG1 & IgM, against SSI were established, and the epitopes were analyzed by NMR; X-ray diffraction data on the crystals of SSI (wild and mutant)-subtilisin BPN' complexes were obtained with syncrotron radiation, and structural refinement was made; Base sequence of the gene of a thermophilic proteinase, Aqualysine I, was determined, and the structure and function of the enzyme was studied; Optimal conditions were searched for extra-cellular production of SMPI by making use of a cloned gene; Methodology of Orotein NMR by using stable isotope labelling was established and the effects of site-specific amino acid replacement on the higher structure of SSI was analyzed; Fluctuation in SSI molecular structure was analyzed by deuterium NMR; A sensitive electro-chemical method for micro-analysis of SH/SS compounds was established; Interactions of SSI mutants-subtilisin BPN' and of SMPI-thermolysin were analyzed statistically and kinetically; The primary structure of Marinostatin D, a novel serine-proteinase inhibitor isolated from a marine micro-organism, was determined; The primary structure of Tyrostatin, a novel inhibitor of Pepstatin-insensitive carboxyl-proteinases, was determined; Unique properties of a novel, thermophilic carboxyl-proteinase and its strict substrate specificity were shown.
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