| Project/Area Number |
60300001
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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 | Hokkaido University |
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Principal Investigator |
ISHII Shin-ichi Faculty of Pharmaceutical Sciences, Hokkaido University Professor, 薬学部, 教授 (90001031)
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| Co-Investigator(Kenkyū-buntansha) |
MITSUI Yukio Faculty of Pharmaceutical Sciences, University of Tokyo Instructor, 薬学部, 助手 (40012637)
MIURA Kin-ichiro Faculty of Engineering, University of Tokyo Professor, 工学部, 教授 (30000227)
HIROMI Keitaro Faculty of Agriculture, Kyoto University Professor, 農学部, 教授 (50025425)
KAINOSHO Masatsune Faculty of Science, Tokyo Metropolitan University Associate Professor, 理学部, 助教授 (20137029)
TSURU Daisuke Faculty of Pharmaceutical Sciences, Nagasaki University Professor, 薬学部, 教授 (90039652)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1986: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1985: ¥3,000,000 (Direct Cost: ¥3,000,000)
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| Keywords | Protease inhibitor / Streptomyces protease / Subtilisin / Metalloprotease / Site-directed mutagenesis / Gene cloning / X-Ray analysis / 核磁気共鳴 |
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| Research Abstract |
Comparative structure-function studies were carried out in multiple ways on various proteinaceous protease inhibitors produced by Actinomycetes. The results were as followings.
(1) Chemical structure of SMPI, a metalloprotease inhibitor produced by Streptomyces nigrescens TK-23, was determined. The bacterium cultivated under improved conditions was now able to supply us with a larger amount of SMPI, and it turned out to promote our further studies on the reactive site of this inhibitor and its mode of interaction with thermolysin.
(2) Streptomyces subtilisin inhibitor (SSI) inhibited not only subtilisin-like proteases and chymotrypsin-like proteases originated from Actinomycetes, but also a metalloprotease from the same origin. Thus, SSI was shown to have an unexpectedly broad inhibitory spectrum, as far as the proteases from Actinomycetes were concerned.
(3) Successful results were obtained in the cloning of SSI genomic DNA, the primary structure determination of this DNA, and its expression in Streptomyces lividans. Various new SSI species were now able to be artifically prepared by introducing site-directed mutagenesis on the genomic DNA, for example at the reactive site ( <Met^73> -> <Lys^73> ).
(4) Interaction of several proteases with <Lys^73> -SSI (an artifial mutant) and plasminostreptin (a natural mutant), as well as with various SSI derivatives prepared by enzymatical and chemical treatment, were comprehensively compared by kinetical, spectroscopical, and thermal measurements.
(5) X-Ray crystallographic data of SSI in its free form and in the subtilisin-bound form were refined, the results of which indicated the occurrence of structural change in the SSI molecule upon it interaction with substilisin. Analyses by NMR spectroscopy further suggested that a portion of the molecule shows structural flexibility in solution. Such behavior might be favorably correlated with the broad inhibitory spectrum of SSI mentioned in 2.
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