| Project/Area Number |
07044218
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Tohoku University |
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Principal Investigator |
TAMURA Shinri Institute of Development, Aging and Cancer, Tohoku University Professor, 加齢医学研究所, 教授 (20124604)
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| Co-Investigator(Kenkyū-buntansha) |
ABE Sumiko Fukushima Medical College, 助手 (50136975)
YANAGAWA Yuchio Institute of Development, Aging and Cancer, Tohoku University, 加齢医学研究所, 助手 (90202366)
KOBAYASHI Takayasu Institute of Development, Aging and Cancer, Tohoku University, 加齢医学研究所, 助手 (10221970)
COHEN Philip University of Dundee, 医学部, 教授
LYNCH Kevin.R University of Virginia, 医学部, 準教授
LARNER Joseph University of Virginia, 医学部, 教授
平賀 章 東北大学, 加齢医学研究所, 助教授 (80134047)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 1996: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1995: ¥3,700,000 (Direct Cost: ¥3,700,000)
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| Keywords | insulin action / insulin mediator / chyloinositol glycan / protein phosphatase / domain structure / enzyme activity / deletion mutant / point mutant / イノシトールプロテオグリカン / ゲノムDNA / プロモーター領域 |
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| Research Abstract |
In this study we performed a biochemical study on the elucidation of the domain structure of PP2C molecule and on the effect of chyloinositol glycan (CIG), which has been proposed to be an insulin mediator by Dr.Joseph Larner of University of Virginia and other groups, on the enzyme activities of the PP2C isoforms and the mutant molecules derived from PP2Cbeta-1.
We constructed the plasmids of the wild type of PP2C isoforms and the various deletion mutants and point mutants of PP2Cbeta-1 for expression in E.coli cells. We purified the rocombinant proteins from the E.coli cell extracts and determined the protein phosphatase activities. Deletion of up to 78 amino acids from the carboxy-terminal end did not affect the enzyme activity, whereas deletion of 100 amino acids totally eliminated the activity. On the other hand, deletion of 11 amino acids from the amino-terminal end caused a 97% loss of enzyme activity and further deletions caused a total loss of activity. Substitution of any of t
… More
he 6 specific amino acids (E38, E60, H62, R179, R200 and E243) among the 16 tested in this study caused 98-100% loss of enzyme activity. These observations suggested that PP2Cbeta is composed of at least two distinct functional domains, an amino-terminal catalytic domain of about 310 amino acids and the remaining carboxy-terminal domain, which is involved in determination of substrate specificity.
Then, we determined the effect of CIG on the enzyme activities of wild type PP2C isoforms and the deletion mutants and point mutants. It is has been established that a high concentration (Ka for Mg^<2+>,1mM) of Mg^<2+> is required for the standard assay of PP2C activity. However, full activity was obtained even if the Mg^<2+> concentration was as low as 50muM when CIG was co-present in the assay mixture. This stimulatory effct of CIG was observed with all the six PP2C isoforms. The presence of CIG stimulated the activities of the deletion mutants and the point mutants as far as they had Mg^<2+> -dependent protein phosphatase activities. However, the mutants, which did no have Mg^<2+> -dependent activity, did not show any activity even in the presence of CIG.These results suggest that CIG acts as an activator of PP2C isoforms and that the point of action of CIG is in the catalytic domain of PP2C molecule. Less
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