| Project/Area Number |
01480147
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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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 |
NARUMIYA Shuh Professor Department of Pharmacology Kyoto University Faculty of Medicine, 医学部, 教授 (70144350)
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| Co-Investigator(Kenkyū-buntansha) |
MORII Narito ibid ibid, 医学部, 助手 (80220036)
OGOROCHI Toshiya instructor ibid, 医学部, 助手 (80204171)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1990: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1989: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | botulinum exoenzyme / ADP-ribosylation / GTP binding protein / rho.gene product / cytoskeleton / cell adhesion / actin filament / ras gene / ボツリヌスC_3酵素 / Gb / GTPase活性化蛋白質(GAP) / 情報伝達 / ボツリヌス毒素 / rho遺伝子 / 培養細胞 |
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| Research Abstract |
1. An ADP-ribosyltransferase was purified from culture fultrate of type C Clostridium botulinum and identified as a 24 kDa protein named botulinum C3 exoenzyme. This enzyme gene was cloned based on the partial amino acid sequence of the purified protein and expressed in Esherichia coli . This study revealed that this enzyme is a protein of 244 amino and secreted after the signal peptide of N-terminal 40 amino acid peptide is clipped.
2. Gb, a subsrate GTP-binding protein, was purified from the cytosol of bovine adrenal glands, and its cDNA was cloned based on the partial amino acid sequences. By these procedures, it was identified as a product of rho genes, one of the ras oncogene-related genes. Using the purified subsrate, the amino acid site for ADP-ribosylation was determined as an asparagine residue located at the 41st position from the amino terminal of the protein.
3. Using purified or recombinant C3 exoenzyme, cellular function of the rho gene product (Gb) was analyzed in various types of cultured cells as well as blood platelets. C3 exoenzyme added to these cells was time-dependently incorporated into the cells and ADP-ribosylated the rho protein in the cells, and concomitant with this ADP-ribosylation the cells underwent marked morphological changes. From these analyses, we concluded that the rho protein is involoved in assembly of actin filament in the cells and in cell-substrate adhesion.
4. Using catalytic activities of the rho protein as parameters, molecules interacting with this protein was screened and rho-specific GTPase activating protein was identified and purified to apparent homogeneity.
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