| Project/Area Number |
05558092
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Developmental biology
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
HOSHI Motonori Tokyo Inst.Tech.Life Science Profecesser, 生命理工学部, 教授 (20012411)
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| Co-Investigator(Kenkyū-buntansha) |
ITOH Yoshinobu Nikon Corporation Manager, 光機設計部, マネージャー
SAKURAI Katsuyoshi Seikagaku Corporation Chif, 東京研究所, 次長
CHIBA Kazuyoshi Tokyo Inst.Tech.Life Science Assistant Prof., 生命理工学部, 助手 (70222130)
山本 久夫 生化学工業(株), 東京研究所, 室長
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| Project Period (FY) |
1993 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥13,200,000 (Direct Cost: ¥13,200,000)
Fiscal Year 1995: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1994: ¥9,000,000 (Direct Cost: ¥9,000,000)
Fiscal Year 1993: ¥3,000,000 (Direct Cost: ¥3,000,000)
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| Keywords | Protease / Enzyme Activity / Microinjection / 蛍光物質 / ヒトデ / 卵成熟 / 酵素活性 |
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| Research Abstract |
A method was investigated for monitoring an activity of protease (s) in cytosol of a single starfish oocyte using succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid as the substrate to be injected into the cell. After preincubation of immature oocytes with a proteasome inhibitor, N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norvalinal, the initial hydrolysis of the substrate was remarkably inhibited. The inhibitor blocked 1-methyladenine-triggered cyclin degradation which is known to be mediated by proteasome. Similar results were obtained when Carbobenzoxy-leucyl-leucyl-leucinal was applied to the oocytes. These results suggested that the protease activity measured by this method is mainly attributable to cytoplasmic proteasome. Indeed, calpain inhibitor E-64 had no effect on the hydrolysis of the substrate.
The hydrolysis of the substrate was partially inhibited by bestatin, suggesting that the substrate was cleaved by aminopeptidase. Thus, the initial velocity of hydrolysis of the substrate (V0) by proteasome was assayd in a living oocyte after preinjection of bestatin. The values of V0 increased gradually after 1-methyladenine addition and reached to a maximum level at the time corresponding to cyclin degradation. The calculated maximum velocity of a mature oocyte was approximately three times higher than that of an immature oocyte. The Michealis-Menten constant value was also higher in the mature oocytes than in the immature one. These results suggest that proteasome-dependent proteolysis is regulated not only by ubiquitination of substrates as generally believed but also by the proteasome activity by itself.
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