| Project/Area Number |
02454544
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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 |
代謝生物化学
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| Research Institution | Gifu University |
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Principal Investigator |
NOZAWA Yoshinori Gifu University, School of Medicine, Biochemistry Professor, 医学部, 教授 (10021362)
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| Co-Investigator(Kenkyū-buntansha) |
BANNO Yoshiko Gifu University, School of Medicine, Biochemistry Research Associate, 医学部, 助手 (50116852)
KANAHO Yasunori Gifu University, School of Medicine, Biochemistry Research Associate, 医学部, 助手 (00214437)
OKANO Yukio Gifu University, School of Medicine, Biochemistry Associate professor, 医学部, 助教授 (10177066)
中島 茂 岐阜大学, 医学部, 助手 (60188935)
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| Project Period (FY) |
1990 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1992: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1991: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1990: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Keywords | Human Platelets / Phosphatidylinositol-phospholipase C / GTP-binding protein / Signaltransduction / 情報変換酵素 / ホスホリパ-ゼC / GTP結合蛋白質 / 分子多様性 |
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| Research Abstract |
It has generally been known that the stimulation of phosphoinositide turnover after the agonist-receptor interaction is initiated by the activation of phosphoinositide-specific phospholipase C (PI-PLC) through putative GTP-binding protein in plasma membranes. Blood platelets have frequently been used as a potentially useful model for studying transmembrane signaling. The purpose of this study was to clarify the mechanism of activation of thePI- PLC in human platelets, where PI-PLC isozymes and GTP-binding proteins were purified and reconstituted with adequate combination. The effective resolution of PI-PLCs of human platelet cytosolic and membrane fractions revealed five distinct activity peaks. The results of Western blotting analysis with various antibodies against PI-PLC isozymes showed that PLC-beta, PLC-gamma and PLC-delta isozymes were identified and two other unidentified activity peaks were separated from the cytosolic fraction and PLC-beta was mainly conatined in the membranes
… More
. Furthermore, unidentified mPLC-II was purified from the membrane fractions. Two heterotrimeric GTP-binding proteins, Gi2 (main component) and Gi3 (miner component) were purified and identified as substrates of pertussis toxin from human platelet membranes. The purtussis toxin-insensitive GTP-binding protein (Gq) was detected by Western blotting analysis with anti-GLalpha antibody. Various types of low molecular weight GTP- binding proteins were purified from cytosol and membrane of human platelets (c21KG, c25KG, m22KGI,II). These are identified to be smg21A (Krev-1), rap1B and ralA. The cDNA of novel c25KG was obtained and namedas ram. GTP-binding proteins have been known to be involved in thrombin and thromboxane A2-mediated production of inositol phosphates in human platelets. It has been recently reported that thromboxane A2 receptor bound pertussis toxin-insensitive GTP-binding protein (Gq) and PLC-beta 1 was specifically activated by the Gq alpha . We previously observed that human platelet membrane-associated PLC was activated by Gi and Go. The PLC-beta2 isozyme was activated beta gamma subunit of GTP-binding protein. Thrombin receptor coupled with two different GTP-binding proteins (Gi and Gq). These results suggested that thromboxane A2-stimulation activate PLC-beta1 via Gq and thrombin induced the PLC (beta2) activation mediated by beta gamma subunit of Gi2. We investigated that PLC-gamma was associated with actin-gelsolin complex in cytosolic fraction and suggested that gelsolin may play a role in regulation of PLC activity in human platelets. Less
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