| Project/Area Number |
10308028
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
TANIGUCHI Kazuya Hokkaido Univ., Grad. Sch. of Sci., Prof., 大学院・理学研究科, 教授 (40028204)
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| Co-Investigator(Kenkyū-buntansha) |
IMAGAWA Toshiaki Hokkaido Univ., Grad. Sch. of Sci., Asso. Prof., 大学院・理学研究科, 助教授 (20142177)
KAYA Shunji Hokkaido Univ., Grad. Sch. of Sci., Asso. Prof., 大学院・理学研究科, 助教授 (90186023)
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| Project Period (FY) |
1998 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥24,350,000 (Direct Cost: ¥23,600,000、Indirect Cost: ¥750,000)
Fiscal Year 2001: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
Fiscal Year 2000: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1999: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1998: ¥15,300,000 (Direct Cost: ¥15,300,000)
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| Keywords | Na / K-ATpase / H / K-ATPase / phosphorylation / phosphatases / kinase / oligomer / Hポンプ / フォスファターゼ |
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| Research Abstract |
Reversible phosphorylations of Tyr^7 and Tyr^<10> of pig stomach H/K-ATPase α-chain were initially demonstrated in vivo in rat, rabbit and. These data and others also suggest that some important enzyme systems are present in the apical membrane and in sufficiently proximity to participate in the reversible phosphorylation of Tyr^7 and Tyr^<10> and Se^<27> residues of the α-chain of H/K-ATPase. The tyr-kinase is recognized by anti-c-Src antibody. The Ser-kinase is recognized by antibodies against PKCα and PKC βII. The presence of protein phosphatase-1 was also immunologically detected. Column chromatographic separation of CHAPS solubilized G1 membrance and others indicate the appearance molecular weight of the Src-kinase to be 〜60 kDa, the PKCα and/or PKCβII to be 80 kDa, the Tyr-phosphatase to be 200 kDs and PP-1 to be 〜35 kDs.
The maximum amount of phosphorylated intermediate (E^<32>P) / mol of α-chain of pig stomach H/K-ATPase from [γ-^<32>P]ATP was found to be 〜0.5, which was half of that formed from ^<32>Pi. The maximum ^<32>P binding for the enzume during turnover in the presence of [γ-^<32>P]ATP was due to 0.5 mol of E^<32>P + 0.5 mol of and acid labile enzyme bound [γ-^<32>P]ATP (EATP). The H^+ -ATPase activity/(EP + EATP), was very close to the apparent rate constants for EP breakdown and Pi liberation. The ratio of the amount of Pi liberated to that of EP that disappeared, increased from 1to 〜 2 with increasing concentrations of ATP. This represents the first direct evidence, for the case of a P-type ATPase in which 2 mol of Pi liberation occurs simultaneously from 1 mol of EP for half of he enzyme molecules and 1 mol of EATP for the other half, during ATP hydrolysis involving in crosstalk.
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