| Project/Area Number |
62480456
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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 | The University of Tokyo |
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Principal Investigator |
KAWAKITA Masao University of Tokyo, College of Arts and Sciences, Professor, 教養学部, 教授 (00012740)
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| Co-Investigator(Kenkyū-buntansha) |
HATTORI Seisuke University of Tokyo, College of Arts and Sciences, Instructor, 教養学部, 助手 (50143508)
KAWATO Suguru University of Tokyo, College of Arts and Sciences, Associate Professor, 教養学部, 助教授 (50169736)
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| Project Period (FY) |
1987 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1987: ¥4,700,000 (Direct Cost: ¥4,700,000)
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| Keywords | Sarcoplasmic Reticulum / Calcium Transport / ATPase / Active Transport / Na / H Antiporter / Ion Transport / カルシウム輸送ATPア-ゼ / 運動輸送 / ATPアーゼ / カルシウム / 親和性標識 / Hアンチポーター / カルシウムポンプ |
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| Research Abstract |
1. The following results were obtained concerning the structure and the reaction mechanism of Ca^<2+>-transporting ATPase of sarcoplasmic reticulum. (1) Thermolytic digestion of sarcoplasmic reticulum membranes followed by HPLC was shown to be effective in identifying target sites of fluorescence- and affinity-labeling reagents. (2) IAEDANS and NEM(ANM) were shown to be specifically attached to Cys674 and Cys344/364, respectively. (3) Timeresolved fluorescence anisotropy measurements revealed independent flexible motion of submolecular domains of ATPase labeled with IAEDANS and ANM. (4) Products of limited tryptic digestion were purified and analyzed. Susceptibility of Lys218, Lys234 and Arg236 was profoundly affected by binding of Ca^<2+> and AMP-P(NH)P to the ATPase molecule due to a conformational change around these residues. This particular region seems to be intimately involved in energy coupling of the Ca^<2+>-transport, and also is supposed to be the site of contact between Ca^
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<2+>-binding and ATP-binding domains. The present results thus suggest that interactions between submolecular domains may be important for proper coupling between ATP splitting and Ca^<2+>-transport. (5) An affinity labeling reagent, ATP-pyridoxal was utilized to map the ATP binding site. Lys684 and Lys492 were labeled in the absence of Ca^<2+>, but only the former was labeled in its presence, indicating a Ca^<2+>-dependent conformational change in the ATP-binding site. A close vicinity of Asp351, Lys492 and Lys684 was also suggested. (6) Paramagnetic interactions between covalently attached spin labels and Gd^<3+> (a paramagnetic Ca^<2+> analogue) was analyzed and the Ca^<2+>-binding site was located in the intramembranous region of the ATPase molecule, not being very far from the membrane surface.
2. Na^+/H^+ antiporter was reconstituted into proteoliposome by cholatedialysis procedure. Partially purified preparation of the antiporter was obtained from the blush border membranes of bovine kidney through gel filtration and DEAE-Sephacel chromatography. Less
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