| Project/Area Number |
62480458
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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 | Osaka University |
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Principal Investigator |
FUTAI Masamitsu Professor The Institute of Scientific and Industrial Research, Osaka University, 産業科学研究所, 教授 (50012646)
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| Co-Investigator(Kenkyū-buntansha) |
MAEDA Masatomo Research Assistant The Institute of Scientific and Industrial Research, Osaka Un, 産業科学研究所, 助手 (80190297)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1988: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1987: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | H^+-ATPase / H^+ / K^+ ATPase / H^+ / K^+ ATPase / H^+輸送性ATPase / K^+輸送性ATPase / ATP / adenosine triphosphopyridoxal |
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| Research Abstract |
Ion-translocating atpases transport ions such as H^+, Na^+, K^+ and Ca^<2+> coupling with hydrolysis of ATP. Detailed structure of the ATP binding (catalytic) site, mechanism of hydrolysis of ATP coupling with ion-transport and mechanism and structure of the ion-transport pathway are still unknown in molecular level. In this project we studied H^+-ATPase (F_1F_O) mainly from Escherichia coli and H^+/K^+ ATPase from hog and human.
H^+-ATPase is formed from F_1 sector (alpha, beta, gamma, delta and epsilon subunits) and F_O (a__-, b__- and c__- subunits). We analyzed ATP binding site(s) and H^+ pathway by conbined approach of random and site-directed mutagenesis. ATP binding site was located near 149 - 156 residues and Tyr-285 residue (numbered from the amino-terminus) of the beta subunit. Lys-155 was located closely near the gamma phosphoryl moiety of aTP. Importance of residues between Gln-269 and Thr-277 of the gamma subunit for ATPase activity was shown. About half of the epsilon subunit was shown to be dispensible. We analyzed H^+ pathway (F<@2O<@D2) using the similar approach: one of the essential amino acid residues for H<@D1+@>D1 translocation was located within 20 residues from the carboxyl terminus of the a<@D5-@>D5 subunit. Two residues from the carboxyl-terminus of the b<@D5-@>D5 subunit were essential for assembly of F<@D2O@>D2.
We cloned cDNA (pig) and genomic DNA (human) of H^+/K^+ ATPase and determined their nucleotide sequences. The amino acid sequence of H^+/K^+ ATPase was closely homologous to other ion-translocating ATPases such as Na^+/K^+- and Ca^<2+>-ATPase. We also showed that pyridoxal phosphate binds to Lys-497 of the H^+/K^+ ATPase and this lysine residue formed ATP binding site. We proposed a model of catalytic site and H^+ pathay of the enzyme.
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