| Project/Area Number |
06045012
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | University-to-University Cooperative Research |
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| Research Institution | TOKYO Inst.Technology |
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Principal Investigator |
YOSHIDA Masasuke Tokyo Institule of Technology, Professor, 資源化学研究所, 教授 (90049073)
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| Co-Investigator(Kenkyū-buntansha) |
MONTAL Mauricio Univ.of California, SanDiego, Prof., Univ. of California・San D, 教授
MUNEYUKI Eiro Tokyo Inst.Technology.Asst.Prof., 資源化学研究所, 助手 (80219865)
ALLISON William S Univ.of california, SanDiego, Prof., Univ., 教授
福森 義宏 東京工業大学, 生命理工学部, 助教授 (60135655)
OKAMURA Melv UCSD, Dept. of Physics, 教授
伊中 浩治 東京工業大学, 資源化学研究所, 助手 (30240758)
SAIER Milton UCSD, Dept. of Biology, 教授
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| Project Period (FY) |
1994 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1996: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1995: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1994: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | ATPase / F1-ATPase / FoF1-ATPase / ATP synthase / H^+-ATP synthase / Reconstitutioin / Single-site catalysis / Chase-promotioin / ユニサイト活定 / F1-ATPase / 光親和性標識 / 触媒部位 / エネルギー共役 / 光親和性修飾 / アジドATP / アジドADP / ATP水解 / 触媒カルボキシル基 / 部位特異的変異 |
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| Research Abstract |
In order to know how many functional catalytic sites are necessary for ATPase activity of F1-ATPase from a thermophilic Bacillus PS3, a new method to isolate homogeneous prepartion of the alpha3beta3gamma complex with 1,2, or 3 incompetent catalytic sites was developed. Ten glutamic acids (Glu・Tag) were linked to C-terminus of the catalytically incompetent beta (E190Q) subunit. Glu・Tag itself did not affect ATPase activity of the complexes. Two kinds of alpha3beta3gamma complexes, one containing beta (wild-type) and the other Glu・Tag-linked beta (E190Q), were mixed, urea-denatured, dialyzed, and alpha3beta3gamma complexes were reconstituted. Each of the complexes containing different number of Glu・Tag-linked beta (E190Q) was separated by anion-exchange chromatography and analyzed. The results were as follows. 1) Normal steady-state ATPase activity requires three intact catalytic sites. 2) Chase-acceleration, a catalytic cooperativity, requires at least two intact catalytic sites. 3) Single-site catalysis can be mediated by a single intact catalytic site alone. Re-scrambling of subunits between complexes could occur when the complex was aged under some condition and this might be one of the reasons of the previous contradictory result (Miwa et al. (1989) J.Biochem. (Tokyo) 106,730-734).
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