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Molecular mechanism of Energy conversion on the biomembrane

Research Project

Project/Area Number 06045012
Research Category

Grant-in-Aid for international Scientific Research

Allocation TypeSingle-year Grants
SectionUniversity-to-University Cooperative Research
Research InstitutionTOKYO Inst.Technology

Principal Investigator

YOSHIDA Masasuke  Tokyo Institule of Technology, Professor, 資源化学研究所, 教授 (90049073)

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
Project Period (FY) 1994 – 1996
Project Status Completed (Fiscal Year 1996)
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)
KeywordsATPase / 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
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).

Report

(4 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • 1994 Annual Research Report
  • Research Products

    (25 results)

All Other

All Publications (25 results)

  • [Publications] Noji,H.,Yasuda,R.,Yoshida,M.,Kinosita,K.: "Direct observation of the rotation of F_1-ATPase" Nature. (in press). (′97)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Matsui,T.,Dou,C.,Allison,W.S.,Yoshida,M.: "Catalytic activity of the α_3β_3γ complex of F_1-ATPase without noncatalytic nucleotide Binding Site" J.Biol.Chem.272(in press). (′97)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Jault,J.M,Dou,C.,Allison,W.S.,Yoshida,M.: "The α_3β_3γ subcomplex of the F_1-ATPase with the βT165S substitution does not entrap inhibitory MgADP" J.Biol.Chem.271. 28818-28824 (′96)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Amano,T.,Hisabori,T.,Muneyuki,E.,Yoshida,M.: "Catalytic activity of α_3β_3γ complexes of F_1-ATPase with 1,2,or 3 catalytic sites" J.Biol.Chem.271. 17-17, 343-348 (′96)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Noji, H., Yasuda, R., Yoshida, M., Kinoshita, K.: "Direct observation of the rotattion of F_1-ATPase" Nature. (in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Matsui, T., Muneyuki, E., Honda, M., Dou, C., Allison, W.S., Yoshida, M: "Catalytic activity of the alpha_3beta_3gamma complex of F_1-ATPase without noncatalytic ncleotide bindirg site" J.Biol.Chem.271(in press). (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Jault, J-M., Dou, C., Matsui, T., Yoshida, M.: "The alpha_3beta_3gamma subcomplex of the F_1-ATPase with the betaT165S Substitution does not entrap inhibitory MgADP in a catalytic site during turnover" J.Biol.Chem.271. 28818-28824 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Amano, T., Hisabori, T., Muneyuki, E., Yoshida, M.: "Catalytic activigty of alpha_3beta_3gamma complexes of F_1-ATPase with 1,2, or 3 incompetent catalytic sites" J.Biol.Chem.271. 17439-17444 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Naji,H.,Yasuda,R.,Yoshida,M.,Kinosita,K.: "Direct observation of the rotation of F_1-ATPase" Nature. (in press). (′97)

    • Related Report
      1996 Annual Research Report
  • [Publications] Matsui,T.,Dou,C.,Allison,W.S.,Yoshida,M.: "Catalytic activity of the α_3β_3γ complex of F_1-ATPase without noncatalytic nucleotide Binding Site" J.Biol.Chem. 272. (in-press) (′97)

    • Related Report
      1996 Annual Research Report
  • [Publications] Jault,J-M,Dou,C.,Allison.W.S.,Yoshida,M.: "The α_3β_3γ subcomplex of the F_1-ATPase with the βT165S substitution does not entrap inhibitory MgADP" J.Biol.Chem. 271. 28818-28824 (′96)

    • Related Report
      1996 Annual Research Report
  • [Publications] Amano,T.,Hisabori,T.,Muneyuki,E.,Yoshida,M.: "Catalytic activity of α_3β_3γ complexes of F_1-ATPase with 1,2,or3 catalytic sites" J.Biol.Chem. 271. 17343-17348 (′96)

    • Related Report
      1996 Annual Research Report
  • [Publications] Miyauchi,M.: "F_1ATPase α-subunit made up from two fragments(1-395,396-503)is stabilized by ATP and complexes containing it obey altered kinetics." Biochim. Biophys. Acta. 1229. 225-232 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Saika,K.: "A minimum catalytic unit of F_1ATPase shows non-cooperative ATPase activity inherent in a single catalytic site with a Km 70 μM." FEBS Lett.368. 207-210 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Matsui,T.: "Expression of the wild-type and the Cys-/Trpless α_3β_3γ complex of thermophilic F_1-ATPase in Escherichia coli." Biochim. Biophys. Acta. 1231. 139-146 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Kato,Y.: "Analysis of time dependent change of Escheri-chia coli F_1-ATPase activity and its relationship with appatent negative cooperativity." Biochim. Biophys. Acta. 1231. 275-281 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Jault,J-M.: "The α_3β_3γ of the F_1-ATPase from thermophilic Bacillus PS3 containing the αD_<261>N substitutioin fails to dissociate inhibitory MgADP" Biochimistry. 34. 16412-16418 (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Kaibara,C.: "Stuctural asymmetry of F1-ATPase caused by the γ subunit generates a high affinity nucleotide binding site." J. Biol. Chem.271. 2433-2438 (1996)

    • Related Report
      1995 Annual Research Report
  • [Publications] Jault,J-M.: "Probing the specificity of nucleotide binding to the F_1-ATPase from thermophilic Bacillus PS3 and its isolated α and β subunits with 2-N_3-[β,γ-_<32>P]ATP." Arch.Biochem.Biophys.310. 282-288 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Odaka,M.: "Tyr-341 0f the β subunit a major Km-determining residue of TF1-ATPase;Parallel effect of its mutations on Kd(ATP)of the β subunit and on the Km of the α3β3γ complex." J.Biochem.115. 789-796 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Nakamura,J.: "Two Types of Proton-modulated Calcium Binding in the Sarcoplasmic Reticulum Ca^<2+>-ATP-ase" J.Biol.Chem.269. 30818-30821 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Yokoyama,K.: "lsolation of prokaryotic VoV1-ATPase from a thermophilic eubacterium Thermus thermophilus." J.Biol.Chem.269. 12248-12253 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Muneyuki,E.: "Catalytic cooperativity of beef heart mitochondrial F1-ATP-ase revealed by using 2',3'-O-(2,4,6-trinitrophenyl)-ATP as a substrate,an indication of mutually activating catalytic sites" Biochim.Biophys.Acta.1188. 108-116 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Yamada,T.: "Structural and Functional Analyses of Arg-Gly-Asp Sequence lntroduced into Human Lysozyme" J.Biol.Chem.268. 10588-10592 (1993)

    • Related Report
      1994 Annual Research Report
  • [Publications] 吉田賢右: "タンパク質の運命-生成から崩壊まで:蛋白質-この絶妙なる設計物" 日本生物物理学会, 26 (1994)

    • Related Report
      1994 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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