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Subunit structure and ion transport mechanism of vacuolar ATPase

Research Project

Project/Area Number 11672158
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Biological pharmacy
Research InstitutionChiba University

Principal Investigator

KAKINUMA Yoshimi  Chiba University, Faculty of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (80134394)

Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
KeywordsVacuolar ATPase / Subunit / Ion transport / Mechanism
Research Abstract

(i) We succeeded in the large-scale purification of Enterococcus hirae V-ATPase achieving column chromatographies ; about 30 mg of purified enzyme comprising nine subunits, A, B, C, D, E, F, G, I, K, was obtained from 20 liter culture. SDS-PAGE revealed that seven subunits, A, B, C, D, E, F and G, are releasable as the V1 subunit form thew V0V1 complex by incubation with EDTA ; the V0 portion consists of I and K subunits.
(ii) E.hirae V-ATPase catalyzes translocation of Na+ or Li+ coupled with ATP hydrolysis. It is suggested that the glutamic acid residue (Glu139) of NtpK proteolipid subunit of this multisubunit enzyme is the binding site of these ions for translocation. We established a complementation system from the ntpK gene with its deletion mutant, and found that the ATPase activity disappeared upon replacement of Glu139 to aspartic acid. The side-chain length of this acidic residue of NtpK is thus important for this ATPase reaction.
(iii) We investigated the ion binding step to E.hirae Na+-translocating ATPase. The kinetics of Na+ binding to purified V-ATPase suggested 6 Na+ bound/enzyme molecule, with a single high affinity (Kd (Na+) =15 microM.The number of cation binding sites is consisitent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. This suggest that the Na+ binding sites of the Na+-ATPase are readily accessible from the aqueous phase.

Report

(3 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • Research Products

    (30 results)

All Other

All Publications (30 results)

  • [Publications] Kakinuma,Y.,: "Structure and function of vacuolar Na+-translocating ATPase in Enterococcus hirae"Journal of Bioenergetics and Biomembranes. 31. 7-14 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Murata,T.: "Properties of the V0V1 Na+-ATPase from Enterococcus hirae and its V0 moiety"Jounal of Biochemistry (Tokyo). 125. 414-421 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kakinuma,Y.: "Potassium/proton antiport system is dispensable for growth of Enterococcus hirae at low pH"Bioscience Biotechnology and Biochemistry. 63. 875-878 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Takase,K.: "Indispensable glutamic acid residue-139 of NtpK proteolipid in the reaction of Na+-translocating ATPase in Enterococcus hirae"Bioscience Biotechnology and Biochemistry. 63. 1125-1129 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kakinuma,Y.: "Isolation and properties of Enterococcus hirae mutants defective in potassium/proton antiport"Journal of Bacteriology. 181. 4103-4105 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ikegami,M.: "Enterococcus hirae vacuolar ATPase is expressed in response to pH as well as sodium"FEBS Letters. 454. 67-70 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kawano,M.,: "Two major potassium uptake systems, KtrI and ktrII, in Enterococcus hirae"FEMS Microbiology Letters. 176. 449-453 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ikegami,M.,: "Sodium ATPase and sodium/proton antiporter are not obligatory for sodium homeostasis of Enterococcus hirae at acid pH"Bioscience Biotechnology and Biochemistry. 64. 1088-1092 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Murata,T.,: "Na+binding of V-type Na+-ATPase in Enterococcus hirae"Journal of Biological Chemistry. 275. 13415-13419 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kawano,M.,: "Evidence for Ma+ influx via the NtpJ protein of the KtrII K+uptake system in Enterococcus hirae"Journal of Bacteriology. 182. 2507-2512 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kakinuma, Y.et al.: "Structure and function of vacuolar Na+-translocating ATPase in Enterococcus hirae"Journal of Bioenergetics and Biomembranes. 31. 7-14 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Murata, T.et al.: "Properties of the V0V1 Na+-TAPase from Enterococcus hirae and Its V0 moiety"Jounal of Biochemistry (Tokyo). 125. 414-421 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kakinuma, Y.et al.: "Potassium/proton antiport system is dispensable for growth of Enterococcus hirae at low pH"Bioscience Biotechnology and Biochemistry. 63. 875-878 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Takase, K.et al.: "Indispensible glutamic acid residue-139 of NtpK proteolipid in the reaction of Na+-translocating ATPase in Enterococcus hirae"Bioscience Biotechnology and Biochemistry. 63. 1125-1129 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kakinuma, Y.et al.: "Isolation and properties of Enterococcus hirae mutants defective in potassium/proton antiport"Journal of Bacteriology. 181. 4103-4105 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ikegami, M.et al.: "Enterococcus hirae vacuolar ATPase is expressed in response to pH as well as sodium"FEBS Letters. 454. 67-70 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kawano, M.et al.: "Two major potassium uptake systems, KtrI and KtrII, in Enterococcus hirae"FEMS Microbiology Letters. 176. 449-453 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Ikegami, M.et al.: "Sodium ATPase and sodium/proton antiporter are not obligatory for sodium homeostasis of Enterococcus hirae at acid pH"Bioscience Biotechnology and Biochemistry. 64. 1088-1092 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Murata, T.et al.: "Na+ binding of V-type Na+-ATPase in Enterococcus hirae"Journal of Biological Chemistry. 275. 13415-13419 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kawano, M.et al.: "Evidence for Na+ influx via the NtpJ protein of the KtrII K+ uptake system in Enterococcus hirae"Journal of Bacteriology. 182. 2507-2512 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kawano,M.,: "Two major potassium uptake systems, KtrI and KtrII, in Enterococcus hirae"FEMS Microbiology Letters. 176. 449-453 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Ikegami,M.,: "Sodium ATPase and sodium/proton antiporter are not obligatory for sodium homeostasis of Enterococcus hirae at acid pH"Bioscience Biotechnology and Biochemistry. 64. 1088-1092 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Murata,T.,: "Na+ binding of V-type Na+-ATPase in Enterococcus hirae"Journal of Biological Chemistry. 275. 13415-13419 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kawano,M.,: "Evidence for Na+ influx via the NtpJ protein of the KtrII K+ uptake system in Enterococcus hirae"Journal of Bacteriology. 182. 2507-2512 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kakinuma, Y.: "Structure and function of vacuolar Na+translocating ATPase in Enterococcus hirae"Journal of Bioenergetics and Biomembranes. 31. 7-14 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Murata, T.: "Properties of the V0V1 Na+-ATPase from Enterococcus hirae and Its V0 moiety"Journal of Biochemistry(Tokyo). 125. 414-421 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kakinuma, Y.: "Potassium/proton antiport system is dispensable for growth of Enterococcus hirae at low pH"Bioscience Biotechnology and Biochemistry. 63. 875-878 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Takase, K.: "Indispensible glutamic acid residue-139 of NtpK proteolipid in the reaction of Na+-translocating ATPase in Enterococcus hirae"Bioscience Biotechnology and Biochemistry. 63. 1125-1129 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kakinuma, Y.: "Isolation and properties of Enterococcus hirae mutants defective in potassium/proton antiport"Journal of Bacteriology. 181. 4103-4105 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Ikegami, M.: "Enterococcus hirae vacuolar ATPase is expressed in response to pH as well as sodium"FEBS Letters. 454. 67-70 (1999)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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