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Structure and function of membrane-spanning cytochrome b561 in neuroendocrine secretary vesicles

Research Project

Project/Area Number 10680638
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionHimeji Institute of Technology

Principal Investigator

TSUBAKI Motonari  Himeji Institute of Technology, Faculty of Science, Associate Professor, 理学部, 助教授 (00145046)

Project Period (FY) 1998 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
Keywordsvitamin C / ascorbic acid / cytochrome b561 / noradrenaline / catecholamine / chromaffin vesicle / adrenal medulla / heme / 結晶化
Research Abstract

1. Pulse Radiolysis Analysis of Electron Transfer Reactions of Cytochrome b561 : Pulse radiolysis analysis of electron transfer reactions of cytochrome b561 was performed. MDA radical oxidized rapidly (in msec time domain) the reduced heme b of cytochrome b561. In following time phase (in sec time domain), AsA re-reduced the oxidized heme b center. The rate of the oxidation of heme b with MDA radical showed a maximum at pH 5.5, whereas the reduction of heme b with AsA occurs at neutral pH. Further, only half of heme b could be reduced in the presence of excess MDA radical concentration.
2.Inhibition of Electron Transfer with DEPC-Treatment and Analysis of the DEPC-Modification Sites with MALDI-TOF-MS Spectrometry : Treatment of oxidized cytochrome b561 with DEPC, a reagent specific for HIS residue, caused an inhibition of electron transfer from AsA. Pulse radiolysis study showed that oxidation phase of reduced of heme b with MDA radical was similar to that observed for the control sampl … More e. However, the following reduction phase with AsA was completely absent for the DEPC-treated sample. The modification sites were analyzed with MALDE-TOF-MS spectrometry using tryptic and V8 protease digests. Three major modification sites, His88, His161, and Lys85, were identified. It is considered that modification of His88 and His161, which were though as the ligands of heme b at the cytosolic side based on the membrane spanning model, caused the inhibition of the electron acceptance from AsA.
3. cDNA-cloning of Planarian Cytochrome b561. Planarian cytochrome b561 : cDNA was cloned and its deduced amino acid sequence was analyzed. Two fully conserved sequences were also conserved and were thought to be functionally essential. One of the 6 histidyl residues was found to be replaced with Asn residue. This lead to the suggestion that His88 and His161 are indeed the ligands of heme b at the cytosolic side.
4. cDNA-cloning of Arabidopsis thaliana Cytochrome b561 : Arabidopsis thaliana, a plant, showed to express a cytochome b561-like protein based on the EST-tag. We obtained two cDNA clones from an A.thaliana cDNA library. Analysis of the deduced amino acid sequences showed that the conserved sequence located at the intravesicular side, which which may play a role for the interaction with MDA radical, was well-conserved in plant also. The other conserved sequence for cytochrome b561 in animals was not conserved. In addition, one of the 6 conserved histiine residues showed a substitution to Gln. This residue corresponds indeed to the same site with the one where the substitution to occur in planarian cytochrome b561. Plant cytochrome b561 may evolved differently with a distinct physiological function from those of cytochrome b561 in animals. Less

Report

(3 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] Tsubaki, M. et al.: "Fourier-transform infrared studies on azide-binding to the binuclear center of the Eschericha coli bo-type ubiquinol oxidase"FEBS Lett.. 449. 191-195 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsubaki, M, et al.: "Fluoride-binding to the Escherichia coli bd-type ubiquind oxidase studied by visible absorption and EPR spectroscopies"J. Biochem,. 126. 98-103 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsubaki, M, et al.: "Azide-and cyanide-binding to the Escherichia coli bd-type ubiquinol oxidase studeid by visible abvsorptior, EPR, and FTIR spectroscopies"J. Biochem.. 126. 510-519 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 鍔木基成: "神経内分泌小胞膜に特異的に存在する電子伝達系の構造と機能"財団法人 ひゅうご科学技術協会 平成10年度学術研究支援事業 研究成果報告集. 33-41 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsubaki, M.: "Heme-steroid interaction in cytochrome p450_<c21> studied byEPR and FTIR spectroscopies"Molecular Steroidogenesis (Universal Academy Press). 69-72 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsubaki, M. et al.: "Diethylphyrocarbonate-modification abolishes fast electron aceepting ability of cytochrome b561 from ascorbate but does not influence on electron"Biochemistry. (in press). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsubaki M., M.Mogi, and H.Hori: "Fourier-transform infrared studies on aside binding to the binuclear center of the Escherichia coli bo-type ubiquinol oxidase"FEBS Lett.. 449. 191-195 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsubaki M., M.Mogi, and H.Hori: "Fluoride-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption and EPR spectroscopies"J. Biochem.. 126. 98-103 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsubaki M., M.Mogi, and H.Hori: "Azide- and cyanide-bindings to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption, EPR and FTIR spectroscopies"J. Biochem.. 126. 510-519 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsubaki M.: "Heme-steroid interaction in cytochrome P450c21 studied by EPR and FTIR spectroscopies"In "Molecular Steroidogenesis" M.Okamoto, Y.Ishimura, H.Nawata, editors, Universal Academy Press, Inc., Tokyo, Japan. 69-72 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsubaki M., K.Kobayashi, T.Ichise, F.Takeuchi and S.Tagawa: "Diethylpyrocarbonate-modification abolishes fast electron accepting ability of cytochrome b561 from ascorbate but does not influence on electron donation to monodehydroascorbate radical : Identification of the modification sites by mass spectrometric analyses."Biochemistry. (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tsubaki,M.et al.: "Fourier-transform infrared studies on azide-birding to the binuclear center of the Escherichia coli bo-type ubquinol oxidase"FEBS Lett.. 449. 191-195 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tsubaki,M.et al.: "Fluoride-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption and EPR spectroscopies"J.Biochem.. 126. 98-103 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tsubaki,M.et al.: "Azide-and cyanide-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption,EPR,and FTIR spectroscopies"J.Biochem.. 126. 510-519 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 鍔木 基成: "神経内分泌小胞膜に特異的に存在する電子伝達系の構造と機能"財団法人 ひょうご科学技術協会平成10年度学術研究支援事業研究成果報告集. 33-41 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tsubaki,M.: "Heme-steroid interaction in cytochrome P450_<c21> studied by EPR and FTIR spectroscopies"Molecular Steroidogenesis(Universal Academy press). 69-72 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tsubaki,M.et al.: "Diethylpyrocarbonate-modification abolishes fast electron accepting ability of cytochrome b561 from ascorbate but does not influence on electron"Biochemistry. (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Okuyama,E.et al.: "Structural basis for the electron transter across the chromaffin vesicle membranes catalysed by cytochrome b561" Biochim.Biophys.Acta. 1383. 269-278 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kobayashi,K.et al.: "Distinct roles of two heme centers for transmembrane electron transfer in cytochrome b561 from bovine adrenal chromaffin vesicles by pulse radiolysis" J.Biol.Chem.273. 16038-16042 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Mogi,T.et al.: "Two terminal guinol oxidases families in Escherichia coli. Variations on molecular machinery for dioxygen reduction" J.Biochem.Mol.Biol.Biophys.2. 79-110 (1998)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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