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A rapid quenching a apparatus permitting to mix for different microvolume solutions

Research Project

Project/Area Number 03558026
Research Category

Grant-in-Aid for Developmental Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 分子遺伝学・分子生理学
Research InstitutionHokkaido University

Principal Investigator

TANIGUCHI Kazuya  Hokkaido Univ., Faculty of Science, Prof., 理学部, 教授 (40028204)

Co-Investigator(Kenkyū-buntansha) OKAZAKI Fumiyasu  Kitami dept.Enver.Assistant Institute Tech., Engineer., 工学部, 助手 (10213927)
分部 健二  メディカルエンジニアリング(株), 開発研究員
白崎 雄治  メディカルエンジニアリング(株), 部長
Project Period (FY) 1991 – 1992
Project Status Completed (Fiscal Year 1992)
Budget Amount *help
¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1992: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1991: ¥3,400,000 (Direct Cost: ¥3,400,000)
KeywordsNa^+, K^+-Pump / H^+, K^+-Pump / rapid quenching / Stopped flow / P-type ATPase / Tyr phosphorylation / H^+ーポンプ / ラビッドクエンチング / H^+-ポンプ / ラピッドクエンチング
Research Abstract

A preparation of pig kidney Na^+ K^+-ATPase showed changes in fluorescence energy transfer between probes bound to the alpha-subunit. Excitation of BIPM probe, which was covalently bound to Cys-964, and excitation of FITC probe at Lys-501 gave different FITC fluorescence intensity changes at 520 nm in BIPM-FITC doubly labeled enzyme accompanying formation of reaction intermediates. The data suggest that Fluorescence energy transfer from the BIPM to the FITC probe accompanies the processes of migration of Na^+ and K^+ in the pump molecules. The dynamic fluorescence changes after phosphorylation and dephosphorylation seem to reflect changes in the binding state or the process of migration of these ions.
The Lys-480 in the alpha-subunits of Na^+, K^+-ATPase from pig kidneys was specifically modified with PLP or AP2PL probes in the presence of NaCl. The data show that these probes can sense molecular events related to the formation of phosphoenzymes induced by AcP and presumably to the form … More ation of Mg-Na-ATP-enzyme complex. The data also suggest that PLP or AP_2PL probes at Lys-480 in the presence of Na^+ and Mg^<2+> do not affect the transphosphorylation from AcP to Asp-369 to form phosphoenzymes byt that they inhibit the transphosphorylation from the gamma-phosphoryl group of ATP and also ATP binding in the absence of Mg^<2+>.
When pig stomach membrane H^+, K^+-ATPase preparations were incubated with [gamma-^<32>P]ATP and Mg^<2+> with vanadate, ^<32>P was incorporated into the alpha-chain of H^+, K^+-ATPase to a steady-state level of approximately 0.7 mol of phosphotyrosine (Tyr (P)) /mol of phosphoenzyme intermediates. Mild tosylphenylalanyl chloromethyl ketone-trypsin treatment solubilized ^<32>P-containing peptides from the alpha-chain almost completely. A reverse-phase column chromatography of the supernatant gave two peaks of ^<32>P-peptide with similar total radioactivities.
The comparison of the recovery of amino acid from each Edman cycle showed that the phosphorylation of Tyr^<10> occurred preceding the phosphorylation of Tyr^7. These data and others suggested the presence of a novel membrane bound enzyme system to participate in reversible phosphorylation of boty Tyr residues in the alpha-chain of H^+, K^+-ATPase. Less

Report

(3 results)
  • 1992 Annual Research Report   Final Research Report Summary
  • 1991 Annual Research Report
  • Research Products

    (48 results)

All Other

All Publications (48 results)

  • [Publications] Togawa,K.: "Reversible phobphorylation of both Tyr-7 and tyr-10 in the α-chain of pig stomach H^+,K^+-ATPase by a membrane-bound kinase and a phosphatase" Journal of Biological Chemistry. 270. 15475-15478 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Kaya,S.: "Pyridoxal 5'-phosphate Probe at Lys-480 Can Sense the Binding of ATP and the Formation of Phosphoenzymes in Na^+,K^+-ATPase." Journal of Biological Chemistry. 269. 7419-7422 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Yamazaki,A: "An extraphosphorylation of Na^+,K^+-ATPase by Paranitorohenyl phosphate (PNPP) : Evidence for the oligemeric nature of the enzyme." Journal of Biochemistry. 116. 1360-1369 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Tosa,H.: "Estimation of distance changes between Cys-964 and Lys-501 in Na^+/K^+-ATPase intermediates and the orientation of transmembrane segment containing Cys-964." The Sodium Pump. 649-652 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Yamazaki,A.: "Phosphorylation of Na^+/K^+-ATPase by P-nitrophenyl phospate an and other phosphatase sub-strates." The Sodium Pump. 645-648 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Nakamura,Y.: "Different Susceptibility of Phospholipase A2 Treatment of the Fluorescence Intensity Changes in the Vicinity of Cys-964 and Lys-501 in the α-Chain of Probe-Labeled Na^+/K^+-ATPase." Journal of Biochemistry. 115. 454-462 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Kaya,S.: "Pyridoxal 5'-phosphate Probe at Lys-480 can monitor conformational events induced by acetyl phosphate in Na^+/K^+-ATPase." The Sodium Pump. 641-644 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Taniguchi,K.: "Changes in the conformational State of probe labeled Na^+/K^+-ATPase in real time." The Sodium Pump. 581-592 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Taniguchi,K.: "Reversible Chages in the Fluorescence energy Transfer Accompanying Formation of Reaction Intermediates in ProbeLabeled (Na^+,K^+)-ATPase." Journal of Biological Chemistry. 268. 15588-15594 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Eguchi,E.: "Phosphorylation of Half and All Sites in H^+,K^+-ATPase Results in Opposite Changes in Tryptophan Fluorescence." Biochemical and Biophysical Resarch Communication. 196. 249-300 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Adachi,Y.: "Na^+,K^+-ATPase Based Bilayer Lipid Membrane Sensor for Adenosine 5'-Triphoshate." Anal.Chem.Acta.281. 577-584 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Taniguchi,K.: "Estimation of Distance Changes between Cyr-964 and Lys-501 in Na^+/K^+-ATPase Intermediates." Biol.Chem.Hoppe-Seyler. 374. 600 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Kaya,S.: "Pyridoxal 5'-Phoshate Probe at Lys-480 can Monitor Confromational Events Induced by Acetylphosphate in Na^+,K^+-ATPase." Biol.Chem.Hoppe-Seyler. 374. 599 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Taniguchi,K.: "Conformation Change of Probe Labelld Na^+,K^+-ATPase in Real Time." Biological Chemistry Hoppe-Seyler. 374. 556 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Taniguchi,K.: "Conformation Change Accompanying Formation of OligomycinInduced Na Bound Forms and Their Conversion to ADP-Sensitive Phospho-enzyme in Na,K-ATPase." Journal of Biochemistry. 109. 299-306 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Sasaki,T.: "The Binding Sates before and after Accumulation of ADP-Seensitive Phosphoenzyme in Na,K-ATPase." The Na Pump. 413-417 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Shinoguchi,E.: "Fluorescence energy Transfer Between N-p(2-benzimidazolyl) phenyl maleimide (BIPM),N-(1-anilinonaphytyl-4) maleimide (ANM),Fluorescence 5'-isothiocyanate (FITC) and Anthroylouabain (AO) in Na^+,K^+-ATPase." The Na Pump. 363-367 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] 谷口和彌: "Na^+,K^+-ATPaseの機能と構造新タンパク質応用工学" フジテクノシステム, (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] 谷口和彌: "Na^+,K^+-ATPaseタンパク質化学 9巻:脳神経系タンパク質接着タンパク質" 廣川書店, 266-271 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] 谷口和彌: "Na^+,K^+-ATPaseの真時間での構造変化:新生化実験講座 6" 東京化学同人, 427-437 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Togawa, K.: "Reversible phophorylation of both Tyr 7 and Tyrio in the alpha-chain of pig stomach H^+, K^+-ATPase by a membrane-bound kinase and a phosphatas phosphatase." J.Biol.Chem.270. 15475-15478 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Kaya, S.: "Pyridoxal 5'-phosphate Probe at Lys-480 Can Sense the Binding of ATP and the Formation of Phosphoenzymes in Na^+, K^+-ATPase." J.Biol.Chem.269. 7419-7422 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Yamazaki, A.: "An Extraphosphorylation of Na^+, K^+-ATPase by Paranitorophenylphosphate (PNPP) : Evidence for the oligemeric nature of the enzyme." J.Biochem.116. 1360-1369 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Tosa, H.: "Estimationof distance changes between Cys-964 and Lys-501 in Na^+/K^+-ATPase intermediates and the orientation of transmembrane segement containing Cys-964." The Sodium Pump. 649-652 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Yamazaki, A.: "Phosphorylation of Na^+/K^+-ATPase by P-nitrophenyl phosphate and other phosphatase sub-strates" The Sodium Pump. 645-648 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Nakamura, Y.: "Different Susceptibility of Phospholipase A_2 Treatment of the Fluorescence Intensity Changes in the Cicinity of Cys-964 and Lys-501 in the alpha-Chain of probe-Labeled Na^+, K^+-ATPase." J.Biochem.115. 454-462 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Kaya, S.: "Pyridoxal 5'-phosphate probe at Lys-480 Can monitor conformational events induced by acety lphosphate in Na^+/K^+-ATPase." The Sodium Pump. 641-644 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Taniguchi, K.: "Changes in the conformational sate of probe labeled Na^+/K^+-ATPase in real time." The Sodium Pump. 581-592

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Taniguchi, K.: "Reversible Changs in the Fluorescence Energy Transfer Accompanying Formation of Reaction Intermediates in Probe-Labeled (Na^+, K^+)-ATPase" J.Biol.Chem.268. 15588-15594 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Eguchi, E.: "Phosphorylation of Half and All Sites in H^+, K^+-ATPase Results in Opposite Changes in Tryptophan Fluorescence." Biochem.Biophys.Res.Comm.196. 294-300 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Adachi, Y.: "Na^+, K^+-ATPase Based Bilayr Lipid Membrane Sensor for Adenosine 5'-Triphosphate." Anal.Chem.Acta.281. 577-584 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Taniguchi, K.: "Estimation of Distance Changes between Cys-964 and Lys-501 in Na^+, K^+-ATPase Intermediates." Biol.Chem.Hoppe-Seyler. 374. 600 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Kaya, S.: "Pyridoxal 5'-Phosphate Probe at Lys-480 can Monitor Confromational Events Induced by Acetylphosphate in Na^+/K^+-ATPase." Biol.Chem, Hoppe-Seyler. 374. 599 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Taniguchi, K.: "Conformation changes of Probe Labelled Na^+, K^+-ATPase in Real Time." Biol.Chem, Hoppe-Seyler. 374. 556 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Taniguchi, K.: "Conformation change Accompanying Formation of O1igomycin-Induced Na Bound Forms and Their Conversion to ADP-sensitive Phospho-enzyme in Na, K-ATPase." J.Biochem.109. 299-306 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Sakaki, T.: "The Binding Sates before and after Accumulation of ADP-sensitive PhoPhosphoenzyme in Na, K-ATPase." The Na Pump.413-417 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] Shinoguchi, E.: "Flurescence energy Transfer Between N-p-(2-benzimidazolyl) phenyl maleimide (BIPM), N-(1-anilinonaphtyl-4) maleimide (ANM), Fluorescen Flurescence 5'-isothiocyanate (FITC) and Anthroylouabain (AO) in Na^+, K^+-ATPase." The Na Pump.363-367 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1992 Final Research Report Summary
  • [Publications] K.Taniguchi: "Conformation Change Accompanying Formation of Oligomycin-Induced Na Bound Forms and Their Conversion to ADP-Sensitive Phosphoenzyme in Na,K-ATPase" Journal of Biochemistry. 109. 299-306 (1991)

    • Related Report
      1992 Annual Research Report
  • [Publications] K.Taniguchi: "Reversible Changes in the Fluorescence Energy Transfer Accompanying Formation of Reaction Intermediates in Probe-Labeled Na^+,K^+-ATPase" Journal of Biological Chemistry. (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] 谷口 和弥: "新生化学実験講座" 日本生化学会, 10 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 中村 洋介: "Na^+、K^+-ATPaseのα鎖Cys-964及びLys-501に導入した蛍光プロ-ブの蛍光強度変化のリン脂質依存性" 生化学. 63. 773- (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] 谷口 和弥: "蛍光プロ-ブでラベルしたNa^+,K^+-ATPaseにおける蛍光エネルギ-移動:Oligomycinの効果" 生化学. 63. 773- (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] 山崎 明雅: "Na^+,K^+-ATPaseのPhosphatase基質による構造変化" 生化学. 63. 774- (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] 江口 博明: "ATPとアセチルリン酸によるH^+,K^+-ATPaseの構造変化" 生化学. 63. 775- (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] 牛田 吉彦: "オリゴマイシンのH^+,K^+-ATPaseに及ぼす効果" 生化学. 63. 775- (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] 谷口 和弥: "Na^+,K^+-ATPaseとH^+,K^+-ATPaseのアセチルリン酸による構造変化とリン酸化:オリゴマイシンの効果" 生体エネルギ-研究会第17回討論会講演要旨集. 17. 158-159 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Eiji Shinoguchi: "The Sodium Pump:Recent Developments" The Rockfeller University Press, 4 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Toshio Sasaki: "The Sodium Pump:Recent Developments" The Rokfeller University Press, 5 (1991)

    • Related Report
      1991 Annual Research Report

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Published: 1991-04-01   Modified: 2016-04-21  

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