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1993 Fiscal Year Final Research Report Summary

Studies on Structure and Function of the ATP-Binding sites of the Sarcoplasmic Reticulum Calcium Pump.

Research Project

Project/Area Number 04670134
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field General medical chemistry
Research InstitutionAsahikawa Medical College

Principal Investigator

SUZUKI Hiroshi  Asahikawa Medical College, Department of Biochemistry, Assistant Professor, 医学部, 助教授 (50183421)

Co-Investigator(Kenkyū-buntansha) DAIHO Takashi  Asahikawa Medical College, Department of Biochemistry, Assistant, 医学部, 助手 (90207267)
Project Period (FY) 1992 – 1993
KeywordsSarcoplasmic Reticulum / Calcium Pump / ATP-Binding Sites / Fluorescein 5-Isothiocyanate
Research Abstract

In an attempt to establish the stoichiometry of the phosphorylatable catalytic site to the specific fluorescein 5-isothiocyanate (FITC) binding site (Lys-515) in the Ca^<2+>-ATPase of sarcoplasmic reticulum vescicles (SRV), labeling by FITC or phosphorylation by ATP (or Pi) was : performed with SRV under the conditions in which almost all of the specific FITC binding sites or phosphorylatable catalytic sites can be labeled or phosphorylated. The resultant SRV were solubilized in lithium dodecyl sulfate, and then the Ca^<2+>ATPase was purified by using a size exclusion high performance liquid chromatography. The contents of bound FITC and phosphoenzyme in the ATPase isolated as above were determined. Peptide maapping of the tryptic digests and sequencing showed that Lys-515 of the Ca^<2+>-ATPase was exclusively labeled with FITC.This specific labeling was completely prevented by ATP.The content of phosphoenzyme (4.57 and 4.94 nmol/mg of protein from ATP and Pi respectively) was approximately half that of the specific FITC binding site (8.16-8.19 nmol/m of protein) and also half that of the llO-KDa Ca^<2+>ATPase chain (9.06 nmol/mg of protein) calculated on the assumption that the isolated ATPase chain was pure. These findings are consistent with a possible half-of-the-sites reactivity, being in favor of a dimeric structure of the Ca^<2+>ATPase in SRV.The extent of the specific FITC binding required for complete inhibition of ATP-induced phosphorylation corresponded to 6.4 nmol of bound FITC/mg of protein. This finding suggests that FITC somewhat preferentially binds to one half of the ATPase chains in SRV and that this binding is primarily responsible for the observed inhibition of phosphorylation.

  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] 鈴木 裕: "筋小胞体Ca^<2+>-ATPaseの触媒部位とFITC結合部位のstoichiometry" 生化学. 64. 889 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 鈴木 裕: "筋小胞体Ca^<2+>-ATPアーゼ" 生化学. 64. 1444-1449 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 中村 哲史: "筋小胞体Ca^<2+>-ATPaseの8-thiocyano-ATPによる親和修飾" 生化学. 65. 948 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 鈴木 裕: "筋小胞体Ca^<2+>-ATPaseのリン酸化中間体形成における構造変化と基質メタルイオンの影響" 生体エネルギー研究会 第19回討論会講演要旨集. 140-141 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Suzuki Hiroshi, Nakamura Satoshi, and Kanazawa Tohru: ""Stoichiometry of the Phosphorylatable Catalytic Site to the Specific Fluorescein 5-Isothiocyanate-Bindig Site in the Ca^<2+>-ATPase of Sarcoplasmic Reticulum Vesicles."" Seikagaku. 64. 889 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Suzuki Hiroshi: ""Sarcoplasmic Reticulum Ca^<2+> -ATPase."" Seikagaku. 64. 1444-1449 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Nakamura Satoshi, Suzuki Hiroshi, and Kanazawa Tohru: ""Affinity Labeling of the Sarcoplasmic Reticulum Ca^<2+> -ATPase by 8-Thiocyano-ATP."" Seikagaku. 65. 948 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Suzuki Hiroshi, Nakamura Satoshi, and Kanazawa Tohru: ""Effects of Divalent Cations Bound to the Catalytic Site on ATP-Induced Conformational Changes in the Sarcoplasmic Reticulum Ca^<2+>-ATPase."" Abstracts for the 19th Meeting of Japan Bioenergetics Group. 140-141 (1993)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1995-03-27  

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