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Development of analytical method of molecular function in protein kinases by using newly synthesized protein kinase inhibitor - affinity chromatography

Research Project

Project/Area Number 62880018
Research Category

Grant-in-Aid for Developmental Scientific Research

Allocation TypeSingle-year Grants
Research Field 物質生物化学
Research InstitutionNagoya University School of Medicine (1988)
Mie University (1987)

Principal Investigator

HIDAKA Hiroyoshi  Nagoya University School of Medicine, Professor, 医学部, 教授 (80100171)

Co-Investigator(Kenkyū-buntansha) 中 充子  三重大学, 医学部, 助手 (10093139)
田中 利男  三重大学, 医学部, 教授 (00135443)
HAGIWARA Masatoshi  Nagoya University School of Medicine, Staff, 医学部, 助手 (10208423)
Project Period (FY) 1987 – 1988
Project Status Completed (Fiscal Year 1988)
Budget Amount *help
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1988: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1987: ¥5,200,000 (Direct Cost: ¥5,200,000)
KeywordsMyosin light chain kinase / ML-9 / Thyroxine / Affinity chromatography / Protein kinase C / ML-9 / サイロキシン
Research Abstract

We succeed the purification of protein kinase C from rabbit brain and myosin light chain kinase (MLCK) from chicken gizzard and human platelet by using protein kinase inhibitor-affinity chromatography. The purified C kinase was used to determine amino acid sequence. Rabbit complementary DNA clones cording for three disinct types of protein kinase C, named alpha,beta and gam, have been identified and sequenced.Then, homogenous C-kinase were classified into three subtypes, using hydroxylapatite column chromatography. We obtained three types of protein kinase C monoclonal antibodies which selectively interact with hydroxyapatite column chromatographically resolved isozymes, type i, ii and iii of protein kinase. To clarify the role of protein kinase C in vascular smooth muscle contraction, the effects of exogenous protein kinase C was investigated on skinned smooth muscle preparations of the rabbit mesenteric artery. It was clarified that protein kinase C phosphorylation of myosin light chain play inhibit role in contraction of vascular smooth muscle. MLCK is phosphorylated by A kinase (2 mol of phosphate/mol of MLCK). It was recognized by using thyroxine that one site of phosphorylation by A kinase is calmodulin binding site. We clarified interrelationship among each protein kinase in vascular smooth muscle contraction mechanism using protein kinase inhibitor and affinity purified protein kinase. These evidence suggested that protein kinase inhibitor-affinity chromatography can be used to investigate the physiological function of purified protein kinase and to study interrelation among each protein kinase.

Report

(2 results)
  • 1988 Annual Research Report   Final Research Report Summary
  • Research Products

    (27 results)

All Other

All Publications (27 results)

  • [Publications] M.Inakaki et.al.: Archives of Biochemistry and Biophysis. 254. 136-141 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] M.Hagiwara et.al.: Molecular Phormacology. 32. 7-12 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] H.Hidaka et.al.: Method in Enzymology. 139. 570-582 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] H.Hidaka et.al.: Nature. 325. 161166 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] H.Hidaka et.al: Pharmacology (Proceedings of 10th Inte rnational Congress of Pharmacology). 393-399 (1987)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] M.Hagiwara et.al.: The Jou rnal of Biological Chemistry. 263. 6438-6441 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] H.Hidaka et.al: The Journal o f Biological Chemistry. 263. 4523-4526 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] M.Hagiwara et.al.: Biochemical and Biophysical research communications. 152. 270-276 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] M.Hagiwara et.al.: The Journal of Bidogical Chemist ry. 264. 40-44 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] S.Mamiya et.al.: The Journal of Biologic al Chemistry. (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] T.Chijiwa et.al.: The Journal of Biological Chemistry. (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] M. Inagaki; et al.: "Purified rabbit brain protein kinase C relaxed skinned vascular smooth muscle and phosphorylates myosin light chain" Archives of Biochemistry and Biophysics. 254. 136-141 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] M. Hagiwara; et al.: "Selective modulation of calcium-dependent myosin phosphorylation by novel protein kinase inhibitors, isoquinolinesulfonamide derivatives" Molecular Pharmacology. 32. 7-12 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] H. Hidaka; et al.: "Transmembrane Ca^<++> signaling and a new class of inhibitors" Methods in Enzymology. 139. 570-582 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] H. Hidaka; et al.: "Tissue-specific expression of three distinct types of rabbit protein kinase C" Nature. 325. 161-166 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] H. Hidaka; et al.: "Myosin phosphorylation inhibitors and vascular contraction" Pharmacology (Proceedings of 10th International Congress of Pharmacology). 393-399 (1987)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] M. Hagiwara; et al.: "Modulation of thyrosine phosphorylation of p36 and other substrate by the S-100 protein" The Journal of Biological Chemistry. 263. 6438-6441 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] H. Hidaka; et al.: "Cell type-specific expression of protein kinase C isozymes in the rabbit cerebellum" The Journal of Biological Chemistry. 263. 4523-4526 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] M. Hagiwara; et al.: "Thyroid hormones inhibit the Ca^<2+> calmodulin-induced activation of myosin light chain kinase" Biochemical and Biophysical Research Communications. 152. 270-276 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] M. Hagiwara; et al.: "Selective binding of 1-thyroxine by myosin light chain kinase" The Journal of Biological Chemistry. 264. 40-44 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] S. Mamiya; et al.: "Thyroid hormones inhibit platelet function and myosin light chain kinase" The Journal of Biological Chemistry. (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] T. Chijiwa; et al.: "A newly synthesized selective casein kinase I inhibitor, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide and affinity purification of casein kinase I from bovine testis" The Journal of Biological Chemistry. (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1988 Final Research Report Summary
  • [Publications] H.Hidaka;et al.: Pharmacology.(Proceedings of the 10th International Congress of Pharmacology). 393-399 (1987)

    • Related Report
      1988 Annual Research Report
  • [Publications] M.Hagiwara;et al.: Biochemical and biophysical research communications. 152. 270-276 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] M.Hagiwara;et al.: The Journal of Biological Chemistry. 264. 40-44 (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] S Mamiya;et al.: The Journal of Biological Chemistry. (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] T.Chijiwa;et al.: The Journal of Biological Chemistry. (1989)

    • Related Report
      1988 Annual Research Report

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Published: 1987-04-01   Modified: 2016-04-21  

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