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Physiological function of lipid-phosphorylating enzymes and their mechanism of action.

Research Project

Project/Area Number 61570119
Research Category

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

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

Principal Investigator

KANOH Hideo  Professor, 医学部, 教授 (70045475)

Project Period (FY) 1986 – 1987
Project Status Completed (Fiscal Year 1987)
Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1987: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1986: ¥1,500,000 (Direct Cost: ¥1,500,000)
KeywordsDiacylglycerol / Diacylglycerol kinase / Protein phosphorylation / Protein kinase C / プロティンキナーゼC / 抗体 / 蛋白リン酸化 / C-キナーゼ
Research Abstract

In the agonist-stimulated cells, diacylglycerol(DG) kinase plays a central role in the metabolism of a second messenger, DG, which is released from phosphoinositides upon receptor activation. We intend to characterize this lipid kinase by employing purified enzymes and rabbit antibody raised against 80 kDa DG kinase. The projects and major findings are summarized as follows:
1) Immunochemical Studies on DG Kinase in Different Pig Tissues DG kinase from different pig tissues was examined by immunoblot as well as immunoprecipitation. Among the tissues examined, the immunoreactive 80 kDa kinase previously purified by us from the brain(J. Biol. Chem. 258, 1767, -83) was found only in the thymus and, to a much lesser extent, in the spleen. Other tissues like platelets,heart, kidney and liver, contained little, if any, immunoreactive enzymes. A gel filtration of cytosolic enzymes showed three major activity peaks, corresponding apparently to 280, 120 and 80 kDa. The platelet kinase was, inter … More estingly, consisted almost exclusively of nonimmunoreactive 120 kDa species with an apparent lack of immunoreactive 80 kDa kinase. The nonimmunoreactive 280 kDa kinase was abundant in the lymphoid tissues, and was much more heat-stable when compared with the 80 kDa enzyme. Purification of different kinase forms is required to elucidate their physiological implication in each tissur.
2) In vitro Phosphorylation of DG Kinase by Protein Kinase C DG kinase and protein kinase C(PKC) were highly purified from pig thymus. Since both enzymes showed the same mobility(80 kDa) upon SDS-PAGE, the phosphorylation of DGK had to be confirmed by immunoprecipitation with anti-DGK antibody. We found that DGK serves as a good substrate for PKC, and that almost stoichiometric phosphorylation of DGK was achieved. Phosphorylation of DGK by PKA occurred to a very limited extent. Phosphorylated DGK showed a broad band around pI 6.5. The two protein kinases phosphorylated different sites of DGK molecule. The Vmax of partially phosphorylated DGK increased approximately two-fold. Both DGK and PKC were bound to phosphatidylserine in the system. Less

Report

(2 results)
  • 1987 Final Research Report Summary
  • 1986 Annual Research Report
  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] 加納英雄: J.Biol.Chem.261. 5597-5602 (1986)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1987 Final Research Report Summary
  • [Publications] 加納英雄: FEBS Lett.201. 97-100 (1986)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1987 Final Research Report Summary
  • [Publications] 山田恵子: Biochem.J.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1987 Final Research Report Summary
  • [Publications] 加納英雄: Biochem.J.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1987 Final Research Report Summary
  • [Publications] 加納英雄: "続生化学実験講座No, 3, 膜脂質と血漿リポタンパク質" 東京化学同人, 8 (1986)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1987 Final Research Report Summary
  • [Publications] Kanoh, H., Iwata, T., Ono, T. and Suzuki, T.: "Immunological characterization of sn-1,2-diacylglycerol and sn-2-monoacylglycerol kinase from pig brain." J. Biol. Chem.261. 5597-5602 (1986)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1987 Final Research Report Summary
  • [Publications] Kanoh, H. and Ono, T.: "Phosphorylation of pig brain diacylglycerol kinase by endogenous protein kinase." FEBS Lett.201. 97-100 (1986)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1987 Final Research Report Summary
  • [Publications] Yamada, K. and Kanoh, H.: "Occurrence of immunoreactive 80 kDa and nonimmunoreactive diacylglycerol kinases in different pig tissues." Biochem. J.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1987 Final Research Report Summary
  • [Publications] Kanoh, H. and Yamada, K.: "In vitro phosphorylation of diacylglycerol kinase by protein kinase C." Biochem. J.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1987 Final Research Report Summary
  • [Publications] 加納,岩田,小野,鈴木: J.Biol.Chem.261. 5597-5602 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] 加納,小野: FEBS Lett.201. 97-100 (1986)

    • Related Report
      1986 Annual Research Report
  • [Publications] 加納英雄,他: "続生化学実験講座 膜脂質と血漿リポタンパク質" 東京化学同人, 156-163 (1986)

    • Related Report
      1986 Annual Research Report

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Published: 1987-03-31   Modified: 2016-04-21  

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