1987 Fiscal Year Final Research Report Summary
Physiological function of lipid-phosphorylating enzymes and their mechanism of action.
| Project/Area Number |
61570119
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Sapporo Medical College |
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Principal Investigator |
KANOH Hideo Professor, 医学部, 教授 (70045475)
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| Project Period (FY) |
1986 – 1987
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| Keywords | Diacylglycerol / Diacylglycerol kinase / Protein phosphorylation / Protein kinase C / プロティンキナーゼC |
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| 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
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Research Products
(9 results)
