| Project/Area Number |
06680620
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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 |
Functional biochemistry
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| Research Institution | Sapporo Medical College |
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Principal Investigator |
YAMADA Keiko Sapporo Medical College, School of Health Sciences Associated Professor, 保健医療学部, 助教授 (80045541)
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| Co-Investigator(Kenkyū-buntansha) |
WADA Ikuo Sapporo Medical College, School of Medicine Assistant Professor, 医学部, 講師 (40182969)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1995: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1994: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Diacylglycerol kinase (DGK) / Signal transduction / DGK isozymes / EF-hand / Ca^<2+> binding / T-lymphocyte / Conformational changes |
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| Research Abstract |
1) Role of Diacylglycerol Kinase lsozymes (DGK) on the Proliferation and Differentiation of the cells.
The treatments of HL-60 cells with TPA,DMSO and retinoic acid induced differentiation of HL-60 cells. TPA-and retinoic acid-induced differentiation resulted in increased both DGK activity and expression levels of DGKalpha and DGKgamma.
2) Role of Diacylglycerol Kinase lsozymes of Immune Cells
Jurkat cells and LKT 9 (EBV transformed B cell) were used. We found that a large amount of DGKgamma was expressed in LKT 9 cells. The changes of this enzymes after the stimulation of anti bodies are now studying.
3) The change of phosphatidic acid formation in the expression of DGKalpha
The NIH3T3 cells overexpressed the DGKalpha were grown rapidly than wild type cells. The stimulation of PDGF resulted in the change of the formation of phosphatidic acid from endogenous DG,the convertion to lysophosphatidic acid and the mass of cell-associated DG.
4) Properties of EF-hand motifs in DGK isozymes (DGKs alpha, b and gamma)
The three DGKS alpha, beta and gamma cloned so far contained in common a tandem repeat of EF-hand motifs.The enzyme activities of beta-and gamma-isozymes were apparently independent of added Ca^<2+>whereas the activity of DGKalpha was completely dependent of micromoles concentration os Ca^<2+>. To investigate the intrinsic properties of the three sets EF-hand motifs of DGK isozymes, we bacterially expressed and puritied two consecutive EF-hand motifs. Analysis of calcium-binding abilities by using equilibrium dialysis method indicated that all of the three EF-hand regions bound approximately two mol of Ca^<2+> per mol. The apparent dissociatio constants (Kd) for calcium binding to three isozymes were distinctly different from each other. Conformational changes of all EF-hand regions upon Ca^<2+> binding were detected as a mobility shift in alkaline urea-polyacrylamide electrophoresis. These results were preparing to submit.
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