Role of Diacylglycerol Kinase Isozymes on the Proliferation and Differentiation of the cells

1)DGK在HL-60细胞增殖和分化中的作用TPA和维甲酸诱导分化后，DGK活性和DGKGamma的表达水平均升高。2)免疫细胞二酰甘油激酶同工酶的作用。我们发现在LKT-9细胞中有大量的DGK-γ的表达。3)DGKalpha表达中磷脂酸形成的变化NIH3T3高表达DGKalpha的细胞生长速度比野生型细胞快。4)DGK同工酶中EF-Hand基序的性质目前已克隆的三个DGKsα、β和Gamma都包含一个串联的EF-Hand基序。β-和γ-同工酶的酶活性明显不依赖于外加的Ca~(2+)~(2+)和Gt~(2+)，而DGKalpha的活性完全依赖于微摩尔浓度。为了研究DGK同工酶的三组EF-Hand基序的内在性质，我们对两个连续的EF-Hand基序进行了细菌表达和纯化。用平衡透析法对钙结合能力的分析表明，这三个EF-Hand区每摩尔结合约2摩尔的钙离子。钙与三种同工酶结合的表观解离常数(Kd)明显不同。碱性尿素-聚丙烯酰胺凝胶电泳法检测到与Ca~(2+)和Gt~(2+)结合的所有EF-Hand区的构象变化表现为迁移率的变化。这些结果正在准备提交。

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 CellsJurkat 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 DGKalphaThe 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.