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

1)二酰基甘油激酶同工酶（DGK）对HL-60细胞增殖和分化的影响TPA、DMSO和维甲酸处理HL-60细胞可诱导HL-60细胞分化。TPA和视黄酸诱导的分化导致DGK活性和DGK α和DGK γ表达水平的增加。2）免疫细胞二酰基甘油激酶同工酶的作用使用Jurkat细胞和LKT 9（EBV转化的B细胞）。我们发现DGK γ在LKT 9细胞中大量表达。3）DGKa α表达中磷脂酸形成的变化过表达DGKa α的NIH 3 T3细胞比野生型细胞生长迅速。PDGF的刺激导致内源性DG形成磷脂酸、向溶血磷脂酸的转化以及细胞结合DG的质量发生变化（DGKS α、B和γ）三个DGKS α，迄今为止克隆的β和γ的酶活性包含共同的EF-手形基序的串联重复。同工酶明显不依赖于加入的Ca^<2+>，而DGK α的活性则完全依赖于微摩尔浓度的Ca^<2+>。为了研究DGK同工酶的三组EF-手形模体的内在性质，我们在细菌中表达并纯化了两个连续的EF-手形模体。用平衡透析法对钙离子结合能力的分析表明，所有三个EF-手区域每摩尔都结合了大约2摩尔的Ca^2+。钙离子与三种同工酶结合的表观解离常数（Kd）明显不同。在碱性尿素-聚丙烯酰胺电泳中，所有EF-手区域在Ca^2+结合后的构象变化被检测为迁移率的变化。这些结果正准备提交。

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.