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

二酰甘油激酶同工酶对细胞增殖和分化的作用

基本信息

批准号：
06680620
负责人：
YAMADA Keiko
金额：
$ 1.47万
依托单位：
Sapporo Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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.

1)二酰基甘油激酶溶酶（DGK）在细胞增殖和分化中的作用。TPA、DMSO和视黄酸处理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DGKalpha和DGKgamma表达水平升高。2)免疫细胞二酰基甘油激酶脱菌酶的作用：以jurkat细胞和lkt9 （EBV转化的B细胞）为研究对象。我们发现在LKT 9细胞中有大量的DGKgamma表达。目前正在研究这些酶在受抗体刺激后的变化。3)磷脂酸形成对DGKalpha表达的影响。过表达DGKalpha的NIH3T3细胞生长速度快于野生型细胞。PDGF的刺激导致内源性DG生成磷脂酸、向溶血磷脂酸转化以及细胞相关DG质量的改变。4)DGK同工酶（DGKs α、b和γ）中EF-hand基序的性质目前克隆的3个DGKs α、β和γ均含有EF-hand基序的串联重复序列。β -同工酶和γ -同工酶的活性与Ca^<2+>的添加量无关，而DGKalpha的活性则完全依赖于Ca^<2+>的微摩尔浓度。为了研究DGK同工酶的三组EF-hand基序的内在性质，我们通过细菌表达和纯化了两个连续的EF-hand基序。平衡透析法对钙结合能力的分析表明，3个EF-hand区每mol约结合2 mol Ca^<2+>，钙与3种同工酶的表观解离常数（Kd）有明显差异。在碱性尿素-聚丙烯酰胺电泳中检测到所有EF-hand区域在Ca^<2+>结合时的构象变化。这些结果正准备提交。