Role of Diacylglycerol Kinase lsozymes on the Expression of Cell Function.

1.鞘氨醇对完整Jurkat细胞DGK同工酶的影响纯化的80 kDa DGK的激活剂Sphingolight显著激活了Jurkat细胞内源性和外源性DGs的磷酸化。我们认为鞘氨醇增加磷脂酸(PA)的积累可能是对Jurkat细胞中丰富的80 kDa DGK的直接激活。但最近发现，多种同工酶参与了胞内DGs的磷酸化。因此，我们以不同的DGs(花生四烯酸基和十二酰DGs)为底物，采用两种不同的测定方法(辛基葡萄糖苷胶束法和脱氧胆酸悬浮法)，对Jurkat细胞的同工酶组成进行了重新测定。结果表明，至少存在4种dgk同工酶，它们在细胞内定位、对dg分子物种的特异性、对鞘氨醇的反应性和对抗8…的反应性等方面可以相互区分。更多0 kDa DGK抗体(已提交)。我们正在研究DGK在HL 60.2分化过程中的作用。该酶的一级结构包含假定的ATP结合位点、两个富含半胱氨酸的锌指状序列和两个E-F手型基序。我们发现，纯化的DGK以高亲和力结合每摩尔酶2摩尔的钙离子(J.Biol)。化学。266,7096)。为了阐明E-F手序的调控功能，我们构建了该酶在E.Coil或COS-7细胞中表达的几个截断和缺失突变体(Biochem.生物群落。[中英文摘要]Re.Commun.181,1015)。此外，我们还利用杆状病毒表达系统研究了DG、磷脂、佛波酯或DNA的结合情况。其他DGK同工酶的一级结构。我们无法获得编码150 kDa DGK的cDNA克隆。目前已有80 kDa至90 kDa(神经细胞特异性)DGK同源性较高的寡核苷酸被制备出来，并用于聚合酶链式反应。扩增的DNA片段随后用于克隆DGK同工酶的cDNA。较少

1. Effect of sphingosine On Diacylglyceriol kinase(DGK) isozymes in intact jurkat cells.Sphingoshine which is the activator of purified 80 kDa DGK markrdly activated the phosphorylation of both endogenous and exogenous DGs in intact Jurkat cells. We thought that the increased phosphatidic acid(PA) accumulation by sphingosine may be a direct activation of 80 kDa DGK which was enriched in Jurkat cells. But recently, it was suggested that the plural isozymes contributed the phosphorylation of intracellular DGs. So we reevaluated the isozyme composition in Jurkat cells using combination of different DGs (arachidonoyl-and didecanoyl DGs) as the substrate and two different assay methods (octylglucoside micellar and deoxycholate suspension assays). The results suggested the presence of at least four DGK isozymes, which could be distinguwished from each other with respect to intracellular localization, specificity to DG molecular species, responsiveness to sphingosine, and reactivity to anti-8 … More 0 kDa DGK antibody(in submitted)., we are now examining the role of DGK during differenciation of HL 60.2. Primary structure of 80 kDa DG kinase.Primary structure of this enzyme contains the putative ATP-binding sites, two cysteine-rich zinc finger-like sequences and two E-F hand motifs. We found that the purified DGK binds 2 mol of Ca^<2+> per mol of enzyme with high affinity (J. Biol. Chem. 266, 7096). And to elucidate the reguratory function of E-F hand motifs, we constructed the expressed several truncation and deletion mutants of the enzyme in E.Coil or COS-7 cells(Biochem. Biophys. Res. Commun. 181, 1015). Moreover, we investigated the binding for DG, phospholipids, phorbol ester, or DNA using baculovirus expression system(in preparation).3. Primary structure of other DGK isozymes.We could not get the cDNA clone coding for 150 kDa DGK. And now oligonucleotides with high homology between 80 kDa and 90 kDa (nerve cell specific) DGK were prepared and used in the polymerase chain reaction. Amplified DNA fragments was subsequently used to clone the DGK isozyme cDNAs. Less