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

二酰甘油激酶溶菌酶对细胞功能表达的作用。

基本信息

批准号：
03670129
负责人：
YAMADA Keiko
金额：
$ 1.28万
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：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

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

1. 鞘氨醇对完整jurkat细胞DGK同工酶的影响。Sphingoshine是纯化的80 kDa DGK的激活剂，在完整Jurkat细胞中显著激活内源性和外源性dgg的磷酸化。我们认为鞘氨醇增加磷脂酸（PA）积累可能是直接激活Jurkat细胞中富集的80 kDa DGK。但最近有人提出，多重同工酶参与了胞内dg的磷酸化。因此，我们重新评估了Jurkat细胞中同工酶的组成，使用不同的dg（花生四烯酰基和二癸醇基dg）作为底物和两种不同的测定方法（辛基糖苷胶束法和脱氧胆酸悬浮液法）。结果表明，至少存在四种DGK同工酶，它们在细胞内定位、对DG分子种类的特异性、对鞘氨醇的反应性和对抗8…More 0 kDa DGK抗体的反应性方面可以相互区分。我们现在正在研究DGK在HL 60.2分化中的作用。80 kDa DG激酶的一级结构。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手基序。我们发现纯化后的DGK每mol酶结合2 mol Ca^<2+>具有高亲和力（J. Biol.）。化学。266,7096)。为了阐明E-F手基序的调控功能，我们在e.c coil和COS-7细胞中构建了几个表达的E-F手基序的截断和缺失突变体。Biophys。《共同法典》，181,1015)。此外，我们还利用杆状病毒表达系统（正在制备中）研究了其与DG、磷脂、磷脂酯或DNA的结合。其他DGK同工酶的初级结构。我们无法获得编码150kda DGK的cDNA克隆。制备了80 ~ 90 kDa（神经细胞特异性）DGK高同源性的寡核苷酸，并用于聚合酶链反应。扩增的DNA片段随后用于克隆DGK同工酶cdna。少