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The function of DG kinase, a lipid phosphorylating enzyme having both EF-hands and zinc fingers.

DG 激酶的功能，一种具有 EF 手和锌指的脂质磷酸化酶。

基本信息

批准号：
02454157
负责人：
KANOH Hideo
金额：
$ 0.51万
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 (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

We have cloned cDNA encoding porcine 80K diacylglycerol kinase (DGK), thus demonstrating the presence of a novel enzyme having both EF-hands and zinc fingers (Sakane, Nature, 1990). Based on this finding we studied the structure-function relationship of this enzyme using mainly molecular genetics.We showed that the purified enzyme is indeed a high affinity Ca^<2+>-binding protein. In the absence of Ca^<2+> the EF-hand region serves as an autoinhibitory domain by masking a putative phosphatidylserine binding site (s) of this enzyme. Upon Ca^<2+> binding the EF-hands undergoes conformational change that results in the exposure of phospholipid binding site(s). (Sakane, JBC, 1991, Sakane, BBRC, 1991).We cloned cDNA of the rat homolog of the 80K DGK. This enabled us to study the mode of enzyme expression in detail by in situ hybridization and immunostaining. The 80K DGK and its gene were highly expressed in T-lymphocytes, while in the central nervous system their expression is confined to oligodendrocytes. The mode of mRNA formation was linked to the maturation of myelin tissues. We are currently cloning cDNA coding for neuron-specific DGK using the 80K DGK cDNA as a probe. (Goto, submitted). In this respect, three DGK isozymes were purified from human platelets, and they were shown to be distinct from the 80K isozyme (Yada, JBC, 1990).At present the transcriptional control mechanism of the 80K enzyme is being studied using human gene already cloned. The function of zinc finger is also studied by expressing the mutated enzyme in Baculovirus/Sf9 cell system. The function of the 80K enzyme in T-lymphocytes is investigated by immunocytochemical methods.

我们已经克隆了编码猪80 K二酰基甘油激酶（DGK）的cDNA，从而证明了具有EF-手和锌指的新型酶的存在（Sakane，Nature，1990）。基于这一发现，我们主要用分子遗传学方法研究了该酶的结构-功能关系，结果表明，纯化的酶确实是一种高亲和力的Ca^<2+>结合蛋白。在缺乏Ca^2+的情况下，EF-手区通过掩蔽该酶的假定磷脂酰丝氨酸结合位点而充当自身抑制结构域。在与Ca^2+结合后，EF-手发生构象变化，导致磷脂结合位点暴露。（Sakane，JBC，1991，Sakane，BBRC，1991）。我们克隆了80 K DGK的大鼠同源物的cDNA。这使我们能够通过原位杂交和免疫染色详细研究酶的表达模式。80 K DGK及其基因在T淋巴细胞中高度表达，而在中枢神经系统中其表达仅限于少突胶质细胞。mRNA的形成模式与髓鞘组织的成熟有关。我们目前正在克隆cDNA编码的神经元特异性DGK使用80 K DGK cDNA作为探针。(Goto，已提交）。在这方面，从人血小板中纯化了三种DGK同工酶，它们显示出与80 K同工酶不同（Yada，JBC，1990）。通过在杆状病毒/Sf 9细胞系统中表达突变后的锌指蛋白，研究了锌指蛋白的功能。用免疫细胞化学方法研究了T淋巴细胞中80 K酶的功能。