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

我们已经克隆了编码猪80K二酰基甘油激酶(DGK)的基因，从而证明了一种同时具有EF-手和锌手指的新型酶的存在(Sakane，Natural，1990)。基于这一发现，我们主要从分子遗传学的角度对该酶的结构与功能关系进行了研究，结果表明纯化的酶确实是一种高亲和力的钙结合蛋白。在没有Ca^&lt；2&gt；的情况下，EF-Hand区通过掩盖该酶的一个假定的磷脂酰丝氨酸结合位点(S)而起到自抑制结构域的作用。在Ca^&lt；2&gt；结合后，EF-HAND发生构象变化，导致磷脂结合部位暴露(S)。(Sakane，JBC，1991，Sakane，BBRC，1991)。这使得我们能够通过原位杂交和免疫染色来详细研究酶的表达方式。80K DGK及其基因在T淋巴细胞中高表达，而在中枢神经系统中仅在少突胶质细胞中表达。M RNA的形成方式与髓鞘组织的成熟有关。我们目前正在以80K DGK基因为探针，克隆神经元特异性DGK基因的编码基因。(后藤健二提交)。在这方面，从人血小板中纯化了三种DGK同工酶，它们被证明与80K同工酶(Yada，JBC，1990)不同。目前，人们正在利用已经克隆的人类基因来研究80K酶的转录调控机制。通过在杆状病毒/Sf9细胞系统中表达突变的锌指蛋白来研究锌指蛋白的功能。用免疫细胞化学方法研究了T淋巴细胞中80K酶的功能。

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.