IMAI Shin-ichi Sapporo Medical College, Faculty of medicine, Research Associate, 医学部, 助手 (20213209)
SAKANE Fumio Sapporo Medical College, Faculty of medicine, Research Associate, 医学部, 助手 (10183815)
YAMADA Keiko Sapporo Medical College, Faculty of medicine, Assistant Professor, 医学部, 講師 (80045541)
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 o
ligodendrocytes. 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.
ラット80Kキナ-ゼcDNAをクロ-ン化し、in situ hybridizationと免疫組織染色を行なった(投稿中)。本酵素はTーリンパ球に大量に発現しているが、脳ではoligo dendrocytesに限局して発現しており、ミエリンの成熟度に一致した発現調節を受けることが分かった。
80K酵素のヒト遺伝子に関しては、転写開始点を決定し、5'領域のプロモ-タ-活性をCAT assayにより検討している。亜鉛フィンガ-の機能を調べるために、点変異導入酵素をBaculovirus/Sf9細胞で発現中である。Tーcell hybridoma刺激時のDGキナ-ゼの動態を免疫的手法で検討中である。 Less