Physiological function of lipid-phosphorylating enzymes and their mechanism of action.

脂质磷酸化酶的生理功能及其作用机制。

• 批准号: 61570119
• 负责人: KANOH Hideo
• 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: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-61570119/
• 关键词: Diacylglycerol; Diacylglycerol kinase; Protein phosphorylation; Protein kinase C; プロティンキナーゼC; 抗体; 蛋白リン酸化; C-キナーゼ

In the agonist-stimulated cells, diacylglycerol(DG) kinase plays a central role in the metabolism of a second messenger, DG, which is released from phosphoinositides upon receptor activation. We intend to characterize this lipid kinase by employing purified enzymes and rabbit antibody raised against 80 kDa DG kinase. The projects and major findings are summarized as follows:1) Immunochemical Studies on DG Kinase in Different Pig Tissues DG kinase from different pig tissues was examined by immunoblot as well as immunoprecipitation. Among the tissues examined, the immunoreactive 80 kDa kinase previously purified by us from the brain(J. Biol. Chem. 258, 1767, -83) was found only in the thymus and, to a much lesser extent, in the spleen. Other tissues like platelets,heart, kidney and liver, contained little, if any, immunoreactive enzymes. A gel filtration of cytosolic enzymes showed three major activity peaks, corresponding apparently to 280, 120 and 80 kDa. The platelet kinase was, inter … More estingly, consisted almost exclusively of nonimmunoreactive 120 kDa species with an apparent lack of immunoreactive 80 kDa kinase. The nonimmunoreactive 280 kDa kinase was abundant in the lymphoid tissues, and was much more heat-stable when compared with the 80 kDa enzyme. Purification of different kinase forms is required to elucidate their physiological implication in each tissur.2) In vitro Phosphorylation of DG Kinase by Protein Kinase C DG kinase and protein kinase C(PKC) were highly purified from pig thymus. Since both enzymes showed the same mobility(80 kDa) upon SDS-PAGE, the phosphorylation of DGK had to be confirmed by immunoprecipitation with anti-DGK antibody. We found that DGK serves as a good substrate for PKC, and that almost stoichiometric phosphorylation of DGK was achieved. Phosphorylation of DGK by PKA occurred to a very limited extent. Phosphorylated DGK showed a broad band around pI 6.5. The two protein kinases phosphorylated different sites of DGK molecule. The Vmax of partially phosphorylated DGK increased approximately two-fold. Both DGK and PKC were bound to phosphatidylserine in the system. Less

在激动剂刺激的细胞中，二酰基甘油（DG）激酶在第二信使DG的代谢中起着重要作用，DG在受体活化后从磷酸肌醇释放。我们打算采用纯化的酶和兔抗体提出对80 kDa DG激酶的特点，这种脂质激酶。主要研究内容和结果如下：1）猪不同组织DG激酶的免疫化学研究采用免疫印迹和免疫沉淀法对猪不同组织DG激酶进行了研究。在检查的组织中，我们以前从脑中纯化的免疫反应性80 kDa激酶（J. Biol. Chem. 258，1767，-83）仅在胸腺中发现，在脾脏中发现的程度要小得多。其他组织，如血小板、心脏、肾脏和肝脏，几乎不含免疫反应酶。细胞溶质酶的凝胶过滤显示出三个主要的活性峰，显然对应于280，120和80 kDa。血小板激酶， ...更多信息 几乎完全由非免疫反应性的120 kDa物质组成，明显缺乏免疫反应性的80 kDa激酶。非免疫反应性的280 kDa激酶是丰富的淋巴组织中，更热稳定时相比，80 kDa的酶。需要纯化不同形式的激酶以阐明它们在每个组织中的生理意义。2）蛋白激酶C对DG激酶的体外磷酸化从猪胸腺中高度纯化DG激酶和蛋白激酶C（PKC）。由于两种酶在SDS-PAGE上显示出相同的迁移率（80 kDa），因此必须通过用抗DGK抗体进行免疫沉淀来确认DGK的磷酸化。我们发现，DGK作为一个很好的底物PKC，并实现了几乎化学计量的磷酸化DGK。PKA对DGK的磷酸化程度非常有限。磷酸化的DGK在pI 6.5附近显示一条宽带。这两种蛋白激酶磷酸化DGK分子的不同位点。部分磷酸化的DGK的Vmax增加约两倍。DGK和PKC均与磷脂酰丝氨酸结合。少

项目成果

加納,小野: FEBS Lett.201. 97-100 (1986)

卡诺，小野：FEBS Lett.201。

Yamada, K. and Kanoh, H.: "Occurrence of immunoreactive 80 kDa and nonimmunoreactive diacylglycerol kinases in different pig tissues." Biochem. J.

Yamada, K. 和 Kanoh, H.：“不同猪组织中免疫反应性 80 kDa 和非免疫反应性二酰甘油激酶的出现。”

Kanoh, H. and Yamada, K.: "In vitro phosphorylation of diacylglycerol kinase by protein kinase C." Biochem. J.

Kanoh, H. 和 Yamada, K.：“蛋白激酶 C 对二酰甘油激酶的体外磷酸化”。

山田恵子: Biochem.J.

山田惠子：Biochem.J。

Kanoh, H. and Ono, T.: "Phosphorylation of pig brain diacylglycerol kinase by endogenous protein kinase." FEBS Lett.201. 97-100 (1986)

Kanoh, H. 和 Ono, T.：“内源性蛋白激酶对猪脑二酰甘油激酶的磷酸化”。