Development of detection system for signals triggered by hydrolyzing reactions in intracellular compartment

细胞内水解反应触发信号检测系统的开发

• 批准号: 07558213
• 负责人: EMORI Yasufumi
• 金额: $ 2.18万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558213/
• 关键词: hydroysis; intracellular; calpain; phospholipase C; calcium; Drosophila; sigralling; development; ホスホリパーゼ; シグナル伝達

Degradative pathways of proteins and lipids are important as biological processes. However, knowledges about the processes are less than those for the sunthetic pathways of these biological compounds. In this study, we tried to establish the in vivo detection systems for reactions catalyzes by calpain and phosphoinositede-specific phospholipase C (PLC), which we had studeid by biochemical and molecular biological methods. We have developed the methods that detected expression of the activties of the enzymes and the results of the reaction catalyzed by the enzymes, using Drosophila early embryos and specific antibodies. First, we analyzed the localization of these enzymes during very early embryogenesis using anti-calpain and anti-PLC antibodies. Second, we prepared a specific antibody for the cleavage site that was generated by the hydrolytic reaction by calpain. Third, we have developed a system which made it possible to detect calpain, PLC,and their substrates during the embryogenesis. In addition, we have also analyzed the function of p122, which is a dual mediator between PLC and the cytoskeleton which is an activator of PLC.

蛋白质和脂质的降解途径是重要的生物过程。然而，对这些生物化合物的合成途径的了解还不如对这些过程的了解多。本研究通过生物化学和分子生物学的方法，尝试建立钙蛋白酶和磷酸肌醇特异性磷脂酶C（PLC）催化反应的体内检测系统。我们已经建立了利用果蝇早期胚胎和特异性抗体检测酶活性表达和酶催化反应结果的方法。首先，我们使用抗钙蛋白酶和抗PLC抗体分析了这些酶在非常早期胚胎发生过程中的定位。其次，我们制备了针对由钙蛋白酶水解反应产生的切割位点的特异性抗体。第三，我们已经开发了一个系统，它使得有可能检测钙蛋白酶，PLC，和他们的基板在胚胎发生。此外，我们还分析了p122的功能，它是PLC和细胞骨架之间的双重介质，而细胞骨架是PLC的激活剂。

项目成果

A.S.Arora,P.D.Goen,D.E.Croall,Y.Emori & G.J.Gores: "Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation" Journal of Cellular Physiology. 167. 434-442 (1996)

A.S.阿罗拉、P.D.戈恩、D.E.克罗尔、Y.Emori

J.R.Begette,Y.Emori & D.L.Mykles: "Immunological analysis of two calpain-like Ca2_+-dependent proteinases from lobster striated muscles : relationship to mammalian and Drosophila calpains" Archi ves of Biochemistry and Biophysics. 337. 232-238 (1997)

J.R.贝盖特,Y.Emori