STRUCTURE AND FUNCTION OF PHOSPHOLIPASE C ENZYMES

磷脂酶 C 酶的结构和功能

• 批准号: 2187564
• 负责人: PAUL C STERNWEIS
• 金额: $ 21.88万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187564
• 关键词: G protein; chimeric proteins; enzyme activity; enzyme structure; isozymes; laboratory rabbit; phospholipase C; phospholipase inhibitor; protein structure function; radionuclides; synthetic enzyme; tissue /cell culture; ultracentrifugation

The regulation of phosphatidylinositol-specific phospholipase C (PIPLC) activity by hormones controls the evolution of two second messengers, IP3 and diacylglycerol, and their subsequent effects on intracellular free Ca2+ and the activity of protein kinase C. G proteins of the Gq subfamily have been identified as mediators of PLCbeta1 activity. We have extended this regulation to two other isotypes of PLCbeta, PLCbeta2 and PLCbeta3. All three isotypes of PIPLC can be activated to different extents by both alpha-q and beta-gamma subunits. This application is designed to extend these studies in three major directions. First, we propose to determine the structural determinants of the PLCbeta enzymes that are important for their .regulation by G proteins and action in general. This will be done through expression and characterization of mutant proteins with a Baculovirus expression system. Second, we plan to search for other PIPLC enzymes that respond to alpha-q and/or beta-gamma subunits. This will include experiments with crude preparations from tissues and cells as well as characterization of expressed PLCdelta isoforms. Finally, initial evidence suggests that the purified PLCbeta3 contains an inhibitory subunit. Plans are presented to verify this observation and to characterize the inhibitory protein. We also propose to determine if other putative inhibitory subunits exist for PLCbeta1 and PLCbeta2. This work will increase our understanding of G protein dependent regulation of intracellular free Ca2+ and protein phosphorylation by hormones. A better understanding of the mechanisms of PIPLC regulation will help in the development of better tools to manipulate their function. The identification of specific inhibitory subunits for PLCbeta isotypes would establish a potentially powerful tool for dissecting the roles of these individual enzymes in intracellular regulation.

磷脂酰肌醇特异性磷脂酶C的调控