REGULATION OF PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE C

磷酸肌醇特异性磷脂酶 C 的调节

• 批准号: 3467968
• 负责人: Mario J Rebecchi
• 金额: $ 9.94万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3467968
• 关键词: calcium binding protein; calcium channel; crosslink; diacylglycerols; enzyme induction /repression; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; erythrocyte membrane; human tissue; isozymes; laboratory rabbit; membrane activity; membrane lipids; membrane model; membrane structure; molecular site; monoclonal antibody; phosphatidylinositols; phospholipase C

The phosphoinositide-specific phospholipase C (PI-PLC) isozymes comprise a family of proteins which catalyze the cleavage of phosphoinositides to yield the products diacylglycerol (DG) and the corresponding inositol phosphate headgroup. These enzymes play an essential role in the regulation of a wide variety of biological processes ranging from exocytosis to cell division (1-2). We propose to explore the role of the membrane/solution interface in regulation of the PI-PLC isozymes. We propose that the PI-PLC isozymes possess an interfacial recognition site or region, analogous to extracellular lipases, that is essential for stable association with the substrate surface and is distinct from the catalytic site. It is this region that is key to control to these intracellular lipases by calcium-mobilizing receptors. The immediate goals of this study are as follows:1)Experiments to separate and identify both the interfacial recognition region and the catalytic site will be performed. A combination of site-specific chemical reagents and suicide substrate inhibitors will be employed to label the active site. The interfacial recognition region will be tagged using lipid soluble, photoreactive crosslinking reagents. The activity of chemically modified PI-PLC and fragments of this enzyme towards the monomeric PtdIns analogs and PtdIns aggregates will serve as a method of functionally dissecting the catalytic site from an interfacial recognition region. Mapping of these sites using both monoclonal antibodies directed against the protein and sequence-specific antipeptide antibodies is also proposed. 2)We will perform steady state kinetic experiments using molecularly disperse PtdIns analogs. These studies will allow us to separate the effects of calcium, diacylglycerol and phospholipid modulators upon catalysis from effects on the enzyme's interaction with aggregated substrate surfaces. 3)We will study the interaction of the 85 kDa PI-PLC isozyme with artificial phospholipid vesicles and the membranes of human red blood cells. Our experiments are designed to test the hypothesis that the purified PI-PLC can act at the membrane surface in the "scooting" mode through an interfacial recognition site on the protein, provided that sufficient calcium is available. We will use the kinetics of intervesicle exchange as well as direct binding methodologies to determine the requirements and dynamics of this PLC isozyme.

磷酸肌醇特异性磷脂酶C（PI-PLC）同工酶构成A 蛋白质家族催化磷酸肌醇的裂解至 产生产物二酰基甘油（DG）和相应的肌醇 磷酸盐头组。 这些酶在 对各种生物学过程的调节 胞吞作用到细胞分裂（1-2）。 我们建议探索 PI-PLC同工酶调节中的膜/溶液界面。 我们建议PI-PLC同工酶具有界面识别位点 或类似于细胞外脂肪酶的区域，这对于稳定至关重要 与底物表面相关，与催化不同 地点。 控制这些细胞内的是该区域的关键 脂肪酶通过钙润滑受体。 这项研究的直接目标如下：1）分开的实验 并确定界面识别区域和催化位点 将执行。 特定地点的化学试剂和 自杀底物抑制剂将用于标记活性部位。 界面识别区域将使用脂质可溶性标记 光电反应性交联试剂。 化学修饰的活性 PI-PLC和该酶朝向单体Ptdins类似物的片段 PTDINS聚集体将作为功能剖析的一种方法 来自界面识别区域的催化位点。 这些映射 使用两种针对蛋白质的单克隆抗体和 还提出了序列特异性的抗肽抗体。 2）我们会的 使用分子分散PTDINS进行稳态动力学实验 类似物。 这些研究将使我们能够将钙的影响分开 二酰基甘油和磷脂调节剂在催化后对影响的影响 该酶与聚集的底物表面的相互作用。 3）我们会的 研究85 kDa Pi-Plc同工酶与人工的相互作用 磷脂囊泡和人类红细胞的膜。 我们的 实验旨在测试纯化的PI-PLC的假设 可以通过“踏板”模式在膜表面作用 只要足够 钙可用。 我们将使用互换的动力学作为 以及直接的约束方法，以确定要求和 该PLC同工酶的动力学。