BIOCHEMISTRY OF PLATELET PHOSPHOLIPASE C

血小板磷脂酶 C 的生物化学

基本信息

批准号：
3357799
负责人：
Charles O Rock
金额：
$ 6.73万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-04-01 至 1991-03-31
项目状态：
已结题

项目摘要

There is compelling evidence that the generation of diacylglycerol and inositol 1,4,5-trisphosphate is intimately associated with stimulus-response coupling in a wide variety of cell systems. Diacylglycerol enhances the phosphorylation of target proteins by the activation of protein kinase C, and inositol 1,4,5-trisphosphate causes the mobilization of calcium from intracellular stores, thereby switching on a host of calcium-regulated functions. The platelet system represents one of the clearest examples of the involvement of phosphatidylinositol catabolism in cell physiology. A number of platelet activating agents trigger the generation of phosphatidylinositol-derived second messengers followed by an increase in the intraplatelet calcium concentration and protein phosphorylation. This process is catalyzed by a receptor- regulated phsophatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C that is modulated by guanine nucleotides and/or stimulatory ligands. At present there is little biochemical information that corroborates this important signal transduction hypothesis. The putative G-protein that interacts with phospholipase C has not been positively identified or purified and there is considerable speculation about its molecular structure. There is also evidence for G-protein-independent mechanisms for phospholipase C regulation. The phospholipase C activity modulated by G-proteins, receptors, or ligands has not been attributed to a purified protein. This proposal focuses on defining the biochemical mechanisms that regulate PtdIns-P2 phospholipase C activity and developing immunological reagents that will be used to test for the involvement of our purified phospholipase C's in ligand-initiated PtdIns-P2 hydrolysis. Our preliminary results indicate that membrane-associated and cytosolic platelet PtdIns-P2 phospholipase C's are allosteric enzymes which are activated by both homotropic (substrate) and heterotropic (phosphatidic acid) agents. The specific aims are: (1) to purify and characterize platelet phospholipase C's that degrade polyphosphoinositides and determine the relationship between the soluble and membrane-associated forms of the enzyme, and (2) to define the mechanism(s) of substrate, heterotropic activator, receptor and G-protein regulation of platelet PtdIns-P2 phospholipase activity. The results of this work will contribute important new information to our biochemical understanding of phospholipase C regulation in stimulus-response coupling.

有令人信服的证据表明二酰基甘油的生成肌醇 1,4,5-三磷酸与多种细胞系统中的刺激-反应耦合。二酰基甘油通过以下方式增强靶蛋白的磷酸化蛋白激酶 C 和肌醇 1,4,5-三磷酸的激活引起细胞内储存的钙的动员，从而开启一系列钙调节功能。这血小板系统是最明显的例子之一细胞生理学中磷脂酰肌醇分解代谢的参与。许多血小板活化剂会引发血小板生成磷脂酰肌醇衍生的第二信使，随后是血小板内钙浓度和蛋白质增加磷酸化。这个过程是由受体催化的调节磷脂酰肌醇 4,5-二磷酸 (PtdIns-P2) 由鸟嘌呤核苷酸调节的磷脂酶C和/或刺激配体。目前生化研究还很少证实这一重要信号转导的信息假设。假定的 G 蛋白与磷脂酶 C 尚未被明确鉴定或纯化，并且关于其分子结构有很多猜测。还有证据表明 G 蛋白独立机制磷脂酶 C 调节。磷脂酶C活性尚未被 G 蛋白、受体或配体调节归因于纯化的蛋白质。该提案的重点是定义调节 PtdIns-P2 的生化机制磷脂酶C活性和开发免疫试剂这将用于测试我们纯化的参与配体引发的 PtdIns-P2 水解中的磷脂酶 C。我们的初步结果表明膜相关和胞质血小板 PtdIns-P2 磷脂酶 C 是变构的被同向性（底物）和同向性（底物）激活的酶异变性（磷脂酸）剂。具体目标是： (1) 纯化并表征血小板磷脂酶 C 降解多磷酸肌醇并确定其关系可溶性形式和膜相关形式之间酶，以及 (2) 定义底物的机制，异向激活剂、受体和 G 蛋白调节血小板 PtdIns-P2 磷脂酶活性。这样做的结果工作将为我们提供重要的新信息磷脂酶 C 调节的生化理解刺激-反应耦合。