BIOCHEMISTRY OF PLATELET PHOSPHOLIPASE C

血小板磷脂酶 C 的生物化学

• 批准号: 3357797
• 负责人: Charles O Rock
• 金额: $ 7.23万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/3357797
• 关键词: antibody; biological information processing; biological signal transduction; chemical structure function; diacylglycerols; electrofocusing; enzyme induction /repression; enzyme mechanism; enzyme structure; enzyme substrate; gel electrophoresis; gel filtration chromatography; guanine nucleotides; human tissue; hydrolysis; ion exchange chromatography; membrane proteins; molecular biology; phosphatidate; phosphatidylinositols; phospholipase C; phosphorylation; platelets; receptor; solubility; stimulus /response; thrombin; vasopressins

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。