STRUCTURE AND REGULATION OF PHOSPHOPROTEIN PHOSPHATASES

磷酸蛋白磷酸酶的结构和调控

• 批准号: 3235014
• 负责人: ANNA A DEPAOLI-ROACH
• 金额: $ 9.52万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1989-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3235014
• 关键词: antibody; complementary DNA; diaphragm; enzyme complex; enzyme structure; epinephrine; gel electrophoresis; genetic manipulation; hormone regulation /control mechanism; insulin; liver metabolism; muscle metabolism; phosphomonoesterases; phosphorylation; starvation

Covalent phosphorylation of proteins plays a central role in the control of many cellular processes by hormones such as insulin and epinephrine. Part of this regulation involves the enzymes responsible for dephosphorylating proteins, the phosphoprotein phosphatases. This project addresses one of the main classes of protein phosphatase found in cells, what will be termed "type 1" phosphatase (one characteristic of such enzymes is that their catalytic subunit is inhibited by the heat stable phosphatase inhibitor proteins, inhibitor-1 and inhibitor-2). Knowledge of the structure and regulation of these enzymes is incomplete. A primary objective will be to establish the number and structures of the native forms of this class of enzyme. Methods to be applied will include (1) conventional protein purification, (2) immunological methods, and (3) recombinant DNA techniques. Purified high molecular weight forms of type 1 phosphatase will be characterized and compared with known enzymes of simpler structure, namely the isolated catalytic subunit, C1, and the ATP-mg-dependent phosphatase. The functional roles of non-catalytic subunits will be investigated, particularly in relation to control of enzyme activity. Special attention will be paid to phosphorylation of phosphatases since there is good reason to believe that phosphorylation of the inhibitor-2 component of the ATP-Mg-dependent phosphatase is related to its activation. Possible controls mediated by small molecules will be evaluated. Models for the short-term intracellular control of phosphatase activity will employ mouse diaphragms exposed to insulin and/or epinephrine. Immunological methods will be applied to analyze the phosphorylation of inhibitor-2, and other phosphatase components, as well as phosphatase activity. Success in this investigation can have widespread importance since this class of phosphatase is likely to be involved in the control of many cellular processes. Any mechanistic link to the action of hormones, especially insulin, will be of particular interest in understanding how insulin functions normally. Impaired phosphatase, however, could be a determining factor in some types of diabetes.

蛋白质的共价磷酸化在蛋白质的磷酸化过程中起着重要的作用。 胰岛素和肾上腺素等激素对许多细胞过程的影响。 部分 这种调节涉及负责去磷酸化的酶 蛋白质，磷蛋白磷酸酶。 该项目解决了 在细胞中发现的蛋白磷酸酶的主要类别，将被称为 “1型”磷酸酶（此类酶的一个特征是它们的磷酸酶活性是1型）。 催化亚基被热稳定磷酸酶抑制剂抑制 蛋白质，抑制剂-1和抑制剂-2）。 结构知识和 这些酶的调节是不完全的。 主要目标是 建立这一类的原生形式的数量和结构， 酵素 应用的方法将包括（1）常规蛋白质 纯化，（2）免疫学方法，和（3）重组DNA 技术. 纯化的高分子量形式的1型磷酸酶 将被表征并与结构更简单的已知酶进行比较， 即分离的催化亚基，C1，和ATP-mg依赖的 磷酸酶。 非催化亚基的功能作用将是 研究，特别是关于酶活性的控制。 将特别注意磷酸酶的磷酸化， 有充分的理由相信抑制剂-2的磷酸化 ATP-Mg依赖性磷酸酶的组分与其 activation. 由小分子介导的可能的控制将是 评估。 磷酸酶的短期细胞内控制模型 活动将使用暴露于胰岛素和/或 肾上腺素 免疫学方法将用于分析 抑制剂-2和其它磷酸酶组分的磷酸化，以及 磷酸酶活性。 这次调查的成功可能会对 重要性在于这类磷酸酶可能参与 控制许多细胞过程。 任何机械联系的行动， 激素，特别是胰岛素，将特别感兴趣， 了解胰岛素的正常功能。 磷酸酶受损， 然而，这可能是某些类型糖尿病的决定性因素。