CATALYTIC COMPETENCE AND REGULATION OF RECEPTOR KINASE

受体激酶的催化能力和调节

• 批准号: 3233520
• 负责人: JULES Alan SHAFER
• 金额: $ 8.52万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-01 至 1988-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3233520
• 关键词: catalyst; chemical reaction; conformation; enzyme substrate; gel electrophoresis; glucose transport; high performance liquid chromatography; human tissue; immunochemistry; phosphorylation; protein tyrosine kinase; stoichiometry; zinc

The intact phosphorylated and unphosphorylated forms of insulin receptor kinase will be purified from human placenta by chromatography on immobilized anti-O-phosphotyrosyl antibody. The purified forms of the receptor will be used to characterize the multistep reaction pathway for the conversion of the unphosphorylated form of the receptor to the catalytically active phosphorylated form. In these studies the amino acid sequence around the reactive tyrosyl residue(s) in the receptor will be determined and compared to that of other tyrosine kinases. Rate and equilibrium constants will be evaluated for individual steps in the reaction pathway for receptor activation. Stoichiometry and equilibirum constants for the binding of insulin to the phosphorylated and unphosphorylated forms of the receptor will be measured to determine whether insulin binding might cause the unphosphorylated receptor to assume a conformation similar to that of the phosphorylated receptor. The inhibitory effect of zinc ion on individual steps in the reaction pathway for receptor activation will be characterized, since inhibition of autophosphorylation of the insulin receptor by zinc ion might serve to protect the islet cells and other tissues from deleterious effects of high concentrations of insulin at the site of normal or accidental release of the contents of insulin-containing granules. Another set of studies will be directed toward characterization of interactions of the receptor kinase with potential regulators of metabolism. Thus, the possibility will be assessed that insulin receptor kinase catalyzes phosphorylation of the glucose transporter protein and thereby decreases its exit rate from plasma membranes. The phosphorylation of insulin receptor by casein kinase I will be studied to determine whether casein kinase I catalyzed phosphorylation of insulin receptor alters its ability to bind insulin and undergo autophosphorylation, and whether casein kinase I catalyzed phosphorylations of the receptor might account for the phosphorylation at receptor seryl and thereonyl residues seen in whole cells. In a search for endogenous substrates for the insulin receptor kinase, anti-O-phosphotyrosyl antibody will be used to concentrate products of insulin promoted tyrosyl phosphorylations in whole cells. In an attempt to deduce the structural characteristics of natural substrates for the insulin receptor kinase, the substrate specificity of this enzyme toward nonpeptide and peptide substrates will be determined.

完整的磷酸化和非磷酸化形式的胰岛素受体 从人胎盘中通过层析从人胎盘中提纯激酶 固定化抗O-磷酸酪氨酰抗体。纯净形式的 受体将被用来表征多步反应途径 将受体的非磷酸化形式转化为 催化活性的磷酸化形式。在这些研究中，氨基酸 受体中反应性酪氨酸残基(S)周围的序列将是 测定并与其他酪氨酸激酶进行比较。费率和 将计算每个步骤的平衡常量 受体激活的反应途径。化学计量学和平衡法 胰岛素与磷酸化和磷酸化的结合常数 非磷酸化形式的受体将被测量以确定 胰岛素结合是否可能导致未磷酸化的受体 一种类似于磷酸化受体的构象。这个 锌离子对反应途径中各个步骤的抑制作用 受体激活的特性，因为抑制 锌离子对胰岛素受体的自动磷酸化作用可能有助于 保护胰岛细胞和其他组织免受高密度脂蛋白的危害 胰岛素在正常或意外释放部位的浓度 含胰岛素颗粒的含量。另一组研究将 目的在于表征受体激酶的相互作用 潜在的新陈代谢调节剂。因此，可能性将是 评估了胰岛素受体激酶催化的磷酸化。 葡萄糖转运蛋白，从而降低其从血浆中的排泄率 膜。酪蛋白激酶I对胰岛素受体的磷酸化作用 进行研究以确定酪蛋白激酶I是否催化磷酸化 胰岛素受体改变其与胰岛素结合的能力并经历 自动磷酸化，以及酪蛋白激酶I是否催化了磷酸化 可能解释了受体丝氨酸和丝氨酸的磷酸化 在整个细胞中可见的芳基残基。在寻找内生性 胰岛素受体激酶、抗O-磷酸酪氨酰抗体的底物 将用于浓缩胰岛素促进的酪氨酸产品 整个细胞中的磷酸化。试图推断出结构 胰岛素受体激酶天然底物的特性 该酶对非肽和多肽的底物专一性 底物将被确定。