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CATALYTIC COMPETENCE AND REGULATION OF RECEPTOR KINASE

受体激酶的催化能力和调节

基本信息

批准号：
3153797
负责人：
JULES Alan SHAFER
金额：
$ 9.8万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
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/3153797
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

项目摘要

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.

胰岛素受体的完整磷酸化和非磷酸化形式激酶将通过层析从人胎盘中纯化，固定化抗-O-磷酸酪氨酸抗体。净化的形式受体将用于表征多步反应途径，受体的非磷酸化形式转化为具有催化活性的磷酸化形式。在这些研究中，受体中反应性酪氨酰残基周围的序列将是确定并与其他酪氨酸激酶进行比较。率和平衡常数将评估为个别步骤中，受体活化的反应途径。化学计量和平衡胰岛素与磷酸化和将测量受体的非磷酸化形式以确定胰岛素结合是否会导致非磷酸化受体与磷酸化受体的构象相似。的锌离子对反应途径中各个步骤的抑制作用对于受体活化的特征，因为抑制锌离子引起的胰岛素受体自身磷酸化可能有助于保护胰岛细胞和其他组织免受高血糖的有害影响，正常或意外释放部位的胰岛素浓度含胰岛素颗粒的内容物。另一组研究将针对受体激酶相互作用的表征潜在的代谢调节剂。因此，可能性将是评估胰岛素受体激酶催化胰岛素受体的磷酸化葡萄糖转运蛋白，从而降低其从血浆中排出的速率膜。酪蛋白激酶I对胰岛素受体的磷酸化以确定酪蛋白激酶I是否催化磷酸化胰岛素受体的功能改变了其结合胰岛素的能力，自磷酸化，以及酪蛋白激酶I是否催化磷酸化可能是受体丝氨酸磷酸化的原因，在整个细胞中观察到的苏氨酸残基。在寻找内源性胰岛素受体激酶的底物，抗-O-磷酸酪氨酸抗体将用于浓缩胰岛素促进酪氨酰的产品整个细胞中的磷酸化。为了推断出胰岛素受体激酶的天然底物的特性，这种酶对非肽和肽底物特异性将确定衬底。