CATALYTIC COMPETENCE AND REGULATION OF RECEPTOR KINASE

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 催化活性磷酸化形式。 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。

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.