Two systems in vitro that show insulin-stimulated serine kinase activity towards the insulin receptor.
Two systems in vitro that show insulin-stimulated serine kinase activity towards the insulin receptor.
复制标题
两个体外系统显示胰岛素刺激的丝氨酸激酶对胰岛素受体的活性。
作者：
D. Smith;M. J. King;G. J. Sale
描述了两个体外系统，显示胰岛素刺激的胰岛素受体丝氨酸残基的磷酸化。在第一个系统中，通过将细胞直接溶解在 Triton X-100 中并在小麦胚芽凝集素琼脂糖上进行层析，从 Fao 大鼠肝癌细胞中部分纯化胰岛素受体。在存在或不存在胰岛素的情况下用[γ-32P]ATP磷酸化这些制剂导致32P仅掺入磷酸酪氨酸残基中。通过添加 Fao 细胞提取物来重建针对胰岛素受体的丝氨酸激酶活性。细胞预先暴露于胰岛素可刺激提取物中胰岛素受体的丝氨酸激酶活性达 7.2 倍。通过用环AMP类似物处理细胞而增强的受体丝氨酸激酶活性也保留在重构的系统中。在第二个系统中，从人胎盘中共同纯化胰岛素受体和针对胰岛素受体的胰岛素敏感丝氨酸激酶活性。该方案涉及在小麦胚芽凝集素琼脂糖溶解和层析之前制备膜，使用旨在不破坏胰岛素受体和丝氨酸激酶之间潜在不稳定关联的温和程序。这些制剂中针对胰岛素受体的丝氨酸激酶活性被胰岛素刺激高达10倍，并且对于在胰岛素存在下进行的磷酸化，丝氨酸磷酸化的化学计量估计约为0.8mol/mol胰岛素受体。因此，首次描述了以胰岛素依赖性方式在丝氨酸上磷酸化至高化学计量的胰岛素受体制剂。定义并讨论了促进丝氨酸激酶活性恢复和测定的条件。这些系统为表征磷酸化胰岛素受体的胰岛素敏感性丝氨酸激酶的性质，并确定其在胰岛素作用和受体功能控制中的作用提供了基础。
Two systems in vitro are described that show insulin-stimulated phosphorylation of the insulin receptor on serine residues. In the first system, insulin receptor was purified partially from Fao rat hepatoma cells by direct solubilization of the cells in Triton X-100 and chromatography on wheat-germ-agglutinin-agarose. Phosphorylation of these preparations with [gamma-32P]ATP in the presence or absence of insulin resulted in 32P incorporation exclusively into phosphotyrosine residues. Serine kinase activity towards the insulin receptor was reconstituted by adding extracts of Fao cells. Prior exposure of the cells to insulin stimulated serine kinase activity towards the insulin receptor in extracts 7.2-fold. A receptor serine kinase activity enhanced by treatment of cells with cyclic AMP analogues was also retained in the reconstituted system. In the second system, insulin receptor and insulin-sensitive serine kinase activity towards the insulin receptor were co-purified from human placenta. The protocol involved preparation of membranes, before solubilization and chromatography on wheat-germ-agglutinin-agarose, by using gentle procedures designed not to disrupt a potentially labile association between the insulin receptor and the serine kinase. Serine kinase activity in these preparations towards the insulin receptor was stimulated up to 10-fold by insulin, and the stoicheiometry of serine phosphorylation was estimated to be approx 0.8 mol/mol of insulin receptor for phosphorylations performed in the presence of insulin. Thus a preparation of insulin receptor is described for the first time that is phosphorylated to high stoicheiometry on serine in an insulin-dependent manner. Conditions that facilitate recovery and assay of serine kinase activity are defined and discussed. These systems provide a basis for characterizing the nature of the insulin-sensitive serine kinase that phosphorylates the insulin receptor, and defining its role in insulin action and control of receptor function.