THIOL-BIOTINYLATION OF THE INSULIN RECEPTOR

我们检测了胰岛素受体巯基的反应性。 中国仓鼠卵巢细胞中高水平表达的生物素化 人胰岛素受体(CHO/HIRc细胞)。在生物素化之后 反应中，通过免疫沉淀纯化胰岛素受体，并 电转移前的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法 到膜上。酶联链霉亲和素联合应用 一种化学发光技术可以检测硫醇生物素化 受体β亚基，不修饰α亚基。在……里面 表达大量IGF-1受体的细胞，同样的技术 也能够检测到硫醇生物素标记的IGF-1受体。 不适当试剂对完整CHO/HIRc细胞的硫醇烷基化反应 不损害马来酰亚胺二丁酰基生物细胞素(MBB)的能力 细胞后受体β亚基上的生物素化巯基 洋地黄素的渗透作用。相比之下，硫醇-烷基化反应 洋地黄素通透性细胞阻止MBB诱导的受体 生物素化作用。基础胰岛素受体和胰岛素激活的胰岛素受体 显示的胰岛素受体对MBB表现出类似的反应性。 此外，亲和纯化在单体亲和素-琼脂糖凝胶中的应用 使我们了解到生物素化反应即将发生 量化的。MBB对胰岛素结合和受体均无影响 自磷酸化和胰岛素依赖受体激酶活性。 然而，受体激酶活性的基础水平显著 通过硫醇生物素化而升高。此外，在钒酸盐存在的情况下， MBB保留了增强受体激酶活性的能力 通透性细胞，与这种增加的概念一致 外源底物磷酸化不能通过失活来解释 蛋白酪氨酸磷酸酶。肌动蛋白去磷酸化 颗粒蛋白标记的硫醇生物素标记的胰岛素受体 酪氨酸磷酸酶不受影响。这些结果表明， 胞质结构域中反应性巯基的修饰 胰岛素受体β亚基的表达可能在胰岛素中起关键作用 受体功能。

We examined the reactivity of insulin receptor sulfhydryls to biotinylation in Chinese Hamster Ovary cells that express high levels of human insulin receptors (CHO/HIRc cells). Following the biotinylation reaction, the insulin receptor was purified by immunoprecipitation, and resolved by SDS-polyacrylamide gel electrophoresis before electrotransfer to membranes. The use of enzyme-linked streptavidin in conjunction with a chemiluminescent technique allowed the detection of thiol-biotinylated receptor beta-subunit, with no modification of the alpha-subunit. In cells expressing large numbers of IGF-1 receptors, the same technique enabled the detection of thiol-biotinylated IGF-1 receptors as well. Thiol-alkylation of intact CHO/HIRc cells with an impermeant reagent did not impair the ability of maleimidodibutyryl biocytin (MBB) to biotinylate sulfhydryls on the receptor beta-subunit after cell permeabilization with digitonin. In contrast, thiol-alkylation of digitonin-permeabilized cells prevented MBB-induced receptor biotinylation. The basal and insulin-activated insulin receptors exhibited insulin receptors exhibited a comparable reactivity to MBB. Furthermore, the use of affinity-purification on monomeric avidin-agarose enabled us to learn that the biotinylation reaction was near quantitative. MBB had no effect on insulin binding nor on receptor autophosphorylation and insulin-dependent receptor kinase activity. However, basal levels of receptor kinase activity were significantly elevated by thiol biotinylation. Further, in the presence of vanadate, MBB retained the ability to enhance receptor kinase activity in permeabilized cells, consistent with the notion that this increased exogenous substrate phosphorylation was not accounted for by inactivation of protein tyrosine phosphatases. The dephosphorylation of thiol-biotinylated, 32P- labeled insulin receptors by particulate protein tyrosine phosphatases was not affected. These results suggest that modification of reactive sulfhydryls located in the cytoplasmic domain of the insulin receptor beta-subunit may play a critical role in insulin receptor function.