ALKYLATION OF SULFHYDRYL GROUP(S) ON THE INSULIN RECEPTOR ALTER KINASE ACTIVITY

我们研究了巯基修饰剂对胰岛素的影响 毛地黄皂苷透化的中国仓鼠卵巢细胞受体功能 用人胰岛素受体（IR）转染。 丽晶 使用的是马来酰亚胺丁酰生物胞素（MBB），一种巯基特异性生物素化 试剂. 结果表明，MBB对细胞增殖有促进作用， 胰岛素受体酪氨酸激酶对外源底物活性。 在缺乏胰岛素的情况下，MBB引起基础激酶的增加， 活性与单独使用胰岛素时观察到的水平相当。 治疗 用MBB透化的细胞引起受体的进一步2倍刺激 胰岛素的激酶活性。 IR ~-亚基含有巯基 与MBB反应。 IR ~-亚基的生物素化程度 无论细胞是否被胰岛素刺激都是一样的。 L-半胱氨酸可完全抑制~-亚基的生物素化，而L-半胱氨酸对~-亚基的生物素化无影响。 甘氨酸，表明MBB作为硫醇修饰的特定性质 试剂. 几种巯基试剂作为探针进行了测试， 评估受体上可接近的巯基的位置，以及 确定这些反应性巯基是否是邻位的。 似乎 通过MBB对IR巯基的改性不会引起IR巯基的还原， 细胞蛋白酪氨酸磷酸酶对IR的可及性 磷酸化位点。 我们的结论是，反应性的修改 MBB的半胱氨酸导致IR酪氨酸激酶活性增强， 这可能表明细胞氧化这种 胰岛素信号传导中的残基。

We investigated the effect of a sulfhydryl-modifying reagent on insulin receptor functions in digitonin-permeabilized Chinese hamster overy cells that were transfected with human insulin receptors (IR). The regent used was maleimidobutyryl biocytin (MBB), a thiol-specific biotinylation reagent. Results showed that MBB had an enhancing effect on the insulin receptor tyrosine kinase activity toward an exogenous substrate. In the absence of insulin, MBB caused an increase in basal kinase activity comparable to the level seen with insulin alone. Treatment of permeabilized cells with MBB caused further 2-fold stimulation of receptor kinase activity by insulin. IR ~-subunit contains sulfhydryl group(s) that react readily with MBB. The extent of biotinylation of IR ~-subunit was identical whether the cells were stimulated or not with insulin. Biotinylation of ~-subunit was totally inhibited by L-cysteine but not L- glycine, indicating the specific nature of MBB as a thiol-modifying reagent. Several sulfhydryl reagents were tested as probes for assessing the location of accessible thiol(s) on the receptor as well as determining whether these reactive sulfhydryl(s) are vicinals. It appears that modification of IR sulfhydryl(s) by MBB did not cause reduced accessibility of cellular protein tyrosine phosphatases toward IR phosphorylation sites. We conclude that modification of reactive cysteine(s) by MBB results in enhanced IR tyrosine kinase activity, which could suggest an important role played by cellular oxidation of such residues in insulin signalling.