ROLE OF THIOL-DISULFIDE EXCHANGE IN INSULIN BINDING TO ITS RECEPTOR

The effects of reduction by DTT, oxidation by DTNB and treatment with NEM on the thiol contents and insulin binding to its receptor in mice liver membranes were studied. Reduction with DTT leads to a parallel increase in the thiol content and the speciflc binding of insulin to the membrane. Scatchard analysis of the results shows little change in the number of binding sites but a twofold increase of the binding constant. Washing the membrane with bound insulin by a DTT containing buffer results in a more marked increase in the release of bound insulin than washing with buffer alone, suggesting that part of the insulin is bound to its receptor by covalent disulfide linkages through a thIol-disulfide exchange reaction and reduction with DTT leads to a marked increase in this 'disulfide-linked' insulin. Treatment with DTNB or NEM of the DTT-reduced membrane seems to reverse the effect of DTT reduction, although the reaction of the untreated membrane with DTNB or NEM had little or no effect on the specific binding of insulin. It is suggested that initially, part of the thiols responsible for the exchange reaction may not be available for reaction with DTNB and reduction with DTT generates further thiols leading to increased specific binding in general and increased insulin binding to the receptor through covalent disulfide linkages in particular.

胎盘膜胰岛素受体结合活性与巯基和二硫键的关系

部分纯化的人胎盘膜经DTT还原,NEM,DTNB修饰蛋白巯基后,改变了胰岛素受体的结合活性。Scatchard分析表明,当DTT浓度较低时,亲和常数基本不变;高亲和位点数略微升高,较高浓度的DTT处理时,结合位点数和亲和常数均有所下降。DTT还原膜蛋白二硫键后再用NEM,DTNB修饰巯基,胰岛素结合活性进一步下降。NEM或DTNB单独处理结合活性下降较少。胰岛素与受体结合后,用DTT洗后剩余的结合胰岛素比缓冲液洗低,表明有一部分胰岛素以二硫键与受体共价结合。在0—5mmol/L浓度范围内,随着DTT处理浓度的升高,这种以二硫键共价结合胰岛素增加。