| Abstract: | 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. |
