Differential sensitivity of the insulin-receptor kinase to thiol and oxidizing agents in the absence and presence of insulin

The purified human placental insulin-receptor β-subunit autophosphorylating activity was found to be inhibited, in a time- and concentration-dependent manner, by the specific thiol-alkylating agents N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid). The insulin-receptor kinase was observed to be more sensitive to inhibition by N-ethylmaleimide in the presence [IC₅₀ (concn. giving 50% inhibition) = 25 ± 3 μM] than in the absence (IC₅₀ = 73 ± 6 μM) of insulin. Similarly, inhibition by 5,5'-dithiobis-(2-nitrobenzoic acid) occurred with IC₅₀ = 30 ± 6 μM in the presence and 155 ± 35 μM in the absence of insulin. Examination of the exogenous-substrate protein kinase activity demonstrated that the differential sensitivity to N-ethylmaleimide was due to direct inhibition of protein kinase activity, as opposed to blockade of the phospho-acceptor properties of the insulin receptor. In contrast, iodoacetamide had essentially no effect on the insulin-receptor β-subunit autophosphorylating activity and was able to protect partially against the N-ethylmaleimide inhibition in both the presence and the absence of insulin. Consistent with these findings, none of the thiol-specific agents were able to alter significantly insulin binding at concentrations which maximally inhibited the β-subunit autophosphorylation. Further, in the presence of insulin, the insulin-receptor kinase activity was also observed to be more sensitive to oxidation by H₂O₂ and FeCl₃/ascorbate compared with insulin receptors in the absence of insulin. These results indicate that there is a critical thiol group(s) necessary for the β-subunit autophosphorylating activity of the insulin-receptor kinase and that in the presence of insulin is more susceptible to exogenously added thiol and oxidizing agents.