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 beta-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 microM] than in the absence (IC50 = 73 +/- 6 microM) of insulin. Similarly, inhibition by 5,5'-dithiobis-(2-nitrobenzoic acid) occurred with IC50 = 30 +/- 6 microM in the presence and 155 +/- 35 microM 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 beta-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 beta-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 beta-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.
Wilden
P A
PA
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Pessin
J E
JE
D000255:Adenosine Triphosphate / Q000378:metabolism; D001205:Ascorbic Acid / Q000494:pharmacology; D002712:Chlorides; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005290:Ferric Compounds / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D007460:Iodoacetamide / Q000494:pharmacology; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011972:Receptor, Insulin; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
D002712:Chlorides; D005290:Ferric Compounds; D007328:Insulin; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D005033:Ethylmaleimide; D001205:Ascorbic Acid; C024555:ferric chloride; D007460:Iodoacetamide