Modification of the insulin receptor by diethyl pyrocarbonate: effect on insulin binding and action

Abstract

Insulin binding to rat liver plasma membranes is inhibited in a time- and dose-dependent fashion by prior treatment of membranes with the histidine-specific reagent diethyl pyrocarbonate. If all receptors are occupied by unlabeled hormone during diethyl pyrocarbonate treatment, no inhibition of 125I-labeled insulin binding is observed following washout of unlabeled hormone and unreacted reagent. Scatchard analysis of the binding inhibition due to diethyl pyrocarbonate reveals a loss in receptor number rather than a change in receptor affinity for hormone. [Rat] fat cells treated with diethyl pyrocarbonate exhibit a rightward shift in the dose-response relationship for insulin-stimulated glucose oxidation consistent with a loss in receptor number due to the reagent. The pH profile for inhibition of insulin binding by diethyl pyrocarbonate and the partial reversibility of this inhibition by hydroxylamine are consistent with modification of a histidine residue. A histidine residue at or near the receptor binding site apparently is required for formation of the biologically relevant insulin-receptor complex.