The Binding and Degradation of Insulin by Rat Liver Membranes: Demonstration of Three Distinct Insulin-Binding Components in Detergent-Solubilized Material from Liver Plasma Membrane*

Abstract

The components responsible for insulin-binding and insulin degradation were solubilized from purified liver plasma membranes by treatment with the nonionic detergent Triton X-100. The characteristics of specific insulin binding and insulin degradation were similar to those found with the intact membranes. Polyacrylamide gel electrophoresis of the detergent extract separated the insulin-binding activity into three peaks, designated peaks I, II, and D. The majority of the insulindegrading activity was located in peak D; peak I failed to degrade insulin, while only a small amount of degradation was associated with peak II. Scatchard analysis of the insulin-binding data for peak I showed a curvilinear plot, with the initial high affinity portion of the curve having a K_d of 2 × 10⁻¹⁰m. The Scatchard plot for peak II was linear, with a K_d of 8 × 10⁻⁹m. Both peaks bound ¹²⁵I-labeled insulin with high specificity, as determined by competition studies using desoctapeptide insulin, proinsulin, and glucagon. Insulin degradation by peak D was stimulated by glutathione and inhibited by n-ethylmalemide; the apparent K_m for degradation was 1 × 10⁻⁷m. These findings indicate the presence of three insulin-binding components in Triton-solubilized material from liver membranes, of which one (peak D) appears to be responsible for insulin degradation. High affinity insulin-binding activity, therefore, can be physically separated from the insulin-degrading activity. The insulin-binding characteristics of peaks I and II and their respective electrophoretic mobilities are similar to the two insulin-binding peaks which we have recently extracted from fat cell membranes. (Endocrinology106: 179, 1980)