Insulin-degrading enzyme is capable of degrading receptor-bound insulin

Abstract In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ aggulutinin column and monoclonal anti-IDE antibody affinity column, respectively, [ 125 I]insulin-receptor complex was incubated with various amounts of IDE at 0°C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135kd band (insulin receptor α-subunit) decreased, whereas that of 110kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37°C, about half of the [ 125 I]insulin was dissociated from the complex. However, the time course of [ 125 I]insulin degradation in this incubation was essentially identical to that of free [ 125 I]insulin degradation. Cross-linked, non-dissociable receptor-bound [ 125 I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [ 125 I]insulin from insulin-receptor complex incubated with IDE was significantly (p