Identification of insulin-degrading enzyme on the surface of cultured human lymphocytes, rat hepatoma cells, and primary cultures of rat hepatocytes.

Abstract

Antisera specific for insulin-degrading enzyme (IDE) and glutathione-insulin transhydrogenase were used as probes to identify these enzymes on the surface of cells. Three different experimental approaches indicated that the antibodies to IDE were bound to the surface of IM-9 cultured human lymphocytes. First, IDE-antibodies could be visually detected on the surface of the IM-9 cells after incubation with a peroxidase- linked antibody directed against the first antibody and histochemical staining for peroxidase activity. A spectrophotometric assay for peroxidase activity demonstrated that 15 times more peroxidase bound to the IM-9 cells incubated with the IDE-antibodies than co cells incubated with control antibody. Further, the increased binding of the IDE-antibodies to the IM-9 cell was inhibited 80% by absorption of the antiserum with purified IDE. A second approach was to perform flow fluorometry on IM-9 cells incubated first with control antibody or IDE-antibodies and next with a fluorescent antibody directed against the first antibody. These studies demonstrated an increase in the average fluorescence per cell in cells treated with IDE-antibodies. A third approach was to immunoprecipitate with IDE-antibodies extracts of IM-9 cells surface labeled with radioactive iodine. These precipitates contained a 67,000-dalton mol wt component that was not precipitated by control sera. The molecular weight of this component is consistent with the previously reported molecular weight of purified pig muscle IDE. Binding of the IDE-antibodies to the surface of two rat hepatoma cell lines (HTC and H35 cells) and primary cultures of adult rat hepatocytes could also be demonstrated. In contrast to IDE-antibodies, the binding of glutathione-insulin transhydrogenase-antibodies was detected only on the primary cultures of rat hepatocytes and not on other cell types. In summary, our results indicate that IDE is on the surface of various cell types. This plasma membrane IDE may, therefore, play a role in insulin degradation.