Role of disulfide bonds in human growth hormone binding and dissociation in isolated rat hepatocytes and liver plasma membranes.

Abstract

The role of disulfide bonds and sulfhydryl groups in rat hepatocytes and rat liver plasma membranes in the binding of human GH (hGH) has been studied. Since hGH binding involves uptake and irreversible binding, the effect of disulfide reducing agents [dithiothreitol (DTT) and 2-mercaptoethanol (ME)] and an alkylating agent [N-ethylmaleimide (NEM)] on the time course of binding and displacement of [125I]hGH was determined in the hepatocytes and membranes. The time course of binding and displacement of [125I]hGH was similar in membranes and cells, indicating that the irreversible nature of hGH binding is not dependent upon an intact cellular structure. Both 1% ME and 10 mM DTT prevented further binding of [125I]hGH when added at 60 min of the 300-min binding incubation for both hepatocytes and plasma membranes. The ME caused some initial dissociation of bound [125I]hGH, with subsequent binding to levels that were present when the ME was added. Only ME caused an increase in nonspecific binding with the plasma membranes. Both ME and DTT caused an increase in displacement of [125I]hGH in the presence of excess unlabeled hGH. The amount of [125I]hGH remaining bound in the presence of DTT and unlabeled hGH approached nonspecific levels by 240 min of incubation. NEM caused an increase in the total [125I]hGH bound, but this was apparently due to increased nonspecific binding in the presence of NEM. The effect of the reducers on binding was not secondary to an effect on hGH, since the disulfides of [125I]hGH were not reduced under the conditions of binding with either ME or DTT. The effect of the ME or DTT on binding could be reversed or prevented by subsequent or simultaneous addition of an oxidizer such as NAD or oxidized glutathione. The data indicate that disulfide bonds in the membranes are intimately involved in the maintenance of a receptor structure necessary for hGH binding. The disruption of the disulfides also results in increased dissociation and displacement of the bound [125I]hGH, indicating a possible role in the irreversible nature of hGH binding. This represents a partial delineation of the hGH binding process.