The studies on the loss and recovery of the steroid binding ability of rat liver glucocorticoid receptor

Abstract

We examined the mechanism of loss and recovery of steroid binding activity (inactivation and reactivation) of the rat liver glucocorticoid receptor (GR) in the cell free condition. Male Sprague-Dawley rats were adrenectomized 2 days before the hepatectomy for the experiments, and were fed with normal diet and saline. The cytosol was prepared in 10mM phosphate buffer (pH 7.4) containing 10% glycerol (PG buffer). The nuclei were isolated by centrifuging at 800 x g for 20 min at 4 degrees C in 10mM phosphate buffer (pH 7.4) containing 0.32M sucrose, 3mM MgCl2, then centrifuged again at 40,000 x g for 20 min at 4 degrees C in 10mM phosphate buffer (pH 7.4) containing 1.8M sucrose, 3mM MgCl2. The glucocorticoid receptor was partially purified by affinity chromatography with DOC-Sepharose 6B, and column chromatography with Sephacryl S-300 and DEAE-cellulose in the presence of molybdate throughout the procedure. The steroid binding activity of the cytosol prepared in PG buffer was inactivated by following procedures; either 1) treatment with 1% activated charcoal, 2) dialysis against 500 volumes of PG buffer, 3) incubation at 37 degrees C for 30 min, or 4) incubation in high salt condition (0.4M KCl) at 0 degrees C for 16h. When the steroid binding activity was inactivated by incubation at 0 degrees C for 16hr and charcoal treatment, dithiothreitol (DTT) and NADPH were effective on reactivation, while glutathione (reduced form) was ineffective. Antipain increased the reactivated steroid binding activity in these cases. The steroid binding activity, which was inactivated by dialysis, was slightly recovered by DTT or NADPH, but antipain was ineffective. We could not reactivate steroid binding activity which was inactivated by incubation at 37 degrees C for 30 min or high salt (0.4M KCl) treatment with any reagent we tested. Leupeptin, however, partially prevented the high salt inactivation. When the cytosol prepared in PG buffer was labeled with 3H-dexamethasone (Dex) and then incubated at 25 degrees C for 30 min, 3H-Dex labeled steroid receptors was transformed and it also lost its steroid binding activity partially. DTT or NADPH increased steroid binding activity, but did not increase nuclear binding activity. Interestingly, by dialysis, the cytosol glucocorticoid receptors was not transformed in the PG buffer. We also examined the reactivation of steroid receptor using the partially purified receptor. To remove molybdate, the partially purified receptor was dialyzed against PG buffer. Large part of steroid binding activity was lost during the dialysis. DTT, however, reactivated steroid binding activity without molybdate, and also S-value of reactivated receptor was undistinguishable from the untransformed (molybdate stabilized) receptor.(ABSTRACT TRUNCATED AT 400 WORDS)