The activated hepatic glucocorticoid-receptor complex: A simple one-step method for its separation from nonactivated complex

Abstract

Protamine sulfate was found to precipitate completely the nonactivated [ 3H]-dexamethasone-receptor complex of rat liver. This observation was then used as the basis of a method to separate activated from nonactivated complex. Thus, addition of 10 mg/ml of protamine sulfate to the rat hepatic cytosol [ 3H]dexamethasone-receptor complex, incubated at 0–4°C for 2 hr, resulted in the complete precipitation of [ 3H]dexamethasone-receptor complex. The remaining supernatant obtained on centrifugation at 800 g was unable to bind either to nuclei or to DNA-cellulose. An increase in temperature to 25°C or the addition of 10 m m CaCl 2 to the cytosol resulted in the appearance of activated [ 3H]dexamethasone-receptor complex in the supernatant obtained by addition of protamine sulfate. This was determined by characteristic binding to nuclei or DNA cellulose and by p I. Protamine sulfate could not affect the separation of activated [ 3H]dexamethasone-receptor complex at salt concentrations above 100 m m NaCl. This procedure therefore had to be carried out under conditions of relatively low ionic strength. Finally, a one-step rapid method is described for the separation of activated [ 3H]dexamethasone-receptor complex from nonactivated receptor complex. The homogeneous population of activated complex thus obtained should have considerable applicability in studies of the mechanisms of in vitro glucocorticoid-receptor activation.