Abstract
We examined a method for the measurement of total, activated and non-activated glucocorticoid receptors using sodium-p-hydroxymercuribenzoate (PHMB) and dithiothereitol (DTT) developed by Banerji and Kalimi (1981).
Since the concentration of PHMB required for dissociation of the ligand from the receptors varied with the concentration of protein in the reaction mixture and the rate of reassociation of the ligand to the ligand-liberated receptors was sensitive to the concentration of PHMB used, it was necessary to find the minimum concentration of PHMB which was required for complete dissociation of the ligand. When the optimum concentration of PHMB was selected based on the concentration of protein in the cytosol, almost 100% exchange was attained in the non-heated dexamethasone (Dex)-receptor complexes by this method. However when Dex-receptor complexes were heated at 25° for 30min, the amount of 3H-Dex reassociated with the glucocorticoid receptors dropped to 60% of that of the non-heated ones. DEAEcellulose chromatography of the heated sample revealed that approx. 40% of the bound receptors were activated (eluted with 0.05 M KCl) during the heating period. After DEAE cellulose column chromatography of the exchanged 3HDex receptor, complexes reassociated with 3H-Dex were observed only in the fraction of unactivated receptor complexes (eluted with 0.2 M KCl). Furthermore, the fraction eluted with 0.05M KCl in the DEAE cellulose chromatography of liver cytosol bound to unlabelled Dex did not exchange significantly with 3H-Dex with the method used in the present study. These results all consistently indicated that in the activated receptor complexes dissociation of the ligand was performed almost completely, but reassociation was not achieved successfully after treatment with PHMB and DTT.
From these observations, the method described by Banerji and Kalimi does not seem to be suitable in assessing the bound-activated receptor complexes in tissues.
