Examination of exchange assay for glucocorticoid receptor.
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 degrees C for 30 min, the amount of 3H-Dex reassociated with the glucocorticoid receptors dropped to 60% of that of the non-heated ones. DEAE-cellulose 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 3H-Dex 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 0.05 M 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Noguchi
T
T
1st Department of Internal Medicine, Tottori University School of Medicine, Japan.
Yoshida
A
A
Ueda
Y
Y
Mitani
Y
Y
Urabe
K
K
Adachi
T
T
Onoyama
S
S
Okamura
Y
Y
Shigemasa
C
C
Abe
K
K
D000818:Animals; D003600:Cytosol / Q000032:analysis; D003907:Dexamethasone / Q000032:analysis*; D004229:Dithiothreitol; D006902:Hydroxymercuribenzoates; D008099:Liver / Q000032:analysis*; D008297:Male; D008722:Methods; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000032:analysis*
D006902:Hydroxymercuribenzoates; D011965:Receptors, Glucocorticoid; C028697:dexamethasone receptor; D003907:Dexamethasone; D004229:Dithiothreitol