Characteristics of dexamethasone binding to hippocampal cytosol receptors

Abstract This study was designed to clarify an in vitro system which could be used to study the time course, kinetics, and specificity of [ 3 H]-dexamethasone (DM) binding in hippocampal cytosol. Maximal binding, as measured by chromatography through Sephadex LH-20 mini-columns, was attained by 6 h and maintained to 24 h when the incubation medium contained 20 mM Tris, 20% glycerol, 50 mM KC1, 20 mM monothioglycerol, pH 7.8. Cytosol from adrenalectomized pigs exhibited a 50% greater capacity to specifically bind DM than that from unadrenalectomized pigs. Non-specifically bound DM averaged 18 ± 5% of total [ 3 H]-dm bound. The calculated K d was 1.07 × 10 −10 M and the maximum number of binding sites was 3.0 × 10 −14 moles/mg protein. Thirty-three hours were required for the dissociation of half of the specifically bound [ 3 H]-DM. Instability of the complex increased as incubation temperature was raised. Binding activity was diminished by competition with glucocorticoid or anti-glucocorticoid hormones and by incubation with proteolytic enzymes. It was demonstrated that DM binding in porcine serum is limited and that possible contamination of this system with CBG had negligible effects. Thus a glucocorticoid receptor exists in porcine hippocampal cytosol and can be characterized by its high affinity and specificity as different from CBG.