Studies on the Glucocorticoid Receptor in Rat Brain Cytosol

Abstract

The binding of ³H-corticosterone or ³H-dexamethasone to macromolecules in cytosols of the brain has been studied in vitro in adrenalectomized male rats.
The cytosol fraction was incubated with ³H-corticosterone at 2-4°C for 4 h. Steroid bound to cytosol was separated from the unbound fraction by three methods : gel chromatography of Sephadex G-25, dextran coated charcoal absorption and the protamine precipitation procedure.
The binding properties were analyzed by Scatchard plots. The dissociation constant (K) was. found to be 5.4×10^-9M, and the number of binding sites/mg cytosol protein was 0.49 p moles, indicating a binding with high affinity and limited capacity for ³H-corticosterone.
The ontogeny of the cytosol glucocorticoid binding micromolecules was investigated in the various brain regions of 20, 30 and 65 day-old male. The concentration of corticosterone binding sites was higher at 30 days than at other days. The ³H-corticosterone binding capacity was highest in the hippocampus and septum. Chromatography of the cytosol proteins on Sephadex G-200 showed the presence of two stable corticosterone binding proteins. One (A) was eluted at the void volume fraction, suggesting that its molecular weight is greater than 200,000, and another (B) appeared in the portion corresponding to the elution position of transcortin in serum.
When the same procedure was applied to the study on ³H-dexamethasone binding properties, only one peak (C) was eluted at the void volume fraction. The competitive binding studies of each peak indicated that A and C had a high degree of specificity for corticosterone, cortisol and dexamethasone. After absorption by anti-rat serum using the affinity chromatography, peak B was found to be retained on the column, since it dis appeared when the eluate was further applied on a Sephadex G-200 column. The B binder seems, therefore, to be antigenically similar to transcortin in serum.