Studies on the Glucocorticoid Receptor (original) (raw)

Elsevier eBooks, 1981

Abstract

ABSTRACT The [3H]triamcinolone acetonide-labeled glucocorticoid receptor from rat liver cytosol was purified to 85% homogeneity using sequential chromatography on phosphocellulose, DNA-cellulose twice, and Sephadex G-200. Between the two chromatography steps on DNA-cellulose, the receptor was heat activated. The receptor was affinity eluted from the second DNA-cellulose column with pyridoxal 5′-phosphate. After chromatography on the second DNA-cellulose column, the steroid-receptor complex had a Stokes radius of 6.0 nm and a sedimentation coefficient of 3.4 S in 0.15 M KC1. In the absence of KC1, the sedimentation coefficient was 3.6 S. An immunoglobulin (IgG) fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor contained specific antibodies to the receptor. This was shown following incubation of [3H]triamcinolone acetonice (TA)-receptor complex with the IgG fraction by I adsorption of the [3H]TA-glucocorticoid receptor-antibody complex to protein A linked to Sepharose II an increased sedimentation rate of the [3H]TA-receptor-antibody complex compared to that of the [3H]TA-receptor complex, and III an increased molecular size of the [3H]TA-receptor-antibody complex when compared to that of the [3H]TA-receptor complex as judged from gel filtration. The antibodies cross-reacted with the glucocorticoid receptor from various rat tissues (liver, thymus and hippocampus) as well as with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. Binding of the antibodies was not seen to the androgen, estrogen or progestin receptors in rat or to rat serum transcortin. With an indirect competitive ELISA (enzyme linked immunosorbent assay) combined with various separation techniques, based on different physico-chemical principles, it was shown that the glucocorticoid receptor was the only detectable antibody binding protein from rat liver cytosol using this assay system. Preliminary results from immunofluorescence studies on the localization of the glucocorticoid receptor in rat hypothalamus are also presented.

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