Characterization and Quantification of the Androgen and Glucocorticoid Receptors in Cytosol from Rat Skeletal Muscle
Open Access
- 1 October 1980
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 111 (2), 603-616
- https://doi.org/10.1111/j.1432-1033.1980.tb04977.x
Abstract
The binding of the radioactive synthetic hormonal steroids [3H]dexamethasone (9α-fluoro-11β, 17α, 21-trihydroxy-16α-methyl-1, 4-pregnadiene-3,20-dione) and [3H]methyltrienolone (17β-hydroxy17-α-methyl-4,9,11-estratrien-3-one) to cytosol from rat skeletal muscle was studied using dextrancoated charcoal to separate unbound and receptor-bound steroid. The rates of association, dissociation, and degradation of the complexes of dexamethasone and methyltrienolone with receptor were highly dependent on temperature. The temperature dependence of association was greater for dexamethasone, and that of degradation was greater for methyltrienolone. Dissociation rates were insignificant for both steroid-receptor complexes compared to association and degradation rates. The apparent equilibrium dissociation constants for the binding of dexamethasone and methyltrienolone to their receptor binding sites were about 7 and 0.3 nM, respectively, regardless of temperature (0, 15 or 23° C). The lack of influence of temperature on the equilibrium constants indicate that the binding was of hydrophobic character, and the corresponding free energy changes upon binding of dexamethasone and methyltrienolone to their respective binding sites were –41 and –49 kJ mol-1 under equilibrium conditions at 0° C. The apparent maximum number of binding sites determined from Scatchard plots under these conditions was about 1900 fmol/g of tissue, 3500 fmol/mg of DNA or 30 fmol/mg of protein in the case of the dexamethasone receptor, and the corresponding figures for the methyltrienolone were about 100 fmol/g of tissue, 200 fmol/mg of DNA or 2 fmol/mg of protein. The ligand specificities of the binding sites for dexamethasone and methyltrienolone were typical of a glucocorticoid and an androgen receptor, respectively. Both steroid-receptor complexes were retained on DNA-cellulose columns, and were eluted by NaCl at an ionic strength of 0.1. The DNA-cellulose step purified about 20 times, and was used to allow gel exclusion chromatography and electrofocusing. Both steroid-receptor complexes were excluded from a column of Sephadex G-150. Electrofocusing in preparative columns gave reproducible patterns consisting of three peaks for each receptor. The apparent isoelectric points were 5.4, 5.6 and 0.2 for the glucocorticoid receptor, and 5.9, 6.2 and 8.5 for the androgen receptor.This publication has 45 references indexed in Scilit:
- Physical Chemical Studies of the Structure and Function of Dna Binding (Helix-Destabilizing) ProteinsCritical Reviews in Biochemistry, 1980
- Comparison of the R-3327H rat prostatic adenocarcinoma to human benign prostatic hyperplasia and metastatic carcinoma of the prostate with regard to steroid hormone receptorsThe Prostate, 1980
- Partial characterization of [3H]methyltrienolone binding in rat prostate cytosolBiochimica et Biophysica Acta (BBA) - General Subjects, 1979
- A comparison of the glucocorticoid receptor in cytosol from rat liver and hippocampusBiochimica et Biophysica Acta (BBA) - General Subjects, 1979
- Affinity chromatography on columns containing nucleic acidsFEBS Letters, 1978
- Cytosol protein aggregation and dexamethasone binding to isolated rat liver nucleiFEBS Letters, 1976
- Kinetics of progesterone binding to the chick oviduct receptor proteinThe Journal of Steroid Biochemistry and Molecular Biology, 1976
- Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductasesBiochimica et Biophysica Acta (BBA) - Biophysics including Photosynthesis, 1966
- An Improved Diphenylamine Method for the Estimation of Deoxyribonucleic AcidNature, 1965
- THE ATTRACTIONS OF PROTEINS FOR SMALL MOLECULES AND IONSAnnals of the New York Academy of Sciences, 1949