Erik Dahlberg

Characterization of the cytosolic estrogen receptor in rat skeletal muscle

A charcoal assay and the synthetic estrogen [6,7-3H]R 2858 (17 alpha-ethynyl-11 beta-methoxyestradiol-17 beta) were used to show that rat skeletal muscle cytosol contains an estrogen receptor, which was characterized with regard to association and dissociation rate-constants at several temperatures. The degradation kinetics was also studied, and was more rapid in the absence than in the presence of ligand. Association, dissociation and degradation were all temperature-dependent. The apparent equilibrium dissociation constant (Kd), however, was not temperature-dependent, and was about 0.1-1.0 nM, whether calculated from the rate constants or determined by Scatchard analysis. Estradiol-17 beta and R 2858 were compared as ligands for Scatchard analysis; the maximum number of binding sites being about the same (120 and 110 fmol/g tissue, respectively, but the Kd was lower for estradiol-17 beta (0.16 nM) than for R 2858 (0.73 nM). Ligand specificity studies (using [3H]R 2858 as well as [3H]estradiol-17 beta) showed that R 2858 binds to an estrogen receptor in rat skeletal muscle. The estrogen receptor interacted both with heparin and with DNA covalently coupled to agarose, and was eluted from either column by NaCl. Chromatography of crude cytosol on heparin-agarose or DNA-agarose lead to an at least 10-fold or 25-fold purification of the estrogen receptor, respectively. DNA-agarose chromatography or ammonium sulfate precipitation did not separate the estrogen receptor from the androgen or glucocorticoid receptors in rat muscle.

Removal of hydrophobic compounds from biological fluids by a simple method

Abstract A hydrophobic column-packing material (Lipidex 1000) was utilized to remove unbound as well as protein-bound hydrophobic molecules from aqueous solutions in a controlled manner by choice of flow rate and temperature according to the protein-lipid interaction kinetics. The adsorbed compounds were readily recovered upon change of eluant from buffer to methanol. It could be used to eliminate steroids from water solutions or to accomplish separation of protein-bound from unbound steroids at 0–4°C. At 45°C bound steroid ligand was also removed from albumin, corticosteroid-binding globulin, and sex hormone-binding globulin. These proteins retained their capacity to bind ligand after passing the column. Scatchard analysis of steroid hormone receptors in cytosol from pig seminal vesicles suggested the occurrence of “positive cooperativity”; however, chromatography of the cytosol on Lipidex 1000 prior to the incubation with ligand removed lipid droplets in the sample and resulted in a linear Scatchard plot. Since the gel has a high capacity for adsorption of lipids and steroids, large sample volumes can be used in order not to change the ion composition of the sample. Moreover, no adverse affects on the proteins investigated were observed, and the method is therefore likely to be useful not only in lipid research, but also in protein-biochemical work.

Steroid receptors in metastatic carcinoma of the human prostate

Abstract Using a dextran-coated charcoal technique and isoelectric focusing in polyacrylamide gel, metastases from five patients with prostatic carcinoma were analysed for contents of cytosolic androgen, progestin, estrogen and glucocorticoid receptors. Maximum binding capacity (Bmax) and dissociation constant (Kd) were calculated from Scatchard plots. In three cases the patients had received endocrine therapy prior to removal of the specimen. Androgen and progestin receptors were measurable in 4 5 and 2 5 cases, respectively; no specimen contained estrogen receptors. In 3 5 cases large amounts of glucocorticoid receptors were detected.

Androgen and glucocorticoid receptors in human skeletal muscle cytosol

Abstract The binding of the synthetic radioactive steroids [ 3 H]-methyltrienolone (MT) (17β-hydroxy-17α-methylestra-4,9,11-trien-3-one) and [ 3 H]-dexamethasone (DEX) (9α-fluoro-11β,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione) to cytosol from human skeletal muscle was studied using a charcoal adsorption technique and calculating receptor-bound steroids according to Scatchard. The rate of association was more rapid in the case of MT than in the case of DEX. The maximum specific binding of MT and DEX was reached after about 15 and 22 h at 0–4°C, and lasted for at least 30 and 20 h, respectively. The binding sites were specific for androgens (MT) and glucocorticoids (DEX). In most human muscles analyzed, both receptors were detectable. The apparent equilibrium dissociation constants of the androgen and glucocorticoid receptor-binding were 0.07 to 0.70 nM and 1.7 to 9.7 nM, respectively. The maximum number of binding sites ( B max ) ranged from 55 to 240 fmol/g tissue, 1.1 to 4.0 fmol/mg protein or 60 to 450 fmol/mg DNA in the case of the androgen receptor. The B max , of the glucocorticoid receptor varied between 1,200 and 5,600 fmol/g tissue, 23 and 94 fmol/mg protein or 770 and 12,000 fmol/mg DNA. Hence, human skeletal muscle may respond directly to androgens and to glucocorticoids.

Estimation of the blood contamination of tissue extracts

A method based on measurement of absorbance at 540 or 580 nm is described. The results obtained were closely correlated to the actual level of blood present in different tissues, as determined by use of radiolabeled albumin. The present technique is much simpler than earlier methods used to determine the degree of blood contamination of tissue extracts. Hence, it may find wide application in the life sciences.

Effects of ACTH on the ligand-binding properties of the glucocorticoid receptor exerted not only via elevated levels of corticosteroids

Treatment of mice with ACTH not only increased plasma corticosterone levels, but also reduced the ligand-binding capacity of the glucocorticoid receptor in skeletal muscle cytosol. Non-linear Scatchard plots were obtained for the glucocorticoid receptor following ACTH treatment. This upward concave curvilinearity could not be reproduced by simply adding to the cytosol an amount of corticosterone corresponding to the increase, even though this treatment resulted in reduced binding capacity. The addition of corticosterone resulted in a slower formation of ligand-receptor complexes (lower association rate constant), which offered a partial explanation for the low binding. In addition, ACTH treatment was also found to reduce the binding capacity in adrenalectomized mice that did not respond with corticosterone elevation. However, in this case Scatchard plots were linear.

‘Specific receptor binding’ of radioactively labeled products of radiolysis of the estrogen receptor ligand R 2858 (17α-ethylnyl-11β-methoxy-estradiol-17β)

Abstract Radioactively labeled steroids undergo decomposition processes, which are dependent on time, storage conditions (temperature, solvents, etc.), degree of labeling etc. This communication shows that several decomposition products of 17α-ethynyl-11β-methoxy-estradiol-17β (R 2858 0) bind to rat uterine cytosol in a way that would be interpreted as ‘specific receptor binding’ if some of these compounds were present in the ligand solution used for estrogen receptor determination. Thys, the binding was charcoal-resistant and displaceable with an excess of unlabeled R 2858, and the percentage of binding was of significant magnitude to seriously interfere with receptor measurements.