scholarly journals The use of deoxyribonucleic acid–cellulose chromatography and isoelectric focusing for the characterization and partial purification of steroid–receptor complexes

1973 ◽  
Vol 134 (1) ◽  
pp. 113-127 ◽  
Author(s):  
W. I. P. Mainwaring ◽  
R. Irving
Keyword(s):  
Androgen Receptor ◽  
Physicochemical Properties ◽  
Binding Proteins ◽  
Steroid Receptor ◽  
Partial Purification ◽  
Receptor Complex ◽  
Nuclear Chromatin ◽  
Receptor Complexes ◽  
Steroid Binding Proteins ◽  
Steroid Binding

1. Two characteristic properties of the specific high-affinity steroid-binding proteins or receptors, their ability to bind to DNA–cellulose and their relatively acidic isoelectric point, have been exploited as a means of purification. These two fundamental properties distinguish the receptors from the steroid-binding proteins in serum and the non-specific low-affinity steroid-binding proteins in hormone-responsive cells. 2. A significant degree of purification of both cytoplasmic and nuclear steroid–receptor complexes can be achieved with practical facility by these procedures. The purity of the receptor complexes is sufficient to enable studies on their possible control of metabolic processes to be investigated in the future. 3. After extensive purification the physicochemical properties of the cytoplasmic androgen–receptor complex, such as sedimentation coefficient, were unchanged. Further, the purified complex fully retained at least one of its fundamental physiological properties, namely the ability to transfer 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) into chromatin in vitro. 4. The methods may also be employed for studying the changes in the structure and properties of the receptor complexes that are an essential prerequisite for the transfer of cytoplasmic receptor complexes into nuclear chromatin. The temperature-dependence of the binding of androgen–receptor complexes into chromatin is essentially due to a major change in cytoplasmic receptor complex before its attachment to nuclear chromatin. 5. The resolution of these analytical procedures was sufficient to enable a critical comparison of the receptor proteins from different male accessory glands to be undertaken. From these studies, no substantial evidence in support of the tissue specificity of androgen receptors could be established; rather the receptors from different androgen-dependent glands were remarkably similar in physicochemical properties. 6. Although the methods were initially developed for the partial purification of androgen–receptor complexes, they are equally suitable for the prompt and extensive purification of oestrogen–receptor and progesterone–receptor complexes.

scholarly journals Intracellular inactivation, reactivation and dynamic status of prostate androgen receptors

1982 ◽  
Vol 208 (2) ◽  
pp. 383-392 ◽  
Author(s):  
Gian Paolo Rossini ◽  
Shutsung Liao
Keyword(s):  
Protein Synthesis ◽  
Androgen Receptor ◽  
Half Life ◽  
Ventral Prostate ◽  
Receptor Complex ◽  
Nuclear Chromatin ◽  
Cytosol Fraction ◽  
Receptor Complexes ◽  
Rat Ventral Prostate ◽  
Energy Dependent

The dynamic status of the androgen receptor in prostate cells was studied by incubation of rat ventral prostate with radioactive 17β-hydroxy-5α-androstan-3-one (5α-dihydrotestosterone) in the presence and absence of respiratory poisons and inhibitors of protein and RNA synthesis and also by isotope chasing of the radioactive androgen–receptor complexes. The androgen receptor in the prostate appears to go through a dynamic process of recycling between the cytoplasm and the nucleus as well as an inactivation process. The radioactive androgen–receptor complex, however, is maintained at a constant level for at least 2h during incubation at 37°C, even in the absence of new protein synthesis, suggesting that early androgen actions may not require a depletion of a major portion of cellular receptor. In the presence of 2,4-dinitrophenol, the androgen receptor is rapidly deactivated (half life, 2min). The inactive receptor can be reactivated efficiently by an energy-dependent process, even in the absence of protein synthesis. Receptor binding of androgen and nuclear chromatin binding of the androgen–receptor complex are fast processes; half-maximum binding can be achieved within 1 and 10min respectively. On the contrary, the overall process of the release of the receptor complex from nuclear chromatin and its reappearance in the cytosol fraction has a long half life of about 70min. This slow process may reflect the involvement of the steroid–receptor complex in a time-consuming mechanism that is essential for hormone responses. Actinomycin D can increase the nuclear receptor level by 50% or more. This increase is not due to a decrease in the rate of receptor release from nuclei or to inhibition of DNA degradation by the antibiotic.

MEASUREMENT OF STEROID BINDING PROTEINS

1970 ◽  
Vol 65 (1_Suppl) ◽  
pp. S104-S121 ◽  
Author(s):  
E. E. Baulieu ◽  
J. P. Raynaud ◽  
E. Milgrom
Keyword(s):  
Kinetic Parameters ◽  
Binding Proteins ◽  
Binding Specificity ◽  
Binding Parameters ◽  
Target Organs ◽  
Biological Mixtures ◽  
Steroid Binding Proteins ◽  
Steroid Binding ◽  
Steroid Binding Protein

ABSTRACT A brief review of the characteristics of steroid binding proteins found in the plasma and in some target organs is presented, followed by some general remarks on binding »specificity« and binding parameters. Useful techniques for measuring binding parameters at equilibrium are reported, both those which keep the equilibrium intact and those which implicate its disruption. A concept is developed according to which the determination of a specific steroid binding protein is based on the »differential dissociation« of the several steroid binding complexes present in most biological mixtures. Methods which allow determination of the kinetic parameters of the binding systems are also presented. Various representations of the binding and therefore different modes of graphic representation and calculation are discussed, including the recent »proportion graph« method.

Serum Steroid Binding Proteins and the Bioavailability of Estradiol in Relation to Breast Diseases2

1985 ◽  
Vol 75 (5) ◽  
pp. 823-829 ◽  
Author(s):  
Mark S. Langley ◽  
Geoffrey L. Hammond ◽  
Alan Bardsley ◽  
Ronald A. Sellwood ◽  
David C. Anderson
Keyword(s):  
Binding Proteins ◽  
Steroid Binding Proteins ◽  
Steroid Binding

CHARACTERIZATION AND ASSAY OF THE PROGESTERONE RECEPTOR IN RAT UTERINE CYTOSOL

1978 ◽  
Vol 76 (1) ◽  
pp. 21-31 ◽  
Author(s):  
M. T. VU HAI ◽  
E. MILGROM
Keyword(s):  
Progesterone Receptor ◽  
Receptor Complexes ◽  
High Affinity ◽  
Receptor Sites ◽  
Natural Hormone ◽  
Corticosteroid Binding Globulin ◽  
Steroid Binding Proteins ◽  
Steroid Binding ◽  
High Concentrations ◽  
Synthetic Progestogen

SUMMARY The synthetic progestogen R5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) binds with high affinity (Ka = 8·8 × 108 1/mol at 0 °C) to the progesterone receptor from rat uterine cytosol. At nanomolar concentrations, equilibrium is attained in less than 90 min. R5020 has a very low affinity for other specific steroid-binding proteins (corticosteroid-binding globulin and oestrogen receptors) present in relatively high concentrations in the uterine cytosol. The affinity of the receptor for the natural hormone progesterone is remarkably low (Ka= 1 × 108−1·7 × 1081/mol at 0 °C) which explains the instability of progesterone–receptor complexes. Advantage may be taken of this property to remove endogenous progesterone easily by charcoal treatment at 0 °C, a treatment which does not modify the concentration of receptors. A method based on these characteristics is described for the assay of the total number (progesterone-bound and unbound) of receptor sites in uterine cytosol. This assay may be used in various physiological situations where endogenous progesterone is present at unknown concentrations.

408 Characterization of high-affinity, low-capacity steroid binding proteins in human term placental cytosol

1983 ◽  
Vol 19 ◽  
pp. 136
Author(s):  
H.J. Grill ◽  
A. Knichel ◽  
G. Schweikhart ◽  
T. Beck ◽  
B. Manz ◽  
...  
Keyword(s):  
Binding Proteins ◽  
High Affinity ◽  
Steroid Binding Proteins ◽  
Steroid Binding

Plasma Steroid-Binding Proteins in the Cysts of Gross Cystic Disease of the Breast*

1985 ◽  
Vol 61 (1) ◽  
pp. 200-203 ◽  
Author(s):  
WILLIAM ROSNER ◽  
M. SAEED KHAN ◽  
CHARLES N. BREED ◽  
MARTIN FLEISHER ◽  
H. LEON BRADLOW
Keyword(s):  
Binding Proteins ◽  
Cystic Disease ◽  
Steroid Binding Proteins ◽  
Steroid Binding

scholarly journals Nuclear acceptor sites for androgen-receptor complexes in seminal-vesicle epithelium

1980 ◽  
Vol 192 (1) ◽  
pp. 41-47 ◽  
Author(s):  
M J Weinberger ◽  
C M Veneziale
Keyword(s):  
Seminal Vesicle ◽  
Nuclear Dna ◽  
Steroid Receptor ◽  
Assay Method ◽  
Scatchard Analysis ◽  
Receptor Complex ◽  
Receptor Complexes ◽  
Constant Slope

An assay method in vitro was developed and applied to quantify acceptor binding of steroid-receptor complexes in nuclei from isolated epithelium of guinea-pig seminal vesicle. Steroid-receptor complex prepared from 1-day-castrated animals was incubated with purified nuclei from 1-28 day-castrated animals in a medium containing 0.15 M-KCl. Free and bound steroid-receptor complexes were measured and the data were submitted to Scatchard analysis. With nuclei from 1-day-castrated animals the Kd for binding of cytosolic [3H]dihydrotestosterone-receptor complexes was found to be 0.83 × 10(-10) M and the capacity for binding was 0.35 pmol/mg of nuclear DNA. Scatchard analysis consistently disclosed only a single line of constant slope and gave the same kinetic constants for nuclei obtained from animals castrated up to 28 days before assay. Administration of 2 mg of dihydrotestosterone, 3 alpha-androstanediol or androsterone or 100 microgram of oestradiol-17 beta 1 h before killing of the 1-day-castrated animals that provided the nuclei resulted in a significant decrease in nuclear acceptor binding of the steroid-receptor complex compared with untreated animals. Thus our assay method disclosed nuclear acceptor sites that may be involved in responses to androgens (and oestrogens) in vivo. We conclude that there is a class of nuclear accept or sites of high affinity and limited capacity that may be occupied by steroid-receptor complexes in vivo.

Steroid-binding Proteins in Carcinoma of the Human Male Breast

1976 ◽  
Vol 65 (3) ◽  
pp. 360-363 ◽  
Author(s):  
E. Brad Thompson ◽  
Elliott Perlin ◽  
Douglas Tormey
Keyword(s):  
Binding Proteins ◽  
Male Breast ◽  
Human Male ◽  
Steroid Binding Proteins ◽  
Steroid Binding
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