EFFECT OF SODIUM MOLYBDATE ON THE INTERACTION OF ANDROGENS AND PROGESTINS WITH BINDING PROTEINS IN HUMAN HYPERPLASTIC PROSTATIC TISSUE

1982 ◽  
Vol 92 (1) ◽  
pp. 95-102 ◽  
Author(s):  
D. A. N. SIRETT ◽  
J. K. GRANT
Keyword(s):  
Binding Sites ◽  
Hormone Receptors ◽  
Sodium Molybdate ◽  
Nuclear Extract ◽  
Steroid Hormone Receptors ◽  
Prostatic Tissue ◽  
Reliable Measurement ◽  
Nuclear Extracts ◽  
Steroid Binding ◽  
Androgen Binding

A reliable measurement of steroid hormone receptors is essential for attempts to correlate receptor levels with response to endocrine therapy in prostatic carcinoma. Evidence that receptors in many tissues are stabilized by sodium molybdate prompted the examination of the effects of this salt on the measurement of steroid-binding sites in human prostatic tissue. The presence of molybdate (10 mmol/l) during tissue homogenization, cytosol or nuclear extract preparation and binding-site assay led to a threefold increase in the amount of highaffinity androgen binding detected in cytosol, and a slight increase in the number of cytosol progestin-binding sites. The apparent binding affinity for steroids was increased in both cases. No effect of molybdate was observed on androgen-binding sites in nuclear extracts.

Effect of thiol blocking agents on the binding of T3 and T4 in rat liver nuclear extract

1981 ◽  
Vol 98 (1) ◽  
pp. 68-72 ◽  
Author(s):  
J. Knopp ◽  
J. Brtko
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Nuclear Extract ◽  
Blocking Agent ◽  
Binding Affinities ◽  
Structural Configuration ◽  
Blocking Agents ◽  
Nuclear Extracts ◽  
Liver Nuclei ◽  
Apparent Association

Abstract. Liver nuclei bind thyroid hormones (T3 and T4) with different binding affinities. In our studies it was estimated that the apparent association constant (Ka) in the liver nuclei for T3 was 8.8 × 1010 l mol−1 and for T4 was 4.1 × 109 l mol−1. We have found that the binding sites for T4 are also saturated by an excess of unlabelled T4 and that saturation was dependent on the purification step of liver binding proteins. In liver nuclear extracts with 0.4 mol l−1 KCl the specific binding of T3 and T4 was blocked with a typical -SH blocking agent N-ethylmaleinimide (NEM) and by new types of -SH blocking agents such as p-bromphenylisothiocyanate (p-BPI) and 2,3 dicyano-1,4-dithio-9,10 antrachinone (Delan). NEM and p-BPI increased the non-specific binding of T4 and completely abolished specific binding. All these agents blocked the specific binding of T3. These results demonstrate that T3 and T4 binding sites in the liver nuclei may not be altogether identical and that the different effects of -SH blocking agents on the binding of T3 and T4 is probably associated with the structural configuration of these drugs.

The thyroid hormone analogue SKF-94901 and iodothyronine binding sites in mammalian tissues: Differences in cytoplasmic binding between liver and heart

1991 ◽  
Vol 124 (1) ◽  
pp. 37-44 ◽  
Author(s):  
John W. Barlow ◽  
Lorna E. Raggatt ◽  
Chen-Fee Lim ◽  
Emily Kolliniatis ◽  
Duncan J. Topliss ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Cell Membrane ◽  
Binding Sites ◽  
Macaca Fascicularis ◽  
Nuclear Extract ◽  
Hormone Analogue ◽  
Nuclear Extracts ◽  
Significant Difference ◽  
Mammalian Tissues ◽  
Different Response

Abstract. The thyroid hormone analogue, SKF-94901 exhibits greater thyromimetic activity in the liver than in the heart. This difference in activity may reflect heterogeneity in the affinity of SKF-94901 for different forms of the T3 receptor. A difference in extranuclear transport of the analogue could also account for the different response of these two tissues. To distinguish between these possibilities we have examined the binding of SKF-94901 to membrane, cytosolic and nuclear preparations from liver and heart of the primate, Macaca fascicularis. Uptake of SKF-94901 into H4 liver cells was low. Binding of [125I]T3 to cell membrane preparations (Kd ≈3 μmol/l), and to nuclear extracts (Kd ≈0.2 nmol/l was displaceable by SKF-94901 with a potency 2-5% that of T3 in each case. No significant difference was observed between liver and heart for SKF-94901 binding to membranes or nuclear extract. With cytosol, [125I]T3 binding was identical in heart (Kd, 22.7±10.4 nmol/l) and liver tissue (Kd, 30.3±11.1 nmol/l). In liver, and in cardiac cytosol after preliminary washing to remove serum, iodothyronine potency was in the order T3 > T4 > rT3. The ratio of SKF-94901 to T3 concentrations which gave 50% displacement was 15.9±6.8 in the liver; and 152.3±89.1 in the heart (p<0.05). The selective tissue activity of SKF-94901 may be related to a reduced affinity of the analogue for the cytosolic binding proteins in the heart, rather than a difference in affinity for various forms of the T3 receptor.

Characterization of thyroid hormone receptors in human IM-9 lymphocytes

1988 ◽  
Vol 117 (3) ◽  
pp. 327-332 ◽  
Author(s):  
John W. Barlow ◽  
Philippe De Nayer
Keyword(s):  
Thyroid Hormone ◽  
Binding Sites ◽  
Hormone Receptors ◽  
Thyroid Hormone Receptors ◽  
Finite Capacity ◽  
Thyroid Hormone Action ◽  
Nuclear Extracts ◽  
Triton X ◽  
Scatchard Plots

Abstract. Although putatively identified more than 10 years ago, thyroid hormone receptors in human tissues remain poorly characterized. As a first step towards understanding the mechanism of thyroid hormone action in man we have characterized T3 binding sites in nuclei of the human lymphoblastoid line, IM-9 cells. In whole cell experiments at 37°C, nuclear binding of [125I]T3 was saturable (Kd 34 ± 6 pmol/l) and of finite capacity (≈ 350 sites/cell). The binding sites were extracted from a nuclear pellet by treatment with 0.4 mol/l KCl and sonication. Separation of bound from free [125I]T3 in the extracts was achieved using the calcium phosphate matrix, hydroxyapatite at a concentration of 0.3 ml of a 150 g/l slurry. Rectilinear Scatchard plots were obtained only when the hydroxyapatite was washed with a buffer containing 0.5% Triton X-100. Under these conditions T3 binding sites in the nuclear extracts were present at a concentration of 22.4 ± 8.6 fmol/mg protein and showed an affinity of (Kd, room temperature) 140 ± 10 pmol/l. The same assay system was used to determine the hierarchy of affinities for a range of natural and synthetic analogues. Calling T3 100, the order of potencies observed was: Triac, 500; 3,5-diiodo-3′-isopropylthyronine, 89; T4, 32; 35-dimethyl-3′-isopropylthyronine, 2; 3,2-T2, 0.7, rT3, 0.4; 3′5′-T2, < 0.01. These results suggest that the T3 binding sites present in human IM-9 lymphocyte nuclei and extracts thereof are thyroid hormone receptors. These cells may be a useful tool to increase our understanding of human T3 receptors. The use of hydroxyapatite to separate bound from free hormone may be adapted for use with extracts of other tissues to characterize these receptors further.

ANDROGEN BINDING IN CYTOSOLS AND NUCLEI OF HUMAN BENIGN HYPERPLASTIC PROSTATIC TISSUE

1978 ◽  
Vol 77 (1) ◽  
pp. 101-110 ◽  
Author(s):  
D. A. N. SIRETT ◽  
J. K. GRANT
Keyword(s):  
Binding Sites ◽  
Androgen Receptors ◽  
Dissociation Constants ◽  
Nuclear Fraction ◽  
Tissue Samples ◽  
Nuclear Extracts ◽  
Receptor Interactions ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation ◽  
Androgen Binding

Androgen binding sites have been detected in cytosols and nuclear extracts from human benign hyperplastic prostatic (BPH) tissue with exchange assays using [3H]methyltrienolone and [3H]5α-dihydrotestosterone respectively. The concentrations of binding sites and the equilibrium dissociation constants for the [3H]steroid–receptor interactions have been determined and the specificity of the binding has been examined. The observed properties of the binding sites are consistent with those characteristic of androgen receptors. The binding sites are present in nuclear extracts from all BPH tissue samples examined to date. The amount of binding detected in the nuclear fraction is higher than that found in the cytosol.

CONTROL MECHANISMS OF STEROID HORMONE RECEPTORS IN THE REPRODUCTIVE TRACT

1973 ◽  
Vol 74 (Suppl) ◽  
pp. S380-S403 ◽  
Author(s):  
E. Milgrom ◽  
M. Luu Thi ◽  
E. E. Baulieu
Keyword(s):  
Binding Sites ◽  
Hormone Receptors ◽  
Reproductive Tract ◽  
Hormonal Control ◽  
Steroid Hormone Receptors ◽  
Target Cells ◽  
Control Mechanisms ◽  
Target Organs ◽  
Sequential Administration

ABSTRACT The hormonal control of the amount of steroid receptors in the target cells may be a clue for the understanding of hormone receptivity and of integrated hormonal mechanisms (for example cyclic sequences of events). As a model system, the guinea pig uterus progesterone receptor has been studied. After an analysis of the theoretical and practical parameters which should be taken into account in order to measure specifically the receptor binding sites, the protein-RNA synthesis mediated induction of the progesterone receptor by oestradiol is described. The half life of the receptor is about 5 days in hormone deprived animals (in vivo experiments), but in case of progesterone administration it decays very rapidly, due probably to a rise in inactivation rate. Sequential administration of oestradiol and progesterone reproduces the changes observed during the oestrous cycle. The mechanism of the apparent inactivation of the receptor after binding of its own hormonal ligand is unknown. Some information available about the hormonal control of the androgen and oestrogen receptors in their respective target organs is reviewed.

Steroid hormone receptors and the sexual phenotype of the Harderian gland in hamsters

1989 ◽  
Vol 121 (1) ◽  
pp. 149-156 ◽  
Author(s):  
F. Vilchis ◽  
G. Pérez-Palacios
Keyword(s):  
Androgen Receptor ◽  
Binding Site ◽  
Hormone Receptors ◽  
Steroid Hormone ◽  
Sedimentation Coefficient ◽  
Harderian Gland ◽  
Steroid Hormone Receptors ◽  
Progestin Receptors ◽  
Female Gland ◽  
Androgen Binding

ABSTRACT To investigate the participation of intracellular steroid hormone receptors in the sexual transformation process of the Harderian gland, a series of experiments were undertaken in adult golden hamsters. The in-vitro labelling of cytosolic steroid-binding sites with appropriate radioligands revealed the presence of androgen, oestrogen and glucocorticoid but not progestin receptors in the glands from animals of both sexes. The androgen receptor of the female gland was further characterized because it was found to be the predominant intracellular steroid receptor. Studies of binding kinetics using [3H]7α,17α-dimethyl-17β-hydroxy-4-oestren-3-one (DMNT) as ligand, demonstrated a high affinity androgen-binding site with an apparent dissociation constant (Kd) of 0·7 nmol/l and maximal saturation binding capacity of 84·0 ± 3·0 (s.d.) fmol/mg protein. Specificity of the androgen receptor was assessed by displacement analysis; DMNT, 5α-dihydrotestosterone, testosterone and 3α-androstanediol were efficient competitors for the androgen-binding site, while oestradiol-17β, progesterone and dexamethasone exhibited very little, if any, competitive potency. The sedimentation coefficient of the androgen receptor in sucrose density gradients was 8–9 S. These data indicate that the physicochemical characteristics of the androgen receptor from the female gland are similar to those previously described in the male gland. The striking observation of a complete lack of oestrogen-inducible and oestrogen-insensitive progestin receptors in glands cytosol, even after stimulation with cholera toxin, adds further support to the concept that the androgen receptor is the key molecule mediating the hormone-induced sexual transformation of the Harderian gland in this species. Journal of Endocrinology (1989) 121, 149–156

Tamoxifen binding sites heterogeneity in breast cancer: a comparative study with steroid hormone receptors

1991 ◽  
Vol 27 (4) ◽  
pp. 452-456 ◽  
Author(s):  
Giuseppe Leo ◽  
Gabriella Cappiello ◽  
Palmiro Poltronieri ◽  
Carmela Giardina ◽  
Corrado Manca ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Comparative Study ◽  
Binding Sites ◽  
Hormone Receptors ◽  
Steroid Hormone ◽  
Steroid Hormone Receptors

A novel class of inactive steroid-binding sites in female rat liver nuclei

1986 ◽  
Vol 110 (1) ◽  
pp. 27-36 ◽  
Author(s):  
D. M. Bechet ◽  
B. N. Perry
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Specific Binding ◽  
Gel Filtration ◽  
Nuclear Extract ◽  
Female Rat ◽  
Rat Liver Nuclei ◽  
Heat Stable ◽  
Nuclear Extracts ◽  
Liver Nuclei

ABSTRACT Nuclear salt extracts from intact female rat liver showed insignificant levels of progesterone-, oestradiol-, testosterone- or dexamethasone-specific binding. However, brief exposure of nuclear extracts to dextran-coated charcoal (DCC) induced binding for all the above classes of steroids. This 'DCC-effect', which was reproduced neither by gel filtration nor by extensive dialysis of the nuclear extract, could not be ascribed to removal of endogenous free or loosely bound steroids. We show that rat liver nuclei contain a class of secondary binding sites (BsII), which exhibit moderate or low affinity for steroid ligands, positive co-operativity, and cross-reaction between classes of steroids. The capacity of BsII sites to bind steroids depends strictly on prior neutralization by DCC of endogenous heat-stable non-dialysable inhibitor(s). The putative roles of these BsII binding sites are discussed in relation to component(s) probably responsible for inhibitory activity. J. Endocr. (1986) 110, 27–36

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