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

Detection of corticosteroid binding globulin in parotid fluids: evidence for the presence of both protein-bound and non-protein-bound (free) steroids in uncontaminated saliva

1988 ◽  
Vol 119 (1) ◽  
pp. 56-60 ◽  
Author(s):  
F. W. Chu ◽  
R. P. Ekins
Keyword(s):  
Equilibrium Dialysis ◽  
Binding Effect ◽  
Rate Dependent ◽  
Specific Radioimmunoassay ◽  
Corticosteroid Binding Globulin ◽  
Dilution Experiments ◽  
Steroid Binding Proteins ◽  
Steroid Binding ◽  
Mixed Saliva ◽  
Gingival Fluid

Abstract. Corticosteroid binding globulin (CBG) was detected by a specific radioimmunoassay in mixed saliva (25.4 ± 4.0 μg/l, mean ± sem) and in pure, uncontaminated parotid fluids (17.4 ± 2.7 μg/l) at resting flowrates of approximately 500 μl/min and 50 μl/gland per min, respectively. In parotid fluids collected at stimulated flow-rates of between 300–1000 μl/gland per min, CBG could not be detected. This observation suggests the direct flow-rate-dependent transfer/secretion of CBG in saliva. When cortisol was measured (RIA) in dilution experiments in both mixed saliva and parotid fluids using phosphate buffer at pH 7.4 as diluent, a protein-binding effect analogous to that found in plasma samples was observed. However, this effect was abolished if a known CBG inhibitor, phosphate:citrate buffer at pH 4, was used as the diluent in the assay. A bound fraction of cortisol was found in both mixed saliva (14.0 ± 4.0%) and parotid fluid samples (12.3 ± 1.3%) by equilibrium dialysis. These findings appear to contradict the currently accepted notion that specific plasma steroid binding proteins, and hence the protein-bound steroids, are absent in uncontaminated saliva; and that their presence in mixed saliva is the consequence solely of contamination by gingival fluid and/or plasma from mouth or gum abrasions. We conclude that both protein-bound and free steroids are present in uncontaminated saliva and that salivary total and plasma free steroid concentrations are not identical.

Binding of androgens in 5α-reductase-deficient human genital skin fibroblasts: inhibition by progesterone and its metabolites

1982 ◽  
Vol 94 (3) ◽  
pp. 415-427 ◽  
Author(s):  
M. B. Hodgins
Keyword(s):  
Androgen Receptor ◽  
Androgen Receptors ◽  
Cultured Cells ◽  
Reductase Activity ◽  
External Genitalia ◽  
Skin Fibroblasts ◽  
Urogenital Sinus ◽  
Receptor Complexes ◽  
High Affinity ◽  
Receptor Sites

Binding of [3H]testosterone and 5α-dihydro[3H]testosterone ([3H]DHT) to specific androgen-receptor sites of 5α-reductase-deficient human genital skin fibroblasts (five cell-lines) was studied in the intact cultured cells at 37 °C. Under the conditions of the experiments, conversion of [3H]testosterone into [3H]DHT was negligible. Both steroids bound to the same set of high-affinity saturable sites in cytoplasmic and nuclear fractions of the cells. Unlabelled testosterone, DHT and methyltrienolone competed effectively with the labelled steroids. Progesterone and oestradiol were weaker competitors; cortisol did not compete. The dissociation constant (Kd) for high-affinity complexes with [3H]testosterone (0·44 ± 0·035 nmol/l) was higher than that for [3H]DHT complexes (0·20 ± 0·090 nmol/l). Unlabelled DHT was more effective than unlabelled testosterone in competing with either radioactive steroid. Complexes of [3H]DHT and receptor dissociated more slowly than [3H]testosterone-receptor complexes and [3H]DHT bound more extensively to low-affinity non-saturable sites in fibroblasts. As judged by competition with the radioactive androgens, progesterone bound to the androgen receptor with a Kd of about 7 nmol/l. 5α-Pregnane-3,20-dione had an approximately fivefold lower affinity than progesterone for androgen receptors; 3α/β- or 20α-reduction lowered its affinity further. It is suggested that in 5α-reductase deficiency in man, progesterone in amniotic fluid and blood could effectively inhibit testosterone binding to androgen receptors in the male embryonic external genitalia. One function of the high levels of 5α-reductase activity normally found in embryonic external genitalia and urogenital sinus may be to protect these tissues from the potentially antiandrogenic action of progesterone.

scholarly journals Nuclear binding of progesterone in hen oviduct. Role of acidic chromatin proteins in high-affinity binding

1976 ◽  
Vol 156 (2) ◽  
pp. 409-418 ◽  
Author(s):  
R A Webster ◽  
G M Pikler ◽  
T C Spelsberg
Keyword(s):  
Progesterone Receptor ◽  
Binding Sites ◽  
Acidic Protein ◽  
Affinity Binding ◽  
Target Tissue ◽  
High Affinity Binding ◽  
Receptor Complexes ◽  
High Affinity ◽  
Nuclear Binding ◽  
Acidic Proteins

The multiple classes of binding sites for the progesterone-receptor complex in hen oviduct muclei were found to be of chromatin origin. The highest-affinity, and presumably most physiologically important class, is localized in oviduct chromatin and contains approx. 6000-10000 sites per nucleus. None of these sites is detected in spleen chromatin. Two new techniques were used for assaying rapidly the binding of steroid-receptor complexes to soluble deoxyribonucleoproteins in vito. The extent of high-affinity binding by the nucleo-acidic protein fraction from spleen chromatin is as great as that by the nucleo-acidic protein from oviduct chromatin. Consequently the tissue-specific nuclear binding of the progesterone receptor is found not to be a consequence of the absence of the nuclear binding sites (acceptors) from chromatin of non-target tissue (spleen), but rather a result of complete masking of these sites. In the target-tissue (oviduct) chromatin, approx. 70% of the high-affinity acceptor sites are also masked. Acidic proteins, and not histones, appear to be responsible for the masking of these acceptor sites. In addition, acidic proteins represent (or at least are an essential part of) these high-affinity sites in the oviduct nucleus. Pure DNA displays a few high-and many low-affinity binding sites. In support of previous work with immature chicks, the acidic protein fraction of the nucleo-acidic results thus support the hypotheis that protein complexed with DNA, and not DNA alone, represent the high-affinity binding sites for the steroid-receptor complexes in nuclear chromatin. The lower-affinity classes of binding sites may represent DNA and/or other nuclear components.

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 A subclass of glutathione S-transferases as intracellular high-capacity and high-affinity steroid-binding proteins

1986 ◽  
Vol 235 (3) ◽  
pp. 763-768 ◽  
Author(s):  
H Homma ◽  
H Maruyama ◽  
Y Niitsu ◽  
I Listowsky
Keyword(s):  
Binding Capacity ◽  
High Capacity ◽  
Gel Permeation Chromatography ◽  
Hormone Metabolism ◽  
High Affinity ◽  
Glutathione S Transferases ◽  
Steroid Binding Proteins ◽  
Steroid Binding ◽  
Binding Component

The distribution of glucocorticoids incubated with rat liver cytosol preparations or administered in vivo to adrenalectomized rats was analysed by chromatographic procedures. Corticosterone or dexamethasone was co-eluted with Yb-type GSH S-transferases in anion-exchange and gel-permeation chromatography systems, and these glucocorticoids also were bound to Yb forms in analyses by immunoadsorbent and lysyl-GSH affinity matrices. Pretreatment of cytosol with lysyl-GSH to extract GSH S-transferases or incubation with excess bilirubin, which is expected to compete with steroids for binding to the protein, yielded preparations that were devoid of this major steroid-binding component. In mixtures of the multiple rat GSH S-transferases, corticosterone preferentially interacted with Yb forms rather than Ya and Yc subgroups. All of the multiple Yb forms resolved by chromatofocusing procedures retained the steroid-binding capacity. It is suggested that these abundant proteins can account for a considerable share of intracellular glucocorticoid binding and represent a high-affinity non-saturable binding component with potential to function in steroid-hormone metabolism and action.

scholarly journals Unoccupied nuclear oestradiol-receptor sites in normal human endometrium

1980 ◽  
Vol 185 (3) ◽  
pp. 733-738 ◽  
Author(s):  
C Lévy ◽  
R Mortel ◽  
B Eychenne ◽  
P Robel ◽  
E E Baulieu
Keyword(s):  
Significant Proportion ◽  
Human Endometrium ◽  
Endogenous Hormones ◽  
Association Rate ◽  
Affinity Ligands ◽  
Receptor Complexes ◽  
High Affinity ◽  
Receptor Sites ◽  
Normal Human ◽  
Kinetics Of

The existence of unoccupied nuclear oestradiol-receptor sites in normal human endometrium was investigated. Nuclei were prepared from endometrial samples obtained by curettage and exposed to [3H]oestradiol, which became maximmaly bound at 0 degrees C within 1 h. This result contrasted with the binding kinetics of oestradiol-receptor complexes, since the exchange of hormone took at least 3 h at 30 degrees C and no displacement occurred at 0 degrees C. Before concluding that the nuclear sites were unoccupied, the presence of endogenous low-affinity ligands was excluded, because the association rate of oestradiol was unchanged after nuclei were stripped from their putative ligands, and the displacement of oestrone bound to nuclear receptor by oestradiol was very slow at 0 degrees C. The available sites had high affinity for oestradiol (KD 1.3 nM) and binding-specificity characteristics of oestradiol receptors. Similar results were observed with crude and purified nuclear preparations. It was concluded that a significant proportion of nuclear oestradiol receptors in normal human endometrium is unoccupied by endogenous hormones.

CHARACTERIZATION AND ASSAY OF THE PROGESTERONE RECEPTOR IN RAT UTERINE NUCLEI

1978 ◽  
Vol 76 (1) ◽  
pp. 33-41 ◽  
Author(s):  
M. T. VU HAI ◽  
E. MILGROM
Keyword(s):  
Progesterone Receptor ◽  
Nuclear Receptors ◽  
Specific Binding ◽  
Biological Response ◽  
Fold Increase ◽  
Ovariectomized Rats ◽  
Experimental Conditions ◽  
Receptor Complexes ◽  
Synthetic Progestogen ◽  
Nuclear Progesterone Receptor

SUMMARY Rat uterine nuclei containing unlabelled progesterone–receptor complexes were incubated at 0 °C with the synthetic progestogen [3H]R5020 (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione). Complete exchange of steroid bound to the receptor was observed after 5–6 h. For longer times (up to 26 h) there was no further change in the concentration of steroid–receptor complexes, but the non-specific binding was increased. Saturation of nuclear receptors was obtained with a concentration of 5–10 nm-[3H]R5020. Competition with unlabelled steroids showed that only progestogens inhibited the binding of [3H]R5020 to nuclei. Optimum experimental conditions to reduce the non-specific binding to nuclei were established, and a method for the assay of nuclear progesterone–receptor complexes was devised, based on these characteristics. The concentration of nuclear receptors was low in oestradiol-primed ovariectomized rats; adrenalectomy gave rise to slightly lower values. Injection of the rats with progesterone resulted in a 5·5-fold increase in the number of nuclear receptors and a parallel decrease in the number of cytosol receptors. Similar injections of corticosterone and testosterone were without effect. Nuclear receptors were also shown to be stable in uteri kept in liquid nitrogen for up to 3 weeks. This assay may be used to study the correlation of a biological response to progesterone with the extent of receptor occupation in the nuclei.

scholarly journals Functions of MAPR (Membrane-Associated Progesterone Receptor) Family Members As Heme/Steroid-Binding Proteins

2012 ◽  
Vol 13 (7) ◽  
pp. 687-696 ◽  
Author(s):  
Ikuo Kimura ◽  
Yoshiaki Nakayama ◽  
Morichika Konishi ◽  
Kazuya Terasawa ◽  
Mitsuhiro Ohta ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Binding Proteins ◽  
Family Members ◽  
Steroid Binding Proteins ◽  
Steroid Binding ◽  
Receptor Family
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