Steroid Binding Capacity Research Articles

Hydrogen peroxide stabilizes the steroid-binding state of rat liver glucocorticoid receptors by promoting disulfide bond formation.

Hydrogen peroxide and diamide inactivate the steroid-binding capacity of unoccupied glucocorticoid receptors in rat liver cytosol at 0 degrees C, and steroid-binding capacity is reactivated with dithiothreitol. Treatment of cytosol with peroxide or sodium molybdate, but not diamide, inhibits the irreversible inactivation (i.e., inactivation not reversed by dithiothreitol) of steroid-binding capacity that occurs when cytosol is incubated at 25 degrees C. Pretreatment of cytosol with the thiol derivatizing agent methyl methanethiosulfonate at 0 degrees C prevents the ability of peroxide, but not molybdate, to stabilize binding capacity at 25 degrees C. As derivatization of thiol groups prevents peroxide stabilization of steroid-binding capacity and as treatment with dithiothreitol reverses the effect, we propose that peroxide acts by promoting the formation of new disulfide linkages. The receptor in our rat liver cytosol preparations is present as three major degradation products of Mr 40,000, 52,000, and 72,000 in addition to the Mr 94,000 intact receptor. Like the intact receptor, these three forms exist in the presence of molybdate as an 8-9S complex, they bind glucocorticoid in a specific manner, and they copurify with the intact Mr 94,000 receptor on sequential phosphocellulose and DNA-cellulose chromatography. Despite the existence of receptor cleavage products, it is clear that peroxide does not stabilize steroid-binding capacity by inhibiting receptor cleavage.

Time-resolved immunofluorometric assay of sex-hormone binding globulin.

A time-resolved immunofluorometric assay (trlFMA) for human sex-hormone binding globulin (SHBG) is described in which antibody-coated tubes or microliter strip-wells and a europium (Eu) chelate-labeled monoclonal antibody are used. The trlFMA sensitivity is similar to that of other SHBG immunoassays, and other analytical variables compare favorably with an SHBG immunoradiometric assay (IRMA) kit and a steroid binding capacity assay: the interassay coefficient of variation (CV) is less than 8% and the intra-assay CV is less than 6% for concentrations between 6 and 200 nmol/L. The reference intervals (means +/- SD) for SHBG concentrations (nmol/L) in serum from 10 men, 10 women, and 10 pregnant women were 23 +/- 12, 65 +/- 39, and 439 +/- 122, respectively. In 14 hirsute women the mean +/- SD serum SHBG concentration (37 +/- 21 nmol/L) was significantly lower (P less than 0.01) than the mean for an age-matched, nonhirsute female comparison group. The trlFMA is technically simple, requires no centrifugation or separation reagent, and takes a counting time of only 1 s. In addition, the Eu-label is nontoxic, presents no waste-disposal problems, and has a long shelf life.

Relationship between glucocorticoid receptor steroid-binding capacity and association of the M r 90,000 heat shock protein with the unliganded receptor

Treatment of rat liver cytosol with hydrogen peroxide (H 2O 2) or sodium molybdate (MoO 4 2−) inhibits thermal inactivation of glucocorticoid receptor steroid-binding capacity at 25°C. Dithiothreitol (DTT) prevents the stabilization of receptors by H 2O 2. Heating (25°C) of immune pellets formed by immunoadsorption of L-cell murine glucocorticoid receptor complexes to protein-A-Sepharose with an anti-receptor monoclonal antibody (BuGR2) results in dissociation of the M 90,000 heat shock protein (hsp90) from the steroid binding protein. Such thermal-induced dissociation of hsp90 is inhibited by H 2O 2. Pretreatment of immunoadsorbed receptor complexes with the thiol derivatizing agent, methyl methanethiosulfonate (MMTS) prevents the ability of H 2O 2 to stabilize the hsp90-receptor interaction. These data suggest a role for hsp90 in maintaining an active steroid-binding conformation of the glucocorticoid receptor.

The effects of ischemia on estrogen and progesterone receptor profiles in the rodent uterus

Ischemia is considered to invalidate the estrogen receptor (ER) and progesterone receptor (PR) values of tissues available for steroid hormonal analysis. To evaluate the temporal effect of devascularization on steroid receptors, complete uterine ischemia was induced in vivo in 64 female Buffalo rats (80–154 g). Animals were assigned to varying intervals of ischemia (0–90 min) and were maintained at a constant ambient (75°F) and core-body temperature (100–102°F). Following tissue preservation at −80°C, multipoint titration of steroid binding capacity (SBC) was performed with [ 3H]estradiol 17-β (3–0.15 n M) or [ 3H]R5020 (8–0.4 n M) in the presence or absence of a 200-fold excess of an unlabeled ligand. Applying nonlinear regression analysis, ischemia was observed to decrease the binding capacitance for both ER and PR profiles of rodent uterine tissues at 30 min with significant decay over the 90-min interval of devascularization (ER, P < 0.01; PR, P < 0.1). Significant reduction in SBC was evident after 80 min of ischemia for PR ( P < 0.05) and 90 min of tissue ischemia for ER ( P < 0.05) comparative to control ( t 0) valves. The detrimental effect of progressive ischemia on ER and PR values was such that it appears essential to assure rapid and reliable tissue aliquot preservation techniques when organ ischemia ⩾ 90 min is anticipated.

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

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.

Hormone dependency of transformation of rat liver glucocorticoid receptor in vitro: effects of heat, salt, and nucleotides.

A majority of the untransformed glucocorticoid-receptor complexes (GRc) from rat liver cytosol sedimented in the 9S region in 5-20% sucrose gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of the cytosol at 23 degrees C, or at 0 degree C with 10 mM ATP or 0.3 M KCl caused appearance of a slower migrating (4S) form which exhibited an increased affinity toward DNA-cellulose and ATP-Sepharose. Presence of 20 mM Na2MoO4 blocked this 9S to 4S transformation of GRc. A complete conversion of the 9S to the 4S form occurred upon a 2 h incubation of GRc with 10 mM ATP at 0 degree C. Other nucleoside triphosphates (GTP, CTP, and UTP), ADP and PPi (but not AMP or cAMP) were also effective in transforming the 9S form. The heat transformation occurred in a time-dependent manner and was complete within 1 h at 23 degrees C; presence of 10 mM ATP during this 23 degrees C incubation period allowed a complete 9S to 4S alteration in 10-20 min. Addition of ATP also accelerated the rate of salt activation of the GRc; a 50% conversion to the 4S form occurred in 20 min or 3 min in the absence or the presence of 10 mM ATP during the 0 degree C incubation of GRc with 0.15 M KCl. An absolute requirement of the hormone for 9S to 4S transformation of glucocorticoid receptor (GR) was evident, as no conversion of the 9S form to the 4S form could be achieved with the ligand-free GR under any of the above conditions. Incubation of cytosol preparations at 23 degrees C or at 0 degree C with KCl or ATP caused dissociation of the GRc and reduced the steroid binding capacity of GR. Although aurintricarboxylic acid, pyridoxal 5'-phosphate, Na2MoO4, Na2WO4, o-phenanthroline, Rifamycin AF/013 and heparin inhibited the ATP-Sepharose and DNA binding of the GRc, only Na2MoO4 and Na2WO4 selectively blocked the 9S to 4S conversion. We suggest that the 9S to 4S transformation in vitro of rat liver GRc represents an acquisition of DNA and ATP-Sepharose binding ability and may involve a separation of subunits from an oligomeric receptor structure.

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

Serum concentrations of sex hormone binding globulin (SHBG), corticosteroid binding globulin (CBG), and albumin were found to be normal in women with breast cancer (Ca), with benign breast disease (BBD), or with a family history of breast cancer (FHCa). Comparisons between serum steroid binding capacities and immunoassayable SHBG and CBG concentrations did not reveal abnormal forms of either protein. The serum distribution of estradiol (E2) was also determined, and women with Ca were found to have a significantly (P less than .025) higher mean percentage of non-protein-bound E2 than matched controls, but the difference was very small. In general, women with Ca also had proportionately more (P less than .05) albumin-bound E2 and less (P less than .05) SHBG-bound E2 in their sera than the controls, but the serum distributions of E2 in the BBD and FHCa subjects were the same as in controls. The dissociation rates of 5 alpha-dihydrotestosterone and E2 from SHBG in serum appear to increase with time in frozen serum samples, and this factor may effect measurements of the distribution of these steroids in serum.

Plasma sex hormone binding globulin concentration and binding capacity in children before and during puberty

The steroid binding capacity and concentration of plasma sex hormone binding globulin have been compared in 116 children aged between 2 and 14 years. Concentration was measured by electroimmunodiffusion standardised with reference to the mass of the pure protein and binding capacity by quantitating the binding of radiolabelled 5 alpha-dihydrotestosterone. Binding capacity correlated highly with concentration in all subjects and neither differed significantly between the sexes before or during puberty. However, both were significantly lower in pubertal than in pre-pubertal children. These findings suggest the metabolism of the protein is similar in boys and girls and that the fall in its steroid binding capacity at puberty in fact is due to a fall in its concentration rather than to changes in its physicochemical properties.

Heparin-mediated inactivation and transformation of mammary cytoplasmic glucocorticoid receptor

Glucocorticoid receptor of lactating mouse mammary gland cytosol was exposed to heparin when the receptor was either steroid-free or steroid-bound. Heparin caused a dose-dependent and time-dependent loss of steroid binding activity (inactivation) of the steroid-free receptor; this heparin-induced inactivation was inhibited by molybdate. in contrast, steroid-bound receptor maintained its steroid binding capacity in the presence of heparin but the heparin caused transformation of receptor as detected by increased binding to DNA-cellulose and ATP-Sepharose. Heparin also converted steroid-bound receptor from the 7–8S form to the 4S form. Molybdate inhibited both the heparin-induced transformation and associated conversion to the 4S form.

The avian progesterone receptor: Isolation and characterization of phosphorylated forms

Methods have been developed for isolation of the avian progesterone receptor in the nontransformed, molybdate-stabilized state. The final step in this procedure, DEAE-Sephadex chromatography, resolves the receptor into two forms, components I and II. Analysis of these components by polyacrylamide gel electrophoresis under denaturing conditions shows that both contain a peptide with Mr = 90,000. These peptides contain phosphorylated serine residues, as shown by [ 32P]orthophosphate incorporation studies. When the cytosol receptor is treated with alkaline phosphatase, its steroid binding capacity is abolished. These studies show that the nontransformed progesterone receptor is a phosphoprotein and indicate that receptor phosphorylation may be important to the maintenance of steroid binding capacity.

Axonal projections and peptide content of steroid hormone concentrating neurons

The axonal projections of cell groups containing the most dense collections of steroid hormone concentrating cells have been demonstrated with retrograde neuroanatomical tracing methods. Horseradish peroxidase revealed large numbers of neurons in ventrolateral ventromedial nucleus (VL-VM) which project to dorsal midbrain. Wheat germ agglutinin (immunocytochemical recognition method) revealed large numbers of neurons in medial basal hypothalamus (MBH) and particular subdivisions of paraventricular nucleus (PVN) that project to dorsal caudal medulla or spinal cord. Fluorescent dyes revealed that many preoptic area (POA), anterior hypothalamic (AHA), and bed nucleus of the stria terminalis (BNST) neurons project to ventral tegmental area of Tsai (VTA). Also many neurons in POA and BNST project to amygdala. A method which enabled simultaneous demonstration of the steroid binding capacity and axonal projections of neurons in the same tissue section revealed that 26–36% estradiol (E 2) concentrating cells in VL-VM project to dorsal midbrain. E 2 concentrating neurons in POA and BNST project to amygdala and E 2 concentrating POA neurons project to VTA. These neurons, which send their axons to cell groups located in different brain regions, are probably under the genomic-regulatory influence of E 2. Using a method which allows simultaneous demonstration of peptide content and steroid hormone concentrating capacity of cells, many oxytocin-neurophysin and vasopressin-neurophysin containing magnocellular neurons in the caudal PVN were found to concentrate E 2. About 4% of the β-endorphin and about 6% of the dynorphin containing neurons in the MBH concentrate E 2. In contrast, virtually none ( <0.2%) of the LHRH containing hypothalamic neurons concentrate E 2.

A versatile method for the determination of serum cortisol binding globulin and sex hormone binding globulin binding capacities

Cortisol binding globulin (CBG) and sex hormone binding globulin (SHBG) are plasma glycoproteins which bind some of the most important steroid hormones in blood with high affinity and specificity, but limited capacity. Although antisera have been raised against purified preparations of these proteins and radioimmunoassays have been developed to measure their actual serum concentrations [ 1,2], determinations of their steroid binding capacities will continue to be of importance, especially if variants with abnormal steroid binding activities exist [3,4]. In general, most methods currently used to determine the serum or plasma binding capacities of CBG and SHBG yield values which are in close agreement [5,6]. However, some of these methods are not suited for the routine analysis of large numbers of samples [7,8] while others require the use of expensive gels [9,10], or work under cold room conditions [ 111. Ammonium sulphate precipitation is probably the most widely used method to determine serum SHBG binding capacity [ 12141, but this technique is not so easily applied for measurement of CBG binding capacity, and cannot be readily adapted to measure SHBG binding capacity in samples containing low concentrations of protein, such as obtained during purification procedures. It has been our intention therefore to develop a reliable, inexpensive, versatile method that is suitable for the routine determination of both CBG and SHBG in dilute serum samples.

Effects of urea and molybdate on the chick oviduct progesterone receptor

1. 1. The chick oviduct cytosol progesterone receptor, when complexed with ligand, can be exposed to urea concentrations as high as 3 M (at 0°C) without loss of steroid binding capacity. The ligand dissociation rate is increased ≥ 10 fold under these conditions. 2. 2. The “native” 8 S form of the receptor is progressively converted to a 4 S species by urea (> 2M) as seen in ultracentrifugation analysis. This conversion is inhibited by Na 2MoO 4 (5–50 mM) suggesting that molybdate stabilizes the 8 S molecule by direct interaction. 3. 3. At urea concentrations above 2 M, the ligand-free receptor looses progressively its binding capacity. The “transformed”, 4 S receptor was less stable than the 8 S species, and could not be protected by molybdate.

Direct demonstration of glucocorticoid receptor phosphorylation by intact L-cells.

Glucocorticoid-sensitive L-cells were cultured for 18 h in the presence of [32P]orthophosphate and steroid-binding proteins of cytosol were separated by affinity chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Cytosol contains a major phosphoprotein of Mr = 92,000 and a minor phosphoprotein of Mr = 100,000, both of which bind glucocorticoids in a stereospecific, high affinity manner and have the same Mr as glucocorticoid receptor species that have been covalently labeled with the site-specific affinity ligand [3H] 9 alpha-fluoro-16-methyl-11 beta,17 alpha,21-trihydroxypregna-1, 4-diene-3,20-dione 21-mesylate. Cytosol from 32P-labeled, glucocorticoid-resistant L-cells possessing 5% of the steroid-binding capacity of sensitive cells contains very little of the Mr = 92,000 phosphoprotein and none of the Mr = 100,000 phosphoprotein. These observations provide strong evidence that the glucocorticoid receptor is phosphorylated by intact L-cells. The Mr = 92,000 protein is phosphorylated on serine and it can be resolved into two species using isoelectric focusing, consistent with the proposal that there is more than 1 phosphorylated serine/steroid-binding unit. The glucocorticoid-resistant L-cell line produces a unique phosphoprotein of Mr = 104,000 that is recovered in variable amounts after affinity chromatography. It is not known whether this phosphoprotein is a separate gene product or whether it represents a precursor with weak steroid-binding activity that is not cleaved in the resistant cell to the high affinity, Mr = 92,000 mature receptor form.

On the mechanism of estrogen receptor replenishment: recycling, resynthesis and/or processing.

Estrogen receptor replenishment has been extensively studied after a single injection of estradiol-17 beta in the rat. Most studies indicate that replenishment, under these conditions, is due both to recycling and to resynthesis of receptor. In the case of short-acting estrogens, total replenishment occurs in the absence of protein synthesis and loss of nuclear receptor closely corresponds to an increase in cytoplasmic receptor. After estradiol-17 beta injection, there is a loss of nuclear receptor without a corresponding increase in cytoplasmic receptor, leading to a loss in total receptor content or 'processing'. Since little processing occurs with the active, short-acting estrogen, we propose that processing is not essential for estrogen action. Evidence is accumulating to indicate that processing may be due to a reversible inactivation of the steroid binding capacity of the receptor. We discuss a model in which there are two routes for replenishment: a simple equilibrium scheme where no processing occurs and a second route where the receptor is processed to a form with low affinity for estrogen which must be reactivated before binding can occur.

Interaction of rat liver glucocorticoid receptor with sodium tungstate.

Effects of sodium tungstate on various properties of rat liver glucocorticoid receptor were examined at pH7 and pH 8. At pH 7, [3H]triamcinolone acetonide binding in rat liver cytosol preparations was completely blocked in the presence of 10--20 mM-sodium tungstate at 4 degrees C, whereas at 37 degrees C a 30 min incubation of cytosol receptor preparation with 1 mM-sodium tungstate reduced the loss of unoccupied receptor by 50%. At pH 8.0, tungstate presence during the 37 degrees C incubation maintained the steroid-binding capacity of unoccupied glucocorticoid receptor at control (4 degrees C) levels. In addition, heat-activation of cytosolic glucocorticoid-receptor complex was blocked by 1 mM- and 10 mM-sodium tungstate at pH 7 and pH 8 respectively. The DNA-cellulose binding by activated receptor was also inhibited completely and irreversibly by 5 mM-tungstate at pH 7, whereas at pH 8 no significant effect was observed with up to 20 mM-tungstate. The entire DNA-cellulose-bound glucocorticoid-receptor complex from control samples could be extracted by incubation with 1 mM- and 20 mM-tungstate at pH 7 and pH 8 respectively, and appeared to sediment as a 4.3--4.6 S molecule, both in 0.01 M- and 0.3 M-KCl-containing sucrose gradients. Tungstate effects are, therefore, pH-dependent and appear to involve an interaction with both the non-activated and the activated forms of the glucocorticoid receptor.

Binding of sex hormones by male rat liver microsomes

In vitro liver microsomes of adult male rats possess binding sites specific for 17β-hydroxy-5α-androstan-3-one (5α-DHT), estradiol-17β(E 2-17β) and progesterone (Prog), respectively. The characteristics of these microsomal steroid binding were as follows: 1. The equilibrium dissociation constants were estimated to be 35.4, 9.94 and 25.2 nM for 5α-DHT, E 2-17β and Prog, respectively. The maximum numbers of binding sites were estimated to be 0.25, 0.12 and 6.43 pmol/mg of microsomal protein for 5α-DHT, E 2-17β and Prog, respectively. 2. The ligand specificities of microsomal binding for 5α-DHT, E 2-17β and Prog were similar to so-called cytosolic androgen-, estrogen- and progestin-receptors, respectively, with the following two exceptions, namely, unlabeled Prog best inhibited the microsomal [ 3H]-5α-DHT binding and unlabeled DES did not significantly inhibit microsomal [ 3H]-E 2-17β binding. 3. These microsomal steroid binding capacities were not affected significantly by different incubation at 2 and 36°C. The steroid-binding components of microsomes in physiological saline (0.13 M-NaCl) were no longer soluble after 1 M-NaCl extraction. Our data suggest strongly that sex steroids may act on mammalian liver by specific binding to microsomes, which brings about changes of various cytoplasmic events, as well as by their specific binding to so-called cytosolic receptors, which ultimately brings about direct gene activations.

Purification and physicochemical and immunological analysis of chicken alpha-fetoprotein.

Chicken alpha-fetoprotein was isolated from 12 to 13-day-old embryonic chicken serum by column chromatography on CM-Sephadex C-50. Hydroxyapatite and DEAE-Sephadex A-25. The purified protein was homogeneous based on polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The purified protein had the following physicochemical and immunological properties. (1) It was a glycoprotein with a single polypeptide chain. (2) The molecular weight of the protein was estimated at 71,000 by SDS-polyacrylamide gel electrophoresis. (3) The isoelectric point of the protein was 4.90. (4) The amino acid composition of the protein was similar to those of mammalian alpha-fetoproteins. (5) The protein showed no steroid-binding capacity. (6) It was immunologically distinct from mammalian alpha-fetoprotein. (7) No immunological cross-reaction was observed between the protein and chicken albumin.

Androgen, estrogen and progestin binding sites in human leukemic cells.

Sex steroid binding capacity was investigated in malignant cells from 32 patients with acute non-lymphoblastic leukemia (25 patients with acute myeloid leukemia, 4 with subacute leukemia, 3 with chronic myeloid leukemia in blast crisis) and 30 patients with acute lymphoblastic leukemia. Specific binding of labelled steroids was characterized either by competition assay in cytosol fraction or by whole-cell incorporation. In some cases further characterization of the receptor complex was attempted by sucrose gradient centrifugation and gel filtration column. The results show the presence of specific binding sites for dexamethasone (22/32 in non ALL and 30/30 in ALL), for estrogens (11/15 in non-ALL and 5/12 in ALL), for progestins (8/25 in non-ALL and 5/13 in ALL) for androgens when R1881 was used as ligand (8/21 in non-ALL and 5/10 in ALL patients) but only 1/13 non-ALL patients and no ALL patients when labelled 5 alpha DHT was used. These results indicate that the blast cells from patients with acute leukemia contain specific proteins binding steroids with a high affinity. Our results for dexamethasone receptors are similar to those described in the literature in ALL and non-ALL.

Effect of quinoline-type antimalarial drugs on the binding of oestradiol-17 beta and progesterone by rabbit and human uterine cytosols.

Rabbit and human uterine cytosol, prepared and tested in phosphate buffer, bound less oestradiol-17 beta or progesterone than cytosol from the same source prepared and tested in Tris-HCl buffer. Dissociation constants were the same in both buffer systems, and the difference in binding was due to a difference in the number of binding sites. Three quinoline-type antimalarial drugs, chloroquine, quinine and mefloquine, and the quinoline derivative, 4-(4'-hydroxy-1'-methylbutylamino)-7-chloroquinoline, increased the steroid binding capacity of phosphate-buffered cytosol to that of Tris-buffered cytosol, the optimal concentration of quinoline derivative being 1.4-1.6 mM. Tris (50 mM) increased the binding capacity of phosphate-buffered cytosol to that of Tris-buffered cytosol. The effects of Tris and quinoline derivatives were not additive. By gel chromatography and sucrose density gradient centrifugation it was shown that the molecular size and sedimentation behaviour of the oestradiol and progesterone receptors were not affected by the quinoline derivatives. Two types of binding site are proposed, one requiring the presence of low molecular weight, basic compounds. The uterine levels of chloroquine attained by normal pharmacological doses of the drug are potentially capable of influencing the binding of oestradiol-17 beta and progesterone in the uterine cytosol.