Corticosterone binding by rat brain cytosol

Significant quantities of corticosterone were associated with macromolecules of the brain cytosol following intrathecal administration of [³H]corticosterone to adrenalectomized rats. Fifteen times more steroid was found associated with proteins from adrenalectomized rats than from control animals or adrenalectomized animals pretreated with corticosterone. Pretreatment of adrenalectomized rats with 11-dehydrocorticosterone, Cortisol and cortisone decreased the amount of [³H]corticosterone found associated with protein, whereas progesterone, oestradiol and testosterone did not interfere with the association of [³H]corticosterone with macromolecules of the cytosol. Further evidence for protein-steroid interaction was obtained by incubating [³H]corticosterone (B), [³H]cortisol (F), or 11-[³H]deoxycortisol (S) with brain cytosols. The degree of binding was in the order B > F > S.     

Cortisol binding in rat skeletal muscle.

Studies of the reversible binding of [3H]cortisol by rat gastrocnemius muscle cytoplasm in vitro reveal specific binding in the 27,000 times g supernatant fraction at 0 degrees. The [3H]cortisol-binding molecule had an apparant Kd value of 1.7 times 10-7 M and the number of binding sites was 0.99 pmol per mg of cytosol protein. Only a single class of [3H]cortisol-binding sites could be detected, whose protein nature was suggested by its susceptibility to nagarse. The [3H]cortisol-protein complex sedimented at similar to 4 S in a 5 to 20% sucrose gradient either in the presence or absence of 0.3 M KCl. Binding increased more than 2-fold in adrenalectomized rats and was markedly reduced in the muscle of rats pretreated with cortisol. In contrast to the binding of [3H]dexamethasone and [3H]triamcinolone acetonide to receptor proteins in muscle, no correlation was found between the ability of various steroids to complete wtth [3H]cortisol binding and their glucocorticoid potency: [3H]cortisol binding was inhibited by a 1000-fold higher concentration of unlabeled cortisol and progesterone but not by dexamethasone or triamcinolone acetonide. It is therefore suggested that the [3H]cortisol-binding reaction is not directly involved in the biological effects of all potent glucocorticoids in skeletal muscle. The [3H]cortisol-binding protein in muscle cytosol could not be unequivocally distinguished from rat plasma corticosteroid-binding globulin, because both had similar steroid specificity and temperature stability, were not markedly affected by--SH reagents, and displayed similar sedimentation properties.     

Demonstration of 8 S-cytoplasmic oestrogen receptor in rat mullerian duct.

1. High affinity macromolecular binding of the non-steroidal synthetic oestrogen [3H]diethylstilboestrol and of [3H] oestradiol-17beta in cytosol of Müllerian duct and uterus, and in blood plasma of perinatal rats, was investigated by sucrose density gradient sedimentation. 2. While [3H] oestradiol was bound to both the characteristic 8 S uterine cytoplasmic receptor and a 4 S component of uterine cytosol and plasma of 11-day-old rats, [3H] diethylstilboestrol was bound almost exclusively by the 8 S cytoplasmic receptor. 3. The greatly reduced binding of [3H] diethylstilboestrol to the 4 S plasma plasmic receptor in the Müllerian duct (precursor of the uterus) of 20-day-old foetuses.     

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[³H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [³H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur.     

Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible

The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15 degrees C, 30 min), and then returning it to pH 6.5 for a second incubation (15 degrees C, 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide [( 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.     

Multiple estrogen binding sites in the uterus: stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites

Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [3H]IA enantiomers (IA-R and IA-S) and [3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [3H]ligand; DES, IA-Rac, IA-R and IA-S. IA-S enantiomer (Kd = 0.67) binds to the estrogen receptor with the same affinity as DES (Kd = 0.71) and four times higher affinity than IA-R (Kd = 2.56). The number of binding sites for IA-S is approximately the same as estradiol, DES and IA-Rac while IA-R binds far fewer sites than the other ligands. Saturation binding assays indicated that [3H]DES and [3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [3H]DES and [3H]IA compounds bound to the 8S form of the receptor, the same as E2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displaced by the addition of excess unlabeled E2 and DES. Further characterization of the 4S component is described.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

Effect of ATP on the regulation of the steroid binding activity of the oestradiol receptor

We have observed that ATP induces a second type of oestradiol binding site with slightly lower affinity (Ka 3.3 x 10(8) M-1) and lower sedimentation coefficient (4 S) in cytosol from immature lamb uterus and MCF-7 cells. A factor isolated from immature lamb uterine nuclear extract was found to decrease the steroid binding activity of oestradiol receptor that had been purified by heparin Sepharose and oestradiol-Sepharose chromatography. Inhibition of this factor by known phosphatase inhibitors, indicated that this factor may be a phosphatase. Another factor isolated from immature lamb uterine cytosol was found to enhance the effect of ATP on receptor binding in cytosol from immature lamb uterus and MCF-7 cells. The ability of this factor to phosphorylate a partially purified cytosol receptor from immature lamb uterus when incubated with [gamma 32P]ATP, indicates that this factor is a phosphokinase. The phosphorylated products after labeling with [3H]tamoxifen aziridine were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three phosphorylated proteins with molecular weights 150, 97, and 67 kDa bound [3H]tamoxifen aziridine. Ammonium sulphate precipitated cytosol oestradiol receptor from immature lamb uterus was inactivated with receptor inactivating factor and then reactivated with receptor activating factor in the presence of [gamma 32P]ATP and substantially affinity labelled with [3H]tamoxifen aziridine. The affinity labelled oestradiol receptor was immunopurified with the monoclonal antibody JS 34/32. Three proteins with molecular weights 67, 50 and 43 kDa specifically bound [3H]tamoxifen aziridine and only 43 kDa receptor fragment was phosphorylated. The relevance of inactivation/reactivation of oestradiol receptor to the dephosphorylation/phosphorylation of receptor is discussed.     

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.     

Rat uterine progesterone receptor: Stabilization of hormone-binding components for biochemical analyses

A method was developed for quantitative recovery of the labile rat uterine progesterone receptor hormone-binding components. Initial conditions were established by the sucrose gradient procedure. Upon centrifugation through low-salt 5–20% sucrose gradients prepared in 10% glycerol, the well-known 6–8 S progesterone receptor components were observed either when cytosol was prelabeled with [³H]17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-dione ([³H]R5020) or when prelabeled with [³H]progesterone followed by postlabeling the fractions collected after centrifugation with either [³H]progesterone or [³H]R5020. Recovery of progesterone receptor binding was improved by prelabeling with [³H]R5020, by adding 1.5 mm ethylene glycol bis(β-aminoethylether)N,N′-tetraacetic acid (EGTA) to all buffers, and at high tissue concentrations. Under these conditions quantitative conversion of the receptor to specific [³H]R5020-binding 4S components was achieved with 150 or 400 mm KCl. Similar conditions proved unsuitable for receptor analysis by gel filtration (Bio-Rad agarose A0.5M or A1.5M), apparently due to [³H]R5020 dissociation from the receptor in the large volume of elution buffer. However, excellent receptor recovery (97.2 ± 6.7%) was achieved by including 10 nm unlabeled progesterone in all preparation and elution buffers. Receptors were then detected by the addition of 5 nm [³H]R5020 to the column fractions, exchange incubation for 3–6 h at 4 °C, and subsequent separation of bound and free steroid by the hydroxyapatite assay. This method resulted in a consistent elution pattern suggestive of receptor heterogeneity. Identity of the peak(s) as progesterone receptor components(s) was confirmed by the lack of competition by 2 μm cortisol when added either to cytosol or during the post-labeling-exchange process. Neither the qualitative nor quantitative results of the column profiles were changed substantially in the presence of 20 mm molybdate. Although the receptor structure has yet to be established, both statistical analysis of the column profiles by computer curve-fitting procedures and rechromatography of peak fractions suggested that the rat uterine progesterone receptor may be composed of multiple components. This ligand-stabilization/postlabeling-exchange procedure provides a method for further studies of progesterone receptor biochemistry in mammalian systems. Additionally, similar procedures may stabilize other labile ligand-binding proteins for biochemical analyses and/or purification.     

In vivo kinetics and DNA-binding properties of the Ah receptor in the golden Syrian hamster

The in vivo long-term cytosolic-nuclear kinetics and DNA-binding properties of the Ah receptor were examined in liver from the golden Syrian hamster. For the kinetic studies, a dose of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) that has been previously shown to produce maximal and sustained hepatic enzyme induction without substantial toxicity was used. Following an intraperitoneal dose of 10 micrograms/kg of [3H]TCDD, occupied cytosolic receptor levels reached a peak within 8 h and then decreased rapidly to a level that was approximately 2% of the total receptor. Throughout the 35-day period, unoccupied cytosolic receptor represented from 65 to 80% of the total receptor content. At 8 h following dosing, less than 30% of the total amount of receptor was associated with the nuclear fraction; this percentage declined slowly to less than 5% of the total at Day 35. The half-life for the decline in detectable nuclear receptor levels was 13 days and was similar to the half-life for the decline in [3H]TCDD content of the whole liver, cytosol, and nuclear extract. The Ah receptor contained in hamster hepatic cytosol underwent a ligand-dependent transformation in vitro to two forms having affinity for DNA-Sepharose, one of which was isolated from nuclei of animals treated with [3H]TCDD in vivo. A comparison of the specific binding recovered following various analytical procedures revealed that the binding of [3H]TCDD to the form not found in nuclear extracts was more labile under certain experimental conditions. These studies indicate the heterogeneity of the Ah receptor in hamster hepatic cytosol and suggest that DNA binding in vitro and nuclear uptake in vivo occur through a ligand-dependent transformation process. The maintenance of maximal hepatic enzyme induction is, in part, a consequence of the sustained presence in the nucleus of only a small percentage of the total receptor content. The whole-tissue kinetics of TCDD appears to be a major factor regulating the long-term retention of the TCDD-receptor complex in the nucleus.     

In Vitro Gibberellin A(1) Binding in Zea mays L

The first and second leaf sheaths of Zea mays L. cv Golden Jubilee were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [³H]gibberellin A₁ (GA₁) to a soluble macromolecular component present in the cytosol was demonstrated at 4°C by Sephadex G-200 chromatography. The binding component was of high molecular weight (HMW) and greater than 500 kilodaltons. The HMW component was shown to be a protein and the ³H-activity bound to this protein was largely [³H]GA₁ and not a metabolite. Binding was pH sensitive but only a small percentage (20%) appeared to be exchangeable on addition of unlabeled GA₁. Both biologically active and inactive GAs and non-GAs were able to inhibit GA₁ binding. [³H]GA₁ binding to an intermediate molecular weight (IMW) fraction (40-100 kilodaltons) was also detected, provided cytosol was first desalted using Sephadex G-200 chromatography. Gel filtration studies suggest that the HMW binding component is an aggregate derived from the IMW fraction. The HMW binding fraction can be separated into two components using anion exchange chromatography.     

Insulin modulates rat liver glucocorticoid receptor

This investigation used cytosol fraction of rat liver to examine the effects of insulin (INS) on functional properties of glucocorticoid receptor (GR). Male Wistar rats (220-250 g b.wt.) were injected with INS (50 microg/200 g b.wt, i.p.) and 18 h after INS administration used for experiments. INS-stimulated dissociation of G-R complexes was significantly increased by 133% compared to control level. However, INS treatment significantly stimulated stability of GR protein by 138% above control value. Furthermore, results show that INS stimulated activation of formed cytosol [3H] TA-R complexes by 143% in respect to control. [3H]TA-R complexes from INS treated animals could be activated and accumulated at higher rate in cell nuclei of control animals. The physiological relevance of the data was confirmed by INS-related stimulation of Tryptophan oxigenase (TO) activity. It was observed that INS stimulated TO activity while INS injected to adrenalectomized rats, exhibited less effects compared to control. The results indicate that a glucocorticoid hormone (CORT) enhances INS induced stimulation of TO activity, as evidenced by enhanced enzyme activity. Presented data suggest: that INS treatment leads to modifications of the GR protein and the nuclear components and that INS activates the rat liver CORT signaling pathway which mediates, in part, the activity of TO.     

Assessment of progestin receptor polymorphism by various synthetic ligands using HPLC

Tritiated R5020 and [3H]ORG-2058 were utilized to investigate apparent polymorphism of progestin receptors by vertical-tube gradient centrifugation and HPLC in size exclusion (HPSEC), ion-exchange (HPIEC) and chromatofocusing (HPCF) modes. Rapid centrifugation (3 h) following molybdate stabilization (1 h) showed mainly 8-9S receptor species with 90-96% recovery. [3H]R5020 appeared to associate with a receptor isoform sedimenting faster than that bound to [3H]ORG-2058. Excess unlabeled R5020 did not eliminate all of the [3H]R5020 binding by the 8-9S component suggesting some nonspecific association while excess unlabeled ORG-2058 suppressed this binding by either ligand. Separate labeling of cytosol with each ligand and mixing prior to gradient separation showed at least two receptor species isoforms sedimenting in the 8-9S region with mol. wt of 190,000 and 173,000 D. Sephacryl S-300 chromatography revealed two radioactive peaks with either ligand but with slight molecular weight differences. HPSEC confirmed the presence of isoforms with different molecular size and shape as a function of the radioactive ligand employed. HPIEC showed the presence of two labeled receptor species irrespective of the ligand used. The first peak appeared at the void volume of the column (10 mM), co-eluted with free ligand, indicating the possibility of ligand stripping by the column. The second peak bound both steroids specifically and eluted with 100 mM phosphate. HPCF identified a single specific receptor eluting at a pH of 5.6-6.1, but with free steroid in the void volume irrespective of the ligand used. [3H]ORG-2058 appeared to be less prone to the stripping phenomenon than was [3H]R5020. These data suggest these ligands either bind to different progestin receptor species or they modify receptor characteristics in a fashion that allows separation based upon size and shape.     

Differences between cytosol receptor complexes with corticosterone and dexamethasone in hippocampal tissue from rat brain.

The binding of [3H]corticosterone and [3H]dexamethasone to soluble macromolecules in cytosol of the hippocampal region of the brain has been studied in adrenalectomized male rats. Unlabeled dexamethasone appears to be a less effective competitor than corticosterone in the binding of [3H]corticosterone, while both unlabeled steroids compete equally well for the binding or [3H]dexamethasone. Further investigation of macromolecular complexes with [3H]dexamethasone and [3H]corticosterone revealed that they differ from each other in their behavior during ammonium sulfate precipitation, BioRad A-5M gel permeation chromatography, DE-52 anion exchange chromatography and DNA-cellulose chromatography. (1) After exposure to a 33% ammonium sulfate solution relatively more [3H]dexamethasone complex than [3H]corticosterone complex is precipitated. (2) Treatment of the cytosol with 0.3 M KCl gives disaggregation of the supramolecular 3H-labeled corticoid complexes which are seen eluting with the void volume during gel permeation chromatography on Biorad A-5M at low ionic strength. In 0.3 M KCl, the [3H]dexamethasone complex has an elution volume somewhat smaller than that of bovine serum albumin, while the [3H]-corticosterone complex in 0.3 M KCl is too unstable to survive chromatography with A-5M. (3) Chromatography on DE-52 resolved the 3H-labeled corticoid complexes into three binding components. The complex with [3H]dexamethasone contains a higher percentage (85%) of a component less firmly attached (i.e. eluted by 0.15 M KCl) to the anion exchange resin than is observed for the complex with [3H]corticosterone (49%). (4) The complexes with 3H-labeled corticoids display an enhanced affinity for calf thymus DNA adsorbed to cellulose following "activation", warming to 25 degrees C for 15 min. Concurrently, a fraction of the [3H]dexamethasone complex becomes able to more firmly attach to the DE-52 anion exchange resin. These results with the binding of the cytosol hormone-receptor complexes to DNA-cellulose do not explain the marked in vivo preference of hippocampus for the cell nuclear uptake of [3H] corticosterone. However, the other differences in the properties of the complexes formed with the two labeled glucocorticoids support our previous inference that there may be more than one population of adrenal steroid "receptors" in brain tissue.     

Structure and function of the Ah receptor: sulfhydryl groups required for binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to cytosolic receptor from rodent livers

Cytosol from rodent liver was exposed to a variety of sulfhydryl-modifying reagents to determine if the cytosolic Ah receptor contained reactive sulfhydryl groups that were essential for preservation of the receptor's ligand binding function. At a 2 mM concentration in rat liver cytosol, all sulfhydryl-modifying reagents tested (except iodoacetamide) both blocked binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to unoccupied receptor and caused release of [3H]TCDD from receptor sites that had been labeled with [3H]TCDD before exposure to the sulfhydryl-modifying reagent. Exposure of cytosol to iodoacetamide before labeling with [3H]TCDD prevented subsequent specific binding of [3H]TCDD, but iodoacetamide was not effective at displacing previously bound [3H]TCDD from the Ah receptor. The mercurial reagents, mersalyl, mercuric chloride, and p-hydroxymercuribenzoate, were more effective at releasing bound [3H]TCDD from previously labeled sites than were alkylating agents (iodoacetamide, N-ethylmaleimide) or the disulfide compound 5,5'-dithiobis(2-nitrobenzoate). Presence of bound [3H]TCDD substantially protected the Ah receptor against loss of ligand binding function when the cytosol was exposed to sulfhydryl-modifying reagents. This may indicate that the critical sulfhydryl groups lie in or near the ligand binding site on the receptor. Subtle differences exist between the Ah receptor and the receptors for steroid hormones in response to a spectrum of sulfhydryl-modifying reagents, but the Ah receptor clearly contains a sulfhydryl group (or groups) essential for maintaining the receptor in a state in which it can bind ligands specifically and with high affinity.     

Purification and characterization of the androgen receptor from rat ventral prostate

The androgen receptor has been purified from rat ventral prostate cytosol by a combination of differential DNA-Sepharose 4B chromatography and testosterone 17 beta-hemisuccinyl-3,3'-diaminodipropylamine-Sepharose 4B affinity chromatography. Approximately 8 micrograms of protein was obtained from 38 g of rat ventral prostate, with a yield of 24%. The receptor was purified approximately 120 000-fold. Silver nitrate staining of a sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel revealed a major polypeptide band migrating at 86 000 daltons. Affinity labeling of a partially purified receptor preparation with either 17-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one or 17 beta-hydroxy-[1,2,4,5,6,7,16,17-3H8]-5 alpha-androstan-3-one 17-(2-bromoacetate) produced a major band of radioactivity migrating at 86 000 daltons on a NaDodSO4 gel. Under nondenaturing conditions, a Mr of 85 000 was determined by gel filtration (42 A) and sucrose gradient sedimentation analysis (4.5 S). The purified receptor had an isoelectric point of 6.3 [3H]-4,5 alpha-Dihydrotestosterone, bound to the purified receptor, was displaced with 4,5 alpha-dihydrotestosterone greater than testosterone much greater than progesterone greater than 5 alpha-androstane-3 alpha, 17 beta-diol greater than 17 beta-estradiol greater than cortisol. A number of physicochemical properties of the purified receptor were similar to those of the receptor in crude cytosol.     

Pituitary 1,25-dihydroxyvitamin D3 receptors in hyperthyroid- and hypothyroid-rats

The binding of 1 alpha,25-dihydroxy (26,27-methyl-[3H]) cholecalciferol ([3H]1,25-(OH)2D3) to its receptor in cytosol of the anterior pituitary cells was examined in hyperthyroid- and hypothyroid rats, as well as in normal rats. The binding capacity increased by 41% in L-Thyroxine-treated hyperthyroid rats and decreased by 49% in propylthiouracil-ingested hypothyroid rats as compared with normal control rats, whereas the affinity of the receptor for [3H]-1,25(OH)2D3 showed no difference among these 3 animal groups. These findings indicate that the number of 1,25(OH)2D3 receptors in the pituitary may be regulated by thyroid hormone, and further suggest that 1,25-(OH)2D3 may play some role in regulating functions of the anterior pituitary.