Uterine Cytosol Research Articles

Molybdate and the molecular properties of the vervet monkey estrogen receptor

The Vervet monkey ( Cercopithecus aethiops pygerythrus) uterine estrogen receptor was partially characterised. The effect of the molybdate oxyanion on various molecular properties of the receptor was investigated. Molybdate appeared to affect the subunit structure and apparent heterogeneity of the receptor. Anion exchange chromatography of uterine cytosols yielded two ligand binding subunits in a 1:1 ratio in the absence of sodium molybdate, while only a single labelled complex could be demonstrated in cytosols prepared in molybdate containing buffers. Chromatofocussing of the non-stabilized cytosols revealed substantial receptor heterogeniety (7 peaks) while a much simpler pattern (2 peaks) could be observed in the presence of the molybdate. Likewise, iso-electric focussing of labelled cytosols on agarose gels yielded at least 3 high affinity binding components (pI: 6.8, 6.2, 5.9) in the absence and only one major band in the presence of sodium molybdate (pI 5.9).

Fluorescence-based assay of estrogen receptor using 12-Oxo-9(11)-dehydroestradiol-17β

12-Oxo-9(11)-dehydroestradiol-17β (12-oxo-E 2) was used to assay estrogen receptor binding in uterine cytosol preparations by an indirect fluorescence assay. In alkaline solution, 12-oxo-E 2 has a fluorescence excitation maximum at 402 nm (ϵ = 24,000) and an emission maximum at 480 nm ( φ f = 0.57), and its fluorescence can be observed down to 5 × 10 −11 m. The minimum detection limit of 12-oxo-E 2 is 25 fmol by spectrofluorometry and 5 fmol by HPLC-fluorometry. Although this compound is not appreciably fluorescent at neutral pH (i.e., at conditions under which it binds to the estrogen receptor), receptor binding by fluorometry can be measured indirectly: After equilibration of 12-oxo-E 2 with the receptor preparation and removal of excess free ligand, the receptor-12-oxo-E 2 complex is disrupted, and fluorescence measurements are made on the dissociated 12-oxo-E 2 in alkaline medium. This fluorometric assay was validated quantitatively by performing simultaneously, on the same receptor preparation, radiometric and fluorometric assays with [ 3H]E 2 and [ 3H]-12-oxo-E 2. The radiometric determinations with both compounds gave nearly equivalent estimates of receptor site concentrations, but the fluorometric estimate of binding site concentration was somewhat less (70–85%) than that expected on the basis of the [ 3H]E 2 radiometric assay. The use of 12-oxo-E 2 in an indirect spectro- or HPLC-fluorometric assay provides a means for assaying estrogen receptor concentrations by fluorescence with a sensitivity approaching that of radiometric techniques.

Possible mechanism of steroid action of the plant herb extracts glycyrrhizin, glycyrrhetinic acid, and paeoniflorin: Inhibition by plant herb extracts of steroid protein binding in the rabbit

To assess the action of some components of herbal medicine, glycyrrhizin, glycyrrhetinic acid, and paeoniflorin, on steroids, their binding to several classes of intracellular and serum steroid-binding proteins were studied in the rabbit. The affinity (inhibitor constant) for binding of dihydrotestosterone-sex hormone binding globulin in the serum (dissociation constant of 2.0 nmol/L for dihydrotestosterone) was approximately 520 nmol/L, and that for the binding of cortisol-corticosteroid-binding globulin in the serum (dissociation constant of 2.0 nmol/L for cortisol) was approximately 10 μmol/L. In the uterine cytosol, the inhibitor constant value for estradiol receptor binding (dissociation constant of 1.0 nmol/L) was 0.9 μmol/L, and these compounds did not influence progestin receptor binding (dissociation constent of 1.4 nmol/L). The inhibitor constant values for glucocorticoid receptor binding (dissociation constant of 1.0 nmol/L) in the liver cytosol were 3.0 nmol/L for paeoniflorin, 2.0 nmol/L for glycyrrhizin, and 1.7 nmol/L for glycyrrhetinic acid, and those for mineralocorticold receptor binding (dissociation constant = 1.1 nmol/L) in the kidney cytosol were 3.5 nmol/L for paeoniflorin and glycyrrhetinic acid and 3.0 nmol/L for glycyrrhizin. These results suggest that herbal extracts such as the above compounds influence sterold effects by glucocorticoid and mineralocorticoid receptors and to a lesser extent by estrogen receptors or serum sex hormone-binding globulin and corticosteroid-binding globulin.

Competitive binding assay for estrogen receptor in monolayer culture: Measure of receptor activation potency

MCF-7 cells were incubated with [ 3H]estradiol, unlabeled estradiol and various estrogens or antiestrogens to measure their relative binding affinity (whole-cell assay). Comparison of the values with those previously established on uterine cytosol with a DCC assay revealed a good parallelism for both steroid and diphenolic diethylstilbestrol based estrogens. On the contrary, in the whole-cell assay, antiestrogens and weak estrogens of the triphenyl- and gem-diphenylethylene categories always displayed low values which were in the order of magnitude found with weak steroid estrogens. This property was not due to a reduction of binding capacity, nor to the presence in some compounds of an ethoxyaminoalkyl side-chain (source of antiestrogenicity). In view of all these data, it was concluded that the present test would provide an estimate of the ability of a given compound to transform the receptor in a form which interacts with genomic sites involved in the regulation of “estrogenic-induced products“ (“activation”).

Differential effects of molybdate on the hydrodynamic and DNA-binding properties of the non-activated and activated forms of the androgen receptor in calf uterus

Calf uterine cytosol contains an androgen receptor with a relative molecular mass of approx. 90000. In this study we have analysed the structure and aggregation properties of the androgen receptor, using sucrose density gradient centrifugation on a vertical rotor (VTi65). In the presence of 10 mM NaCl the androgen receptor in whole cytosol sedimented at 8 S irrespective of the presence of molybdate. In 400 mM NaCl the receptor dissociated to a 4.3 S entity. In whole cytosol molybdate promoted a partial shift of the 4.3 S receptor into the aggregated 8 S state. The time of exposure of the receptor to molybdate and NaCl determined the proportion of receptor sedimentating at 8 S and 4.3 S. The DNA-binding form of the uterine androgen receptor when analysed under the conditions of the DNA-cellulose binding assay, sedimented at 6.5 S. Increasing concentrations of molybdate shifted its sedimentation coefficient gradually from 6.5 S to 4.5 S and in parallel reduced the DNA-binding capacity. Molybdate added to a partially purified, DNA-binding form of the androgen receptor did not promote receptor aggregation to faster sedimentating forms. This suggests that such preparations are devoid of an androgen receptor-aggregation factor. Indirect evidence for such a factor was obtained from reconstitution experiments with whole cytosol. Our results indicate that the DNA-binding form of the androgen receptor interacts with a cytosol factor to form the 8 S receptor complex. Molybdate has diverse effects: in the presence of the cytosol factor it stabilizes the 8S complex; in its absence molybdate prevents in a concentration-dependent way DNA-binding as well as reaggregation of the monomeric 4.3 S form.

125I]Iododesethyl tamoxifen aziridine: Synthesis and covalent labeling of the estrogen receptor with an iodine-labeled affinity label

Iododesethyl tamoxifen aziridine (I-Tam-Az), an analog of the estrogen receptor-affinity label tamoxifen aziridine (Tam-Az) in which the ethyl group has been replaced by an iodine, has been prepared by two routes: (a) metallation of a bromotriarylethylene system, followed by reaction with iodine, and aziridinylation, and (b) direct iodination of a trimethylstannyl triarylethylene system that is the immediate precursor of I-Tam-Az. The latter method can be used to prepare [ 125I]I-Tam-Az rapidly and in good yield, both at carrier-added and no-carrier-added levels; specific activities greater than 200 Ci mmol have been obtained. In competitive radiometric binding assays with the estrogen receptor, I-Tam-Az has an apparent affinity of ca. 20%, equivalent to that of Tam-Az. It also undergoes rapid and selective time-dependent, irreversible binding to the estrogen receptor. [ 125I]I-Tam-Az reacts covalently with estrogen receptor in uterine cytosol preparations; its attachment is rapid and efficient, but somewhat less selective than that of Tam-Az. Estrogen receptor in intact MCF-7 human breast cancer cells can also be labeled with [ 125I]I-Tam-Az, and autoradiographic analysis of salt extracts of labeled nuclear estrogen receptor on SDS-polyacrylamide slab gels shows highly selective labeling of a 65K protein. [ 125I]I-Tam-Az is an efficient, selective affinity label for the estrogen receptor, available at high specific activity, and should be useful in studies on estrogen receptor structure, dynamics, and chromatin interactions.

Characterization of rabbit uterine estrogen receptor proteins by radioiodination and partial peptide mapping.

Two estrogen binding proteins (Mr = 50,000 and 65,000) were purified from rabbit uterine cytosol using an improved procedure for affinity chromatography on diethylstilbestrol-agarose. The estrogen receptors were radioiodinated while adsorbed to the resin using the lactoperoxidase or Bolton-Hunter techniques. After elution, the labeled receptors were utilized for peptide mapping studies and investigations of receptor function. Partial peptide mapping revealed strong homology between the Mr 50,000 and 65,000 proteins suggesting common structural features. Estrogen receptors labeled by the lactoperoxidase procedure were rendered unable to bind immobilized heparin or hormone; in contrast, the Bolton-Hunter labeling technique yields proteins that retain both their ability to bind hormone and to absorb on heparin-agarose. The development of these iodination methodologies appears useful for the investigation of both the structure and functional properties of the receptor proteins.

Evidence for a single steroid-binding protein in the rabbit progesterone receptor.

The rabbit uterine progesterone receptor copurifies as two molecular weight (Mr) forms of about 105,000 and 78,000. To investigate whether these are different proteins, we have used protease digestion, reversible denaturation, and photoaffinity labeling in studies on the steroid-binding domain of the receptor. Digestion of the Mr 105,000 and 78,000 forms, photoaffinity labeled with [3H]R5020, with Staphylococcus aureus V8 protease revealed identical peptide fragments of Mr 43,000, 39,000, and 27,000-30,000. When receptor in cytosol was denatured, separated by electrophoresis, and then reconstituted, [3H]progesterone bound specifically to a single form at about Mr 105,000. After partial purification, the reversible denaturation procedure revealed both the larger and the smaller progesterone-binding species similar to the photoaffinity-labeled species in this preparation. Receptor in uterine cytosol prepared under mild conditions appeared as a predominant large molecular weight form on photoaffinity labeling with [17 alpha-methyl-3H]R5020, [6,7-3H]R5020, or [3H]RU27987. Further purification of this cytosol showed the generation of a smaller labeled species. These results from three different approaches reinforce the view that the rabbit progesterone receptor contains a single steroid-binding protein.

Competitive Binding of Phytoestrogens in Coffee Extracts with 17β-Estradiol to Uterine Cytosol receptors1

Competitive protein binding assays, employing uterine cytosol from immature ewe lambs, were used to assess the potency of naturally occurring phytoestrogens extracted from coffee. Significant binding of coffee extracts to uterine cytosol receptor proteins was observed in aliquots derived from ethyl ether filtrates of green and roasted coffee beans and instant coffee powders. No activity was observed in extracts derived from ligroin ether. Binding affinites of the coffee extracts parallelled known phytoestrogen and 17β- estradiol standards; however, the in vitro biological potency was much weaker.

Standardization of steroid receptor assays in human breast cancer—IV. Long-term within- and between-laboratory variation of estrogen and progesterone receptor assays

One batch of lyophilized calf uterine cytosol was analyzed for estrogen and progesterone receptor (ER and PgR, respectively) content by 12 members of the EORTC Receptor Group on three different occasions over a total study period of 1 yr: (1) One vial was included with each of 20 consecutive batches of routine tumor analyses between December 1983 and May 1984. (2) Two vials were simultaneously assayed between July and August 1984 (within-run variation). (3) One vial was analyzed at the end of 1984 (November-December). The overall mean ER and PgR values did not change systematically over the total study period of 1 yr. Within the various laboratories, the between-run variations of both ER and PgR assays were considerable and differed from one institution to another (7–26%). For both ER and PgR measurements the average within-run (n = 2) and between-run (n = 20) coefficients of variation were similar (8–9% and 16–17%, respectively). Comparison of the results from multiple sequential assays ( c.v. = 12.9%) with those from single assays ( c.v. = 21.2%) showed that about 60% of the between-laboratory variance in ER could be explained on the basis of the between-run variance. With regard to PgR analysis, however, the between-laboratory variance decreased only 25%. Standardized use of one type of protein assay (Coomassie brilliant blue) and a standard protein solution (human serum albumin) has decreased the between-laboratory variation of the protein analysis results to less than 15%.

3,17 beta-Dihydroxy-20,21-epoxy-19-norpregna-1,3,5(10)-trienes: synthesis, rearrangement, cytotoxicity, and estrogen-receptor binding.

Diastereoisomers of 3,17 beta-dihydroxy-20,21-epoxy-19-norpregna-1,3,5(10)-triene have been prepared as potential antitumor agents. Both isomers undergo the base-catalyzed Payne rearrangement. The isomers were cytotoxic to mammalian cells in culture and were able to displace [3H]estradiol from binding sites in rat uterine cytosols with 1/7 and 1/70 the potency of estradiol. The reasons for this difference are discussed.

Hepatic Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Partial stabilization by molybdate.

In structure and general mode of action, the Ah receptor is very similar to the receptors for steroid hormones. Molybdate previously has been shown to be highly effective at preserving ligand-binding function in steroid receptors during their exposure to elevated temperature or high ionic strength and at stabilizing steroid receptors as high molecular weight oligomeric complexes. Since such stabilization by molybdate can be very useful during characterization and purification of receptors, we tested the effects of molybdate on the Ah receptor to determine if the Ah receptor, like the receptors for steroid hormones, might be stabilized. In hepatic cytosols from C57BL/6N mice and Sprague-Dawley rats, molybdate concentrations up to 30 mM in homogenizing and analysis buffers did not alter the concentration of specific Ah receptor sites detected by binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. However, inclusion of 20 mM molybdate in the homogenizing buffer did significantly protect unliganded Ah receptor from thermal inactivation at 20 degrees C and from KCl-induced loss of ligand-binding ability. In accord with previous reports, 20 mM molybdate in homogenizing and analysis buffers greatly increased the concentration of detectable glucocorticoid receptor in rat hepatic cytosol and estrogen receptor in rat uterine cytosol. Exposure to 0.4 M KC1 caused the glucocorticoid receptor from rat liver to shift sedimentation from approximately equal to 8 S to approximately equal to 4 S and caused a severe loss of specific glucocorticoid binding. Presence of 20 mM molybdate stabilized the glucocorticoid receptor as a single discrete peak sedimenting at approximately equal to 8 S. In contrast, the Ah receptor from rat liver exposed to 0.4 M KC1 in the presence of molybdate sedimented as biphasic peaks; one peak (approximately equal to 9.5 S) corresponded to the form of Ah receptor observed at low ionic strength, while the other peak (approximately equal to 5.5 S) corresponded to the form of Ah receptor seen in cytosol treated with 0.4 M KC1 in the absence of molybdate. Addition of heparin to hepatic cytosols from mice or rats shifted sedimentation of Ah receptor from approximately equal to 9.5 S to approximately equal to 5.5 S. Molybdate, again, provided stabilization in the approximately equal to 9.5 S form, but only for about one-half the total Ah receptor content in both rat and mouse hepatic cytosols. In sum, molybdate is far less effective at stabilizing rodent Ah receptors than it is at stabilizing steroid receptors in the same species.

Induction of progesterone receptor by androgens in the mouse uterus

The comparative abilities of 3 naturally occurring androgens to compete for [ 3H]estradiol-17β ([ 3H]E 2)-binding sites in uterine cytosol and to induce uterotrophic responses in immature female mice have been investigated. 5α-Androstane-3β,17β-diol (3β-diol), 5α-androstane-3α,17β-diol (3α-diol) and 5-androstene-3β,17β-diol (Δ5-diol) are all effective at diminishing cytoplasmic [ 3H]E 2 binding. Their relative effectiveness are in the order of 3β-diol > Δ5-diol = 3α-diol. When these steroids were injected intramuscularly (two 100–200 μg injections) into immature female mice (21 days old), they induced similar elevations of uterine dry weight, water content, cytosolic estrogen (ER) and progesterone (PR) receptor contents, and nuclear ER content as did estradiol-17β (E 2). 3β-Diol was the most effective steroid at inducing cytosolic and nuclear ER, and PR production in mouse uterus. Followed in effectiveness was Δ5-diol and 3α-diol. Unexpectedly, 3β-diol was found to be more potent than E 2 in stimulating uterine ER and PR increases. Nonetheless, the androgens are poorer stimulators of uterine dry weight increase and water retention than E 2 is. These results indicate that the androgens may interact with the ER system in the uterus to bring about the uterotrophic responses and 3β-diol is a more estrogenic steroid than Δ5-diol and 3α-diol in the mouse uterus.

Ribosome-associated estradiol-binding components in the uterus and their relationship to the translational capacity of uterine ribosomes.

Estradiol transiently increases the rate of peptide elongation on uterine ribosomes from ovariectomized mature rats during the first 2 h after hormone injection, suggesting the existence of direct or indirect estradiol receptor interaction with ribosomes. Characterization of estradiol-binding components on isolated uterine ribosomes, microsomes, and cytosol under identical assay conditions indicated that microsomes and cytosol contain estradiol-binding components with similar affinities for estradiol (Kd = 0.5 nM) and sucrose gradient sedimentation characteristics (3.8S and 5.2S for preparations incubated at 0 and 30 C for 1 h, respectively). Those on ribosomes exhibited a higher affinity for estradiol (Kd = 0.14 nM) and had heterogeneous and more dense sedimentation characteristics (5.5-6.0S). The ribosome-associated estradiol binder was clearly different from transformed cytosol and nuclear estradiol receptors based on sedimentation characteristics under identical conditions. Like cytosol and nuclear receptors, microsomal and ribosomal estradiol binding underwent exchange reactions in vitro at 30 C, but not at 0 C. All in vitro bound, but not all in vivo bound, [3H] estradiol could be exchanged from microsomes or ribosomes by estradiol. [3H]Estradiol could be exchanged from ribosomes by a variety of estrogens, but not by progestins, glucocorticoids, or androgens. The amount of estradiol-binding activity on ribosomes decreased after estradiol administration in vivo and was inversely correlated with the rate of peptide elongation by the ribosomes in a cell-free protein synthesis system. These results suggest that accumulation of an estradiol-binding protein, perhaps a nascent estradiol receptor, on ribosomes in the absence of in vivo estradiol may directly or indirectly inhibit the peptide elongation reaction.

Androgen receptor affinity chromatography: Synthesis and properties of 17α-epoxypropyl- dihydrotestosterone sepharose

We have prepared a new affinity chromatography reagent, 17α-epoxypropyl-dihydro-testosterone linked to Thiopropyl-Sepharose, with potential for use in purification of androgen receptor and other specific androgen binding proteins. The linkage is stable, and the ligand has reasonably high affinity for the receptor. Starting with 5α-androstane-3β-ol-17-one, we synthesized in two steps 17α-allyl-dihydrotestosterone, which was then oxidized to 17α-epoxypropyl-DHT yielding 2 diastereomers in about a 4:1 ratio. The 17α-allyl-DHT had about 50% of DHT's affinity for rat uterine androgen receptor, while the affinity of the major epoxide isomer was 9% and that of the minor isomer was 4%. Reaction of the epoxides with Thiopropyl-Sepharose-6B gave about 7 μ mol of covalently bound DHT per ml of beads. These beads took up 83% of the androgen receptor from a rat uterine cytosol in a preliminary study, which more than equalled the performance of identically prepared estradiol beads successfully used for estrogen receptor purification. The use of the new DHT beads in purifications of the androgen receptor and other binding proteins is now being explored by other laboratories.

Cytoplasmic progesterone receptors in uterine tissue of the snapping turtle (Chelydra serpentina).

A high affinity progesterone-binding component was detected in the cytosol of the uterus of the snapping turtle, Chelydra serpentina. Density gradient centrifugation indicated that binding of [3H]progesterone and [3H]promegestone (R5020) was to a fraction with a heavier sedimentation coefficient than bovine serum albumin (BSA) appearing as a broader peak in the 6-7 S region; it was not affected by excess cortisol. Another binding peak, lighter than BSA and appearing with [3H]R5020 and [3H]progesterone near the 4 S region, was affected by excess cortisol. Excess progesterone decreased both the heavier and lighter peaks. Analysis of steroid specificity revealed that, of the natural steroids, progesterone had the highest affinity for the uterine cytosol. This was followed by deoxycorticosterone, 5 alpha-pregnanedione, testosterone, oestradiol-17 beta, corticosterone, 5 alpha-dihydrotestosterone and cortisol. Non-linear regression analysis of saturation data indicated the presence of two classes of high affinity binding sites: progesterone-binding sites (R-sites) with equilibrium association constants (Ka) of 2.9 +/- 0.28 litres/nmol (mean +/- 95% confidence limit) for [3H]R5020 and 0.34 +/- 0.20 litres/nmol for [3H]progesterone, and corticosteroid-binding globulin-like sites (G-sites) with Ka of 4.5 +/- 1.6 litres/nmol for progesterone. The concentration of R-sites was between 0.66 +/- 0.10 and 2.6 +/- 0.55 pmol/mg protein while that of G-sites was between 0.73 +/- 0.05 and 5.0 +/- 0.27 pmol/mg protein. DEAE-cellulose filtration assay also confirmed the presence of R-sites and G-sites in the cytosol. R-sites were detectable without oestrogen priming during the preovulatory and vitellogenic phases (low progesterone, high oestrogen concentrations) when the ovarian follicles are mature (18-22 mm diameter).(ABSTRACT TRUNCATED AT 250 WORDS)

Temporal and receptor correlates of the estrogen response in sheep

Uterine blood flow and uterine cytosol and nuclear estrogen receptors were measured at critical times during estradiol-induced vasodilatation in acute anesthetized and chronic conscious sheep preparations at estradiol bolus injection frequencies from 1 to 24 hours. During acute experiments, the uterine blood flow response was muted and cytosol estrogen receptor replenishment did not occur whereas full replenishment occurred in 12 hours in conscious ewes. In ewes treated with daily estradiol that had a stable daily uterine blood flow response, the uterine blood flow response 24 hours after uterine biopsy was similar to the preoperative one. Analysis of the duration of peak uterine blood flow levels and the rate of uterine blood flow descent from peak levels showed that an interval of 18 hours between estradiol injections was necessary for the uterine blood flow response to approximate that observed after 24 hours. These observations suggest: (1) that uterine vascular receptor replenishment is delayed compared with that of the total uterus; (2) that operative stress compromises cytosol estrogen receptor metabolism and possibly nuclear estrogen receptor function; (3) that the delayed maximum uterine blood flow response to estradiol in ewes previously untreated with estradiol is due to a trophic uterine effect of daily estradiol stimulation.

Binding of 7α,17α-Dimethyl-19-Nortestosterone (Mibolerone) to Androgen and Progesterone Receptors in Human and Animal Tissues*

In rat uterus and prostate, 7 alpha, 17 alpha-dimethyl-19-nortestosterone (DMNT) binds to the androgen receptor specifically and with high affinity. However, this steroid does not bind to glucocorticoid receptors, since it does not displace binding of [3H]triamcinolone acetonide in calf thymus cytosol. In calf uterine and human breast tumor cytosols DMNT binds to the androgen and progesterone receptors, since binding of [3H] DMNT is displaced by unlabeled 16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3, 20-dione triamcinolone acetonide, and 5 alpha-dihydrotestosterone (DHT). Conversely, binding of [3H]16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione is effectively competed for by unlabeled DMNT but not by DHT. The observed differences in binding of [3H]DMNT to rat and calf uterine cytosols suggest the species specificity of progesterone receptors. Unlike DHT, DMNT has no appreciable binding to human sex-steroid binding globulin. These findings suggest DMNT as a suitable ligand for measurement and characterization of androgen receptors in rat and human prostate.

ChemInform Abstract: Synthesis and Evaluation of Potential Radioligands for the Progesterone Receptor.

AbstractGemäß Formelschema werden Iod‐ und Amid‐Steroide (IV), (VI), Und (VIII) dargestellt, die auf Progesteronrezeptor‐Affinität getestet werden.

Characterization of the Calf Uterine Androgen Receptor and Its Activation to the Deoxyribonucleic Acid-Binding State*

The androgen receptor (AR) from calf uterine cytosol has been studied in terms of steroid-binding affinity, hormone dissociation kinetics, and DNA-cellulose-binding capacity. The binding affinity for three androgens, analyzed under conditions where binding to progesterone receptor did not occur, decreased in the order: methyltrienolone greater than 5 alpha-dihydrotestosterone greater than testosterone. Activation of the receptor to the DNA-binding state involved the following changes of the receptor: decrease in dissociation rate for the steroid, disaggregation of the receptor, and increase in affinity for DNA. Dissociation studies with methyltrienolone and 5 alpha-dihydrotestosterone revealed that the AR can exist in two affinity states which differ 13- to 30-fold in their affinity for the steroid. Molybdate (10-20 mM) prevented the formation of the high affinity state. The high affinity state receptor was formed in the absence of molybdate or after ammonium sulfate precipitation (0-40% saturation) of the molybdate-stabilized low affinity state receptor. During formation of the high affinity state, the sedimentation coefficient of the receptor in low ionic strength buffer decreased from 8-9S to 4.5S, indicating receptor disaggregation. DNA-cellulose binding capacity increased from 3 to 65% upon formation of the high affinity state. The DNA-binding form could be eluted from DNA-cellulose at 0.14 M NaCl. After elution the DNA-binding form maintained its sedimentation coefficient of 4.5S and chromatographed as a protein with a Stokes radius of 44 A. From these results it can be concluded that the activated, DNA-binding form of the AR in calf uterus is a protein with a molecular mass of approximately 85,000, which acquires a higher affinity for the ligand upon its formation.