Involvement of a non-hormone-binding 90-kilodalton protein in the nontransformed 8S form of the rabbit uterus progesterone receptor

Nontransformed 8S progesterone receptor (8S-PR) was purified by hormone-specific affinity chromatography from rabbit uterine low-salt cytosol containing 20 mM molybdate. In the eluate obtained with radioactive progestin, sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) showed the presence of several bands, including three that corresponded to approximately 90-, approximately 120-, and approximately 85-kDa proteins. None of these three proteins was found in the eluate of the affinity column when the molybdate-containing cytosol was chromatographed in the presence of nonradioactive progesterone ("mock purification"). Subsequent purification of the affinity eluate by DEAE-Sephacel chromatography gave a single radioactive receptor peak at 0.15 M KCl (approximately 20% yield, 19% purity on the basis of one binding site per approximately 100 kDa) with a sedimentation coefficient of 8.5 S. Silver staining after SDS-PAGE revealed that this purified 8S-PR fraction contained mainly the 120-, 90-, and 85-kDa proteins. [3H]R5020-labeled 8S-PR purified by DEAE-Sephacel column chromatography was UV irradiated, and after SDS-PAGE the 120- and 85-kDa proteins were revealed, but the 90-kDa protein was not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of the molybdate-stabilized 9-10 S estradiol receptor from calf uterus

The molybdate-stabilized calf uterine estradiol receptor has been purified to near-homogeneity by a three-step procedure. Initial purification by heparin-Sepharose chromatography provides a concentrated receptor extract in 40% yield with a 5-10-fold increase in purity. The inclusion of molybdate in phosphate-buffered cytosol enhances 9-10 S receptor stability in high salt and allows elution of the oligomeric receptor complex from heparin-Sepharose with 0.4 M KCl. A second affinity step utilizing estrone carboxymethyloxime coupled to diaminoethyl bis(2-hydroxypropoxy)butane-Sepharose Cl-4B increases purification by a further 1600-fold. High performance liquid chromatography gives homogeneous receptor which migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a polypeptide of Mr approximately 89,000. The purified molybdate-stabilized receptor sediments at 9.3 +/- 0.2 S (n = 4) in glycerol gradients and has a Stokes radius of 74 +/- 3 A (n = 2) giving a calculated Mr approximately 290,000. These properties and the steroid-binding specificity of the purified receptor bear a close similarity to those found for the 9-10 S receptor in crude cytosol.     

Purification of cAMP-specific phosphodiesterase from rat heart by affinity chromatography on immobilized rolipram

Affinity chromatography on a cAMP-specific phosphodiesterase inhibitor related to Rolipram, immobilized to AH Sepharose allowed to perform an efficient purification of the cAMP-specific phosphodiesterase isoenzyme from rat heart cytosol (102-fold purification with a 35% yield in a single step). This affinity chromatography involved a biospecific interaction since a 2 mM cAMP elution step at 30 degrees C was necessary for releasing the cAMP specific form tightly bound on the affinity gel. The cAMP eluate fraction exhibited a high specificity towards cAMP (cAMP/cGMP hydrolysis ratio 5-10), a marked sensitivity to Rolipram inhibition and could be resolved in two cAMP-specific, highly Rolipram-sensitive peaks of pI 6.7 and 4.8 by IEF on polyacrylamide gel plates. Protein stain of the IEF gel revealed a single band at pI 6.7.     

Transformation and phosphorylation of purified molybdate-stabilized chicken oviduct progesterone receptor

The non-transformed, molybdate-stabilized chick oviduct cytosol progesterone receptor was purified approx. 7000-fold using biospecific affinity resin (NADAC-Sepharose), DEAE-Sephacel chromatography and gel filtration on Bio-Gel A-0.5m agarose. The purified preparation contained progesterone receptor which sedimented as a 7.9S molecule, had a Stokes' radius of 7.5 nm, was composed of three major peptides corresponding to Mr 108,000, 90,000 and 79,000. Upon removal of molybdate, the purified [3H]progesterone-receptor complex could be transformed from the 8S form to a 4S form by exposure to 23 degrees C or by an incubation with 10 mM ATP at 0 degrees C. The purified thermally transformed receptor could be adsorbed to columns of ATP-Sepharose. No cytosol factor(s) appeared to be required for the 8S to 4S transformation of purified receptor or for its subsequent binding to ATP-Sepharose. Incubation of purified non-transformed receptor preparation with [gamma-32P]ATP and cAMP-dependent protein kinase led to incorporation of radioactivity in all the three major peptides at serine residues. The results of this study show for the first time that purified 8S progesterone receptor can be phosphorylated in vitro by a cAMP-dependent protein kinase, and that it can be transformed to a 4S form by 0 degrees C incubation with 10 mM ATP.     

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.     

Transformation in vitro and covalent modification with biotin of steroid-affinity-purified rat-liver glucocorticoid-hormone-receptor complex

The molybdate-stabilized rat liver glucocorticoid receptor complex was purified 9000-fold with a 46% yield by steroid-affinity chromatography and DEAE-Sephacel ion-exchange chromatography. The purified glucocorticoid receptor was identified as a 90-92-kDa protein by SDS/polyacrylamide gel electrophoresis. Raising the temperature to 25 degrees C in the absence of molybdate resulted in increased binding of the receptor complex to DNA-cellulose or nuclei, similar to the effect on the cytosolic complex. The purified complex has a sedimentation coefficient of 9-10 S before and after heat treatment in the absence of molybdate. The appearance of smaller 3-4-S species was unrelated to the extent of DNA-cellulose binding of the complex. The process termed 'transformation', i.e. increasing the affinity for DNA, is not concomitant with subunit dissociation or loss of RNA. Highly purified glucocorticoid receptor could be covalently modified with biotin to retain its steroid-binding activity but with a 50% decrease in nuclear binding capacity. The biotin-modified complex reacts with streptavidin in solution without losing its steroid.     

Rat liver glucocorticoid receptor isolated by affinity chromatography is not a Mg2+- or Ca2+-dependent protein kinase

The glucocorticoid hormone receptor (92 kDa), purified 9000-fold from rat liver cytosol by steroid affinity chromatography and DEAE-Sephacel chromatography, was assayed for the presence of protein kinase activity by incubations with [gamma-32P]ATP and the photoaffinity label 8-azido-[gamma-32P]ATP. Control preparations isolated by affinity chromatography in the presence of excess steroid to prevent the receptor from binding to the affinity matrix were assayed for kinase activity in parallel. The receptor was not labeled by the photoaffinity label under photoactivation conditions in the presence of Ca2+ or Mg2+. A Mg2+-dependent protein kinase (48 kDa) that could be photoaffinity labeled with 8-azido-ATP copurified with the receptor. This kinase was also present in control preparations. The kinase could phosphorylate several minor contaminants present in the receptor preparation, including a protein (or proteins) of similar molecular weight to the receptor. The phosphorylation of 90-92-kDa proteins was independent of the state of transformation or steroid-binding activity of the receptor. These experiments provide direct evidence that neither the glucocorticoid receptor nor the 90-92-kDa non-steroid-binding protein associated with the molybdate-stabilized glucocorticoid receptor possesses intrinsic Ca2+- or Mg2+-dependent protein kinase activity.     

An automatic multidimensional chromatography system for purification of human uterine progesterone receptor and induction of polyclonal antibodies

This paper reports on the synthesis of Org2058-bonded microparticulate silicas and their use in affinity chromatography as the first step for the purification of human progesterone receptor. The development of microprocessor-controlled instruments allows all the various steps to be performed automatically. The various steps used for the purification of human progesterone receptor were carried out with the FPLC system: affinity chromatography, desalting of eluate on Sephadex G-25, anion-exchange chromatography using a Mono Q column. With this procedure the receptor was purified approx. 10,000-fold within 24 h. The yield of receptor was generally 85-95%. Investigations with induced anti-progesterone receptor antibodies obtained after the fourth immunization show their immunoreactive behaviour towards progesterone receptor in crude cytosol, which was proved by sucrose density gradient centrifugation and by gel filtration on the FPLC system using a Sepharose 12 column. This implies that progesterone receptor was efficiently purified by our purification procedure.     

Maximizing the purification of the activated glucocorticoid receptor by DNA-cellulose chromatography.

With heat treatment (20 degrees C for 30 min), the glucocorticoid-receptor complex becomes 'activated' and undergoes an increase in affinity for DNA. A two-stage procedure was used to separate sequentially the rat liver glucocorticoid-receptor complex from proteins with high and low affinity for DNA. DNA-cellulose column chromatography of unheated cytosol resulted in the retention of DNA-binding proteins, but not the unactivated receptor complex. Heat treatment of the column eluate resulted in increased affinity of the receptor complex to DNA, and chromatography on DNA-cellulose then yielded receptor complex free from proteins with low affinity for DNA. Removal of DNA-binding proteins during the first chromatographic step was critically dependent on ionic conditions and the ratio of cytosol chromatographed to DNA-cellulose. A purification of 11000-fold (85% yield) was achieved by this procedure. The partially purified receptor complex was taken up by rat liver nuclei.     

Affinity chromatography of the D1 dopamine receptor from rat corpus striatum

The D1 dopamine receptor from rat corpus striatum has been purified 200-250-fold by using a newly developed biospecific affinity chromatography matrix based on a derivative of the D1 selective antagonist SCH 23390. This compound, (RS)-5-(4-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benz azepin-7-o l (SCH 39111), possesses high affinity for the D1 receptor and, when immobilized on Sepharose 6B through an extended spacer arm, was able to adsorb digitonin-solubilized D1 receptors. The interaction between the solubilized receptor and the affinity matrix was biospecific. Adsorption of receptor activity could be blocked in a stereoselective fashion [SCH 23390 greater than SCH 23388; (+)-butaclamol greater than (-)-butaclamol]. The elution of [3H]SCH 23390 activity from the gel demonstrated similar stereoselectivity for antagonist ligands. Agonists eluted receptor activity with a rank order of potency consistent with that of a D1 receptor [apomorphine greater than dopamine greater than (-)-epinephrine much greater than LY 171555 greater than serotonin]. SCH 39111-Sepharose absorbed 75-85% of the soluble receptor activity, and after the gel was washed extensively, 35-55% of the absorbed receptor activity could be eluted with 100 microM (+)-butaclamol with specific activities ranging from 250 to 450 pmol/mg of protein. The affinity-purified receptor retains the ligand binding characteristics of a D1 dopamine receptor. This affinity chromatography procedure should prove valuable in the isolation and molecular characterization of the D1 dopamine receptor.     

The Mr 90,000 heat shock protein-free androgen receptor has a high affinity for steroid, in contrast to the glucocorticoid receptor

Transformed and bacterially expressed glucocorticoid receptors free from Mr 90,000 heat shock protein (hsp90) have a 100-fold lower steroid-binding affinity than the hsp90-bound nontransformed receptor, suggesting that hsp90 is needed for high-affinity steroid binding [Nemoto, T., Ohara-Nemoto, Y., Denis, M., & Gustafsson, J.-A. (1990) Biochemistry 29, 1880-1886]. To investigate whether or not this phenomenon is common to all steroid receptors, we investigated the steroid-binding affinities of bacterially expressed and transformed androgen receptors. The C-terminal portion of the rat androgen receptor containing the putative steroid-binding domain was expressed as a fusion protein of protein A in Escherichia coli. The recombinant protein bound a synthetic androgen, [3H]R1881, with high affinity (Kd = 0.8 +/- 0.3 nM). Glycerol gradient analysis revealed that the recombinant protein sedimented at around the 3S region irrespective of the presence of molybdate, indicating that the receptor is present in monomeric form. The steroid-free transformed androgen receptor was obtained by exposure of rat submandibular gland cytosol to 0.4 M NaCl in the absence of steroid. High-performance ion-exchange liquid chromatography analysis showed that the transformed androgen receptor bound to [3H]R1881 with high affinity. Thus these observations indicate that, in contrast to the glucocorticoid receptor, hsp90 is not required for the high-affinity steroid binding of the androgen receptor. In addition, the hsp90-free androgen receptor prebound with radioinert R1881 was efficiently relabeled with [3H]R1881, while the triamcinolone acetonide-bound, transformed glucocorticoid receptor failed in ligand exchange. The inability to achieve ligand exchange probably reflects the low steroid-binding affinity of this entity.     

Nontransformed rabbit liver glucocorticoid receptor: purification, characterization and transformation

The molybdate-stabilized nontransformed form of the glucocorticoid receptor from rabbit liver has been purified approximately 8,000-fold by a three-step procedure. The first step involved protamine sulfate precipitation which allowed a 5-6-fold purification with 85% yield. The second step, affinity chromatography using a N-(12-dodecyl-amino) 9 alpha-fluoro-16 alpha-methyl-11 beta, 17 alpha-dihydroxy-3-oxo-1,4-androstadiene-17 beta-carboxamide substituted Sepharose gel, purified the receptor 1,500-2,000-fold as calculated by specific radioactivity. The third step involved high performance liquid chromatography resulting in overall purification near 8,000-fold. The final glucocorticoid receptor appeared about 60% pure. The purified nontransformed glucocorticoid receptor had a sedimentation coefficient of 9 S in 0.16 M phosphate containing 5-20% sucrose gradients and the Stokes radius was 6.1-6.3 nm as determined by low pressure gel filtration and HPLC. Binding specificity of the purified receptor was identical to that previously reported in crude rabbit liver cytosol. Isoelectricfocusing and ion-exchange chromatography showed that the purification procedure affected the net charge of the receptor protein. This phenomenon could be related to interactions between the glucocorticoid receptor and cytosolic factors. SDS polyacrylamide gel electrophoresis showed a major Mr = 94,000 protein band which is in good agreement with previously reported values for glucocorticoid receptors. Transformation of the purified receptor was achieved after removal of molybdate by exposure at 25 degrees C to 0.4 M KCl. Characterization of the molecular forms was performed by means of incorporation into isolated nuclei, affinity towards polyanionic exchangers and high pressure size exclusion chromatography. Results show that about 40% of the receptor is in the transformed state.     

Affinity surfactants as reversibly bound ligands for high-performance affinity chromatography

Pyridine was coupled covalently to a nonionic ethoxylated alcohol: octaethylene glycol n-hexadecyl ether. This modified surfactant was found to be a reversible, competitive inhibitor of horse serum cholinesterase. The surfactant bound irreversibly, in aqueous media, to octadecyl-bounded reverse phase silica particles commonly used for high-performance liquid chromatography. The amount of ligand bound was found to be 550 mumol/ml of packing, a concentration that is over 100 times higher than what can be normally bound to agarose affinity chromatography supports. With this packing, a 280-fold purification of cholinesterase from horse serum and a 79-fold purification of human serum cholinesterase were accomplished, with yields greater than 80%, using a 2-cm-long column and a 7-min elution time. The affinity surfactant could be eluted from the column using a 6:4 (v/v) mixture of methanol and isopropanol. This technique should be generally applicable in the development of biospecific supports for high-performance affinity chromatography.     

Purification, characterization, and activation of the glucocorticoid-receptor complex from rat kidney cortex

The unactivated molybdate-stabilized glucocorticoid receptor (GcR) was purified from rat kidney cortex cytosol (RKcC) by using a modification of the procedure previously described by this laboratory for rat hepatic receptor. The purification includes affinity chromatography, gel filtration, and ion-exchange chromatography. The final preparation (approximately 1000-fold pure as determined from specific radioactivity) was used in subsequent physicochemical and functional analyses. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single heavily Coomassie-stained band at 90 kilodaltons. Density gradient ultracentrifugation indicated a sedimentation coefficient of 10.5 +/- 0.05 S (n = 2). Chromatography on an analytical gel filtration column produced a Stokes radius (Rs) of 6.4 +/- 0.07 nm (n = 5). The Rs was unchanged when the molybdate-stabilized GcR was analyzed in the presence of 400 mM KCl or when analyzed in the unpurified (cytosolic) state. In contrast, the hepatic GcR was observed to exist as a larger form in cytosol (7.7 +/- 0.2 nm). Following purification, or upon gel filtration analysis under hypertonic conditions, the Rs was similar to that of the unpurified RKcC GcR. Following removal of molybdate from RKcC GcR and thermal activation (25 degrees C/30 min), DNA-cellulose binding increased 1.5-2-fold over the unheated control. Addition of RKcC or hepatic cytosol (endogenous receptors thermally denatured at 90 degrees C/30 min or presaturated with 10(-7) M radioinert ligand) during thermal activation increased DNA-cellulose binding an additional 2-6-fold beyond the heated control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein purification using a soluble affinity matrix: purification of estrogen receptor with estradiol-polylysine conjugate.

A new strategy for protein purification using a soluble affinity matrix is described. The method was used for purification of estrogen receptor. Cytosols from rat uteri and human fibroid uterine tissue, after fractionation by ammonium sulfate, were treated with estradiol-polylysine conjugate. The highly basic affinity complex was separated from other proteins by DEAE-Sephacel chromatography. After dissociation of the eluted complex with excess estradiol, the receptor was recovered by CM-Sephadex chromatography. A 2000-fold purification of the rat uterine estrogen receptor was obtained with an activity recovery of 35%.     

Partial purification of androgen receptor from hypertrophic human prostate

For purification of androgen receptor from hypertrophic human prostate, solutions used for elution of androgen receptor from DNA Sepharose, affinity labeling of the receptor and ability of affinity gel to retain the receptor were examined. Elution with 20 mM pyridoxal 5'-phosphate of the receptor from DNA Sepharose was more efficient than that with diluted pyridoxal 5'-phosphate, high ionic solution or various concentrations of Mg++, 3H-dihydrotestosterone bromoacetate was applicable to covalent binding with partially purified androgen receptor regardless of the low specificity of the ligand. Affinity gel of thiopropyl-Sepharose 6B coupled to 17 alpha-(2', 3'-epoxy-propyl)-5 alpha-dihydrotestosterone was better than Affigel 102 coupled to N-[3-(3-oxo-5 alpha-androstane-17 beta-yloxycarbonyl) propionyloxy] succimide or aminoethyl-Sepharose 4B coupled to 17 alpha-carboxyethynyl testosterone with respect to the rate of retention of androgen receptor. In view of these observations, the following purification procedures were constructed: Removal of DNA Sepharose-binders from the cytosol, 40% ammonium sulfate precipitation, affinity chromatography using thiopropyl-Sepharose 6B coupled to 17 alpha-(2',3'-epoxypropyl)-5 alpha-dihydrotestosterone, and DNA Sepharose chromatography. After affinity labeling of the receptor thus obtained, the molecular weight was estimated. Some 1300-fold purification with a yield of 0.25% of the androgen receptor was achieved. The molecular weight of the receptor was mainly 45 K with 90 K in a lesser amount. The Stokes radius was calculated as 30 A.     

Isolation of Putative Benzodiazepine Receptors from Rat Brain Membranes by Affinity Chromatography

Abstract: The benzodiazepine receptor from rat brain was solubilised and purified 5200-fold by affinity chromatography. The affinity column contained an immobilized benzodiazepine (delorazepam) and biospecific elution with 6 m m -chlorazepate was achieved. The purified receptor is apparently homogeneous in SDS-polyacrylamide gel electrophoresis. The native protein had a molecular weight of 240,000, and the subunit one of 60,000. The dissociation constant ( K _D) is 8 n m for [³H]diazepam. A correlation exists between the value of affinity obtained for benzodiazepine derivatives and their known pharmacological effectiveness.     

Purification of factor VIII:c coagulant activity from rat liver nonparenchymal cell culture medium by immobilized metal ion affinity chromatography

The purification of factor VIII:c coagulant activity on the basis of its affinity for calcium is described. For this purpose, use was made of a recently introduced chelating matrix, i.e., carboxymethylated aspartic acid agarose, coupled with calcium--thereby creating a gel with specificity comparable with biospecific affinity chromatography. In a single step factor VIII:c activity was purified from rat liver nonparenchymal cell culture medium with a purification factor of 85-fold. The material exhibits a single band on polyacrylamide gel electrophoresis.     

Unique molecular properties of a urea- and salt-stable DNA-binding estrogen receptor dimer covalently labeled with the antiestrogen [3H]desmethylnafoxidine aziridine. A comparison with the estrogen-receptor complex

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafoxidine aziridine, has been used to investigate the salt- and temperature-independent formation of DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or [3H]desmethylnafoxidine aziridine were quantitatively transformed (greater than 90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 M urea at 0 C. The urea effect was hormone-dependent and partially reversible. The transformed receptors were purified (ca 250-fold) by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (Kd = 0.34 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl and displayed no evidence of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr = 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroid-binding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4 and phenyl stationary phases revealed no evidence of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 M urea. Thus, exposure to urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding.     

Evidence that the endogenous heat-stable glucocorticoid receptor-activating factor is thioredoxin

Extraction of rat liver cytosol with 10% charcoal at 4 degrees C inactivates specific glucocorticoid-binding capacity. The steroid-binding capacity of extracted cytosol can be restored by adding dithiothreitol or by incubating with boiled liver cytosol at 20 degrees C in the presence of 10 mM sodium molybdate. Two components of boiled cytosol are required for receptor activation: NADPH and an endogenous heat-stable protein with an apparent Mr of 12,300 by Sephadex G-50 chromatography. This endogenous receptor-activating protein coelutes on Sephadex G-50 chromatography with endogenous thioredoxin activity, and it can be replaced in the activating system by purified Escherichia coli thioredoxin. These observations suggest that glucocorticoid receptors in cytosol preparations are maintained in a reduced, steroid-binding state by a NADPH-dependent, thioredoxin-mediated reducing system.