Isolation and characterization of rat liver nuclear glucocorticoid receptor.

The rat liver nuclear glucocorticoid receptor has a molecular weight of 90 000. Using antibody bound to the stationary matrix, the cytosol and nuclear glucocorticoid receptors from rat liver were purified. The translocation of glucocorticoid receptor from rat liver cytosol into the nucleus was studied using immunoaffinity chromatography. Immediately after the intraperitioneal injection of rats with the hormone, the receptor translocation started and was complete within 10 min. The 90 000 dalton nuclear receptor component is identical to the 90 000 dalton cytosol component. They have identical molecular weights in the same gel electrophoresis system and produce identical peptide fragments after digestion with Staphyolococcal aureus V8 protease. The receptor component enriched by immunoaffinity chromatography from cytosol of adrenalectomised rats contained mainly a 45 000 dalton component.

Specific region in hormone binding domain is essential for hormone binding and trans-activation by human androgen receptor.

Complementary DNA (cDNA) clones encoding human-androgen receptors (haR) were isolated using synthetic oligonucleotides homologous to the human glucocorticoid, estradiol, progesterone, and aldosterone receptors as probes to screen a human testis lambda gt11 cDNA library. One of the receptor proteins (hARa) produced in vitro bound the [3H]dehydrotestosterone ([3H]DHT) with high affinity and selectivity similar to the human androgen receptor present in target tissues and cells. A second cDNA clone (hARb) encoding an identical amino terminal and DNA binding domains, but differing by four amino acids at the hormone binding domain, did not bind [3H]DHT with high affinity when incubated with protein expressed by in vitro transcription-translation. Cotransfection of hARa in an expression vector with mouse mammary tumor virus (MMTV)-bacterial chloramphenicol acetyltransferase chimeric plasmids, followed a hormone-dependent trans-activation, defining the binding affinity of hARa between 5 x 10(-10) and 1 x 10(-9) M for [3H]DHT. A similar cotransfection experiment with hARb indicated a KD of hARb for [3H]DHT to be above approximately 10(-8) M. The deduced primary structures of hARa and hARb contain the viral erbA homologous region found in other steroid, thyroid, and vitamin receptors and is identical to the hAR sequences reported by others. The amino acid sequence differs at the Gly stretch (16 Gly instead of 27, 24 or 23) of the N-terminal domain and in hARb, the sequence reads I.F.F.F.F.L.L (816-822) instead of K.F.F.D.E-L (816-821) in the hARa and other reported hAR sequences. The difference of four amino acids in the steroid binding domain of hARb is associated with altered DHT binding and thus a lack of trans-activation by way of AR responsive elements in MMTV-long terminal repeat. The interaction of hARa and hARb with synthetic responsive elements by gel-retardation assay and their responsiveness in trans-activation by calcium phosphate coprecipitation demonstrates that hARb can inhibit trans-activation by hARa in this system.

GCN5 and ADA adaptor proteins regulate triiodothyronine/GRIP1 and SRC-1 coactivator-dependent gene activation by the human thyroid hormone receptor.

We have used yeast genetics and in vitro protein-protein interaction experiments to explore the possibility that GCN5 (general control nonrepressed protein 5) and several other ADA (alteration/deficiency in activation) adaptor proteins of the multimeric SAGA complex can regulate T3/GRIP1 (glucocorticoid receptor interacting protein 1) and SRC-1 (steroid receptor coactivator-1) coactivator-dependent activation of transcription by the human T3 receptor beta1 (hTRbeta1). Here, we show that in vivo activation of a T3/GRIP1 or SRC-1 coactivator-dependent T3 hormone response element by hTRbeta1 is dependent upon the presence of yeast GCN5, ADA2, ADA1, or ADA3 adaptor proteins and that the histone acetyltransferase (HAT) domains and bromodomain (BrD) of yGCN5 must be intact for maximal activation of transcription. We also observed that hTRbeta1 can bind directly to yeast or human GCN5 as well as hADA2, and that the hGCN5(387-837) sequence could bind directly to either GRIP1 or SRC-1 coactivator. Importantly, the T3-dependent binding of hTRbeta1 to hGCN5(387-837) could be markedly increased by the presence of GRIP1 or SRC1. Mutagenesis of GRIP1 nuclear receptor (NR) Box II and III LXXLL motifs also substantially decreased both in vivo activation of transcription and in vitro T3-dependent binding of hTRbeta1 to hGCN5. Taken together, these experiments support a multistep model of transcriptional initiation wherein the binding of T3 to hTRbeta1 initiates the recruitment of p160 coactivators and GCN5 to form a trimeric transcriptional complex that activates target genes through interactions with ADA/SAGA adaptor proteins and nucleosomal histones.

Glucocorticoid receptors and glucocorticoid effects in rat Sertoli cells.

In this report we demonstrate glucocorticoid receptors in seminiferous tubules of the rat testis, and that these receptors are localized in Sertoli cells and peritubular cells. The receptors had high affinity for [3H]dexamethasone (Kd = 0.5 - 1 x 10(-9) M), and similar Kd values were calculated from equilibrium analysis and from rate studies (k1 = 1.5 x 10(6) M-1 min-1 and k-1 = 1.4 x 10(-3) min-1, O C). Binding specificity was typical for glucocorticoid receptors (affinity: dexamethasone greater than corticosterone greater than cortisol approximately R5020 approximately progesterone greater than aldosterone = R1881 greater than 17 beta-estradiol approximately cortisone approximately testosterone greater than 5 alpha-dihydrotestosterone). The concentration of glucocorticoid receptors in rat seminiferous tubules revealed an age-dependent decrease, coinciding with the increase in the number of germ cells. Glucocorticoid receptor levels were higher in Sertoli cells from immature rats than in cells from adult rats. Cultured peritubular cells from immature rats contained levels of glucocorticoid receptors similar to cultured Sertoli cells from rats of the same age. With a nick-translated human glucocorticoid receptor complementary DNA probe, a messenger RNA (mRNA) species of approximately 7 kilobase was clearly detected in both Sertoli cells and peritubular cells. In peritubular cells, a smaller mRNA species (5 kilobase) was also clearly detectable. In mRNA from whole testis tissue, a similar developmental pattern as for dexamethasone binding was found. Dexamethasone caused a concentration-dependent stimulation of mRNA levels for androgen binding protein and for the cAMP-dependent protein kinase regulatory subunit type II beta in cultured immature rat Sertoli cells. On the other hand, mRNA levels for glucocorticoid receptor decreased, whereas mRNA levels for beta-actin remained constant. This report documents for the first time the presence of glucocorticoid receptors and glucocorticoid effects in rat Sertoli cells, and is also the first demonstration of glucocorticoid receptors in peritubular cells of the rat testis.

Biochemical and morphological characterizations of DU-145 cell mortality in rabbit embryo-fetal fluid.

Rabbit embryo-fetal fluid (EFF) contains regulatory factors of cell proliferation which increase the duration of the cell cycle, induce a quiescent status in some cells and lead up to cell death in others. The objective of this study was to demonstrate which of the two processes, namely necrosis or apoptosis, was responsible for the cell death. Inhibitors of protein synthesis, and nuclease and phospholipase A2 activities did not restore the viability of the cells treated with EFF. Using a combination of DNA labelling and extraction, it was possible to show that a large proportion of DNA was fragmented in the cells released in the supernatant while only a very small portion of DNA was fragmented in the monolayer cells. EFF did not induce fragmentation of DNA into nucleosome-sized subunits as analysed using polyacrylamide gel electrophoresis. Nevertheless, using cytofluorometric analysis, it was possible to demonstrate that 50% of the cells released in the supernatant contained a lower quantity of DNA per cell than in the control cells. This was also observed with EFF-treated monolayer cells but not in the control monolayer cells. The reduction of the DNA content per monolayer cell became significant at 48 h of treatment with EFF. Electron microscopic analysis did not reveal blebbing of the cells. However, depletion of glycogen, condensation of mitochondria and increasing number of lysosomes and residual bodies were observed upon treatment with EFF. From these experiments we conclude that the DU-145 cells treated with EFF do not die by apoptosis, but rather seem to die by necrosis.

Affinity labelling of steroid hormone receptors.

Affinity labelling techniques have proved indispensable for the study of reversible biological recognition systems, since they conserve ligand-receptor interaction by covalent linkage. Using photo- and electrophilic labelling, it has become possible to unequivocally identify steroid hormone receptors and their proteolytic degradation products and it is simple to establish receptor peptide maps even in crude receptor preparations. The isolation of receptor proteins has been greatly simplified, as their integrity can be analyzed at any step of a purification protocol by SDS-PAGE analysis after crosslinking. Moreover, affinity-labelled receptors can be purified under denaturing conditions, e.g., in high-resolving preparative SDS-PAGE, and the material obtained can be efficiently used to generate anti-receptor antibodies. Peptide mapping after crosslinking of related receptors has been used to assess the degree of structural homology between different forms of steroid hormone receptors and receptors of different species. Peptide sequence analysis of purified crosslinked receptor protein and anti-receptor antibodies have provided the basis for cloning corresponding genes. Techniques have been established to demonstrate--via crosslinking--that the cloned DNA sequences correspond to the receptor gene binding the correct ligand. The analytical and preparative crosslinking methods developed for steroid receptors are potentially important for the study of any system in which signal transduction proceeds via the reversible interaction between biological macromolecules.

Distribution of estrogen receptors in the rat pituitary as studied by in situ hybridization.

The localization of estrogen receptors in the rat pituitary gland was performed by in situ hybridization. The probe used was a synthetic oligonucleotide probe labelled with 35S complementary to the mRNA coding for a fragment (1-24) of estrogen receptor. Strong labelling was observed in cells of the anterior and intermediate lobes whereas the posterior lobe remains unlabelled. These results suggest that not only the anterior lobe but also the intermediate lobe is a target for estrogens.

Peptide sequencing of the chick oviduct progesterone receptor form B.

The progesterone receptor form B has been isolated to apparent homogeneity from large scale preparations of laying hen oviduct cytosol. The quantities obtained were sufficient to monitor the separation of tryptic peptides on HPLC columns. Using a multi-dimensional microbore HPLC peptide purification protocol, several peptides were isolated in homogeneous form and sequenced up to 34 steps at the sub-40 pmol level using a gas phase sequenator. One of the peptides showed a striking homology with sequences of the putative steroid binding domain of the human glucocorticoid receptor; this region is also conserved in the human and chick estrogen receptor.

11 beta-Hydroxysteroid dehydrogenase and tissue specificity of androgen action in human prostate cancer cell LNCaP.

Incubation of whole LNCaP cells in suspension with tritium labeled cortisol revealed two major and one minor radioactive product. Of the major products, one migrated with an Rf value identical to cortisol (Kendall's compound "F"), and the second migrated with an Rf value similar to nonradioactive cortisone (Kendall's compound "E"); the third minor product comigrated with 21-acetylated cortisol. The conversion of cortisol to cortisone was linear with respect to cell number, and conversion reached a plateau after 120 min of incubation at 37 degrees C. One half of the cortisol was converted to cortisone within 2 h of incubation at 37 degrees C. This conversion was nicotine amide dinucleotide (NAD) dependent. Low levels of transcription activation by cortisol were documented in LNCaP cells transfected with glucocorticoid and androgen responsive mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene (MMTV-CAT). Hormone binding assay and transactivation analysis revealed the presence of a functional mineralocorticoid receptor in LNCaP cells. Treatment of transfectants with F in the presence of carbenoxolone, a potent inhibitor of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), resulted in a two orders of magnitude increase in measurable CAT activity. The addition of the reduced form of nicotine amide dinucleotide (NADH) in the presence of 10(-7) M E stimulated measurable CAT activity in LNCaP cells. In conferring aldosterone specificity in mineralocorticoid target tissues, 11 beta-HSD may have an important role as "gate keeper" in allowing a specific androgen response in hormone responsive LNCaP prostate cancer cells.

Human glucocorticoid receptor gene promotor-homologous down regulation.

To study the regulation of the human glucocorticoid receptor (hGR), we characterized the promoter region by primer extension, S1 nuclease mapping and by DNA sequencing. We found that the promoter is extremely G + C rich (72% GC content) and contains a "TAATA" and a "CAT" box, eight "GGGCGG", three "CCGCCC" and two "CACCC" motifs and a motif similar to the glucocorticoid responsive element (GRE) which included two interchanged nucleotides "TCTTGT". In contrast to other steroid receptor genes, exon I or GHGR contains the major part of the 5' non-coding sequences of hGR mRNA while exon II contains coding sequences for the first 394 amino acid residues of the A/B region of hGR. The major transcriptional start site was found to be 134 bp upstream of the ATG initiation codon. Transfection of HeLa cells with plasmids containing various deletions of GHGR promoter fused to a promoterless CAT vector suggested the region between -470 and -1030, at the 5' end of the mRNA start site, to contain sequences required for down regulation by hormone.

Interaction of antiandrogen-androgen receptor complexes with DNA and transcription activation.

Human androgen receptor (hAR) is a ligand-dependent transcription factor that mediates androgen-induced actions on target tissues. Transfection studies in receptor deficient monkey kidney cells CV-1 in culture examine the ability of dihydrotestosterone (DHT) and of the antiandrogens hydroxylflutamide (HO-FLU), cyproterone acetate (Cypro.A) and RU 23908-10 to stimulate or to inhibit the transcription activation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase (MMTV-CAT). CV-1 cells cotransfected with wild type hAR (hAR1-910) and MMTV-CAT, were treated with varying concentrations of DHT. DHT stimulated transcription activation of MMTV-CAT gene in a dose-dependent fashion. Cypro.A though only partially, also stimulated the transcription activation of MMTV-CAT. In the absence of steroids, HO-FLU induced the MMTV-CAT transcription in transfectants only 4% above the basal level. RU 23908-10 revealed the least agonistic activity at concentrations between 10 nM and 1 microM. Despite this, 100- to 1000-fold molar excess of all antiandrogens inhibited the agonistic activity of 10 nM DHT in this system. Receptor binding assays confirmed that HO-FLU, Cypro.A and RU 23908-10 competed with [3H]DHT for AR binding with hAR expressed in CV-1 cells. Western blot analysis using AR antipeptide antibodies raised in rabbits revealed the presence of two AR protein bands in extracts prepared from hAR1-910 transfected CV-1 cells. Incubation of labeled synthetic palindromic androgen responsive element (ARE) with the hAR containing CV-1 cell extracts followed by u.v. cross-linking demonstrated the specificity of AR-DNA interaction. Analysis by gel mobility shift assays showed that the interaction of AR-antiandrogen complexes with labeled ARE was specific.

Differential regulation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene by human mineralocorticoid hormone-receptor complexes.

The brain tissues of the rat and mouse express two types of corticosteroid binding proteins, the glucocorticoid (GR) and aldosterone (MR) receptors. Unlike the type II (GR) receptor, type I receptor has a high affinity for aldosterone (ALDO) and corticosterone and is structurally similar to the kidney mineralocorticoid receptor (MR). The results reported in this study provide direct evidence for the interaction of dexamethasone (DEX), triamcinolone acetonide (TA), dexamethasone-21-mesylate (DXM) and 11-deoxycorticosterone (DOC) with human MR expressed in cells by transient co-transfection of a hMR expression vector. The interactions of hMR with DEX, TA, DXM, DOC, promegestone (R5020) and methyltrienelone (R1881) were measured by trans-activation of mouse mammary tumor virus long terminal repeat fused to bacterial chloramphenicol acetyltransferase (MMTV-tk-CAT) in gene co-transfection experiments and by cell free hormone binding assay. The incubation of various steroid hormones in the presence of [3H]ALDO in a competition assay with extracts prepared from HeLa cells co-transfected with hMR expression vector, showed that hMR expressed under these conditions has a high relative affinity for DEX which is similar to ALDO, TA and DOC. Incubation with DXM under these conditions showed very little competition, as was observed with R1881 and R5020. Incubation of the co-transfected cells with DEX, ALDO, DOC, R5020, TA, R1881 and DXM demonstrated that the level of trans-activation did not reflect the previously observed order of binding affinity for the hMR. The level of transactivation was always higher with DEX and TA compared to ALDO and DOC. Analysis of the binding of labeled glucocorticoid regulatory element (GRE) and hMR incubated with DEX, ALDO and DXM by gel shift analysis demonstrated that the trans-activation of MMTV-tk-CAT by hMR is a result of the interaction of hMR with GRE in the MMTV-LTR.

Mapping of estrogen receptor-producing cells in the rat brain by in situ hybridization.

The localization of cells which produce estrogen receptors in the rat central nervous system was performed using an in situ hybridization technique. For this, we used a synthetic oligonucleotide probe labeled with 35S complementary to the mRNA coding for a fragment (1-24) of estradiol receptor which is not common to the other steroid receptors so far characterized. Radioautography revealed that labeling was present in discrete brain areas. The results obtained with the in situ hybridization agree well with previous results obtained with other techniques. This approach should be very useful to study the effects of different factors on estrogen receptor gene expression in specific brain nuclei and individual neurons.

Partial purification of rat liver glucocorticoid binding proteins by affinity chromatography.

Dexamethasone (9-fluoro-16alpha-methyl-11beta,17,21-trihydroxy-1,4-pregnadiene-3,20-dione) binding proteins from rat liver cytosol were purified approximately 6470 fold by the use of an affinity column in which deoxycorticosterone was linked to CH-Sepharose 4B through a disulfide linkage. The receptor proteins were eluted from the column by washing with beta-mercaptoethanol. A preliminary Sephadex G-200 filtration step of the cytosol was necessary in order to separate the dexamethasone binding proteins from other glucocorticoid receptors.

Urokinase-type plasminogen activator: a paracrine factor regulating the bioavailability of IGFs in PA-III cell-induced osteoblastic metastases.

The transplantation of PA-III rat prostate cancer cells onto rat skeleton produces osteoblastic metastases. Therefore w e studied the paracrine interactions between the PA-III cells and osteoblast-derived osteosarcoma cells (UMR 106 cells). A serine protease secreted by PA-III cells hydrolyzed IGF-binding protein-1 and IGF-binding protein-2 (IGFBP-1 and IGFBP-2) detected in the cell culture media (CM) of OMR 106 cells by western ligand blotting. The serine protease of PA-III cell CM was purified using a benzamidine affinity column. This protease was a protein of 45-50 kDa on polyacrylamide gel electrophoresis under non-reducing conditions but generated two protein bands under reducing conditions; a) one of 33-35 kDa possessing protease activity and b) another of 20-25 kDa which was proteinolytically inactive. Sequence analysis identified the amino acid sequence of the a-chain (20-25 kDa band) and of the b-chain (33-35 kDa band) of rat urokinase-type plasminogen activator molecule. Urokinase purified from PA-III cell CM hydrolyzed IGFBPs of UMR 106 cells and stimulated the proliferation of UMR 106 cells in serum-free cultures. Its protease activity was abolished by benzamidine and aprotinin. Its mitogenic activity for osteoblasts was inhibited by anti-IGF-I monoclonal antibody. Northern blot analysis documented the expression of the urokinase-type plasminogen activator gene in the mRNA extracted from PA-III cells. Urokinase expression was inhibited by dexamethasone. Therefore, we conclude that urokinase-type plasminogen activator stimulates osteoblasts via an IGF-I dependent mechanism. Hydrolysis of the IGFBOPs at the sites of PA-III cell-induced bone tumors account for an increased bioavailability of IGFs. This may facilitate the development and the growth of PA-III cell-induced bone tumor and can also mediate the subsequent local osteoblastic reaction.

17 beta-estradiol-regulated expression of protein tyrosine phosphatase gamma gene in cultured human normal breast and breast cancer cells.

BACKGROUND: Protein tyrosine phosphatase gamma (PTP gamma) has been implicated as a potential tumor suppressor gene in kidney and lung adenocarcinomas. We have previously shown that PTP gamma mRNA expression levels are lower in DES-induced kidney tumors than in normal kidneys of Syrian hamsters. The goals of the present study were to determine if PTP gamma mRNA is present in both normal and cancerous human breast cells, and to investigate the estrogenic regulation of PTP gamma mRNA expression in these cell types. METHODS: Primary cultured human breast cells derived from surgical specimens of mammoplasty and breast cancer patients, as well as human breast cancer cell lines were used for the study. RT-PCR and RNase protection assay was utilized to detect and quantify levels of PTP gamma mRNA among the cell types used and between control and 17 beta-estradiol (E2)-treated cells. Transient transfection of human estrogen receptor (ER) into MDA-MB-231 human breast cancer cells was performed to establish the role of ER in the regulation of PTP gamma mRNA expression. RESULTS: The results show that PTP gamma mRNA is expressed in primary cultured human breast cells isolated from mammoplasty and breast cancer patients, as well as in human cancer cell lines, and that E2 significantly inhibits PTP gamma expression in ER-positive human breast cancer cells via an ER-mediated mechanism. We show that PTP gamma mRNA levels are lower in human breast cancer cells than in normal human breast cells. Furthermore, we report that PTP gamma mRNA expression is inhibited by E2 in a dose-dependent manner in primary cultured breast cells. After treatment with 20 nM E2 for 24 hours, PTP gamma mRNA was significantly suppressed in primary cultured cancerous and non-cancerous cells from breast cancer patients, as well as in the ER-positive MCF-7 cell line by 50%, 85%, and 66%, respectively. In contrast, the PTP gamma mRNA expression levels did not change in similarly treated ER-negative MDA-MB-231 cells. Sensitivity to E2-induced suppression could be restored (94% inhibition) by transfecting MDA-MB-231 cells with an ER expression plasmid. CONCLUSIONS: Our results are the first to suggest that PTP gamma is a potential estrogen-regulated tumor suppressor gene in human breast cancer which may play an important role in neoplastic processes of human breast epithelium.

Purification of two dexamethasone-binding proteins from rat-liver cytosol.

Two dexamethasone-binding proteins have been purified from rat liver cytosol. The main purification steps are: precipitation by protamine sulphate, affinity chromatography on CH-Sepharose 4B to which 11-deoxycorticosterone is linked through a disulfide bond and DEAE-cellulose chromatography. Two binding components elute from the DEAE-cellulose column at 0.12 M and 0.2 M NaCl, respectively. By means of dodecylsulphate/polyacrylamide gel electrophoresis it was demonstrated that both components are composed predominantly of a single polypeptide with molecular weights of about 45 000 and 90 000. Antibodies to the two polypeptides have been elicited in rabbits. The antibodies to the 45 000-Mr polypeptide cross react with the 90 000-Mr component. Likewise the antibodies to the 90 000-Mr protein precipitate the 45 000-Mr polypeptide. Either of the two antibody preparations immunoprecipitates the major part (approximately 70%) of the dexamethasone-binding activity of the cytosol.

Characterization of the rat liver glucocorticoid receptor purified by DNA-cellulose and ligand affinity chromatography.

Two rapid and high yield purification methods for the rat liver glucocorticoid receptor based on differential DNA affinity (method A) and ligand affinity (method B) chromatography are described. In method A, the amount of receptor in rat liver cytosol that can be activated and subsequently eluted from a DNA-cellulose column has been increased to 80% by introducing a second heat activation step. Using this method, 1.5 nmol of 25% pure glucocorticoid receptor can be routinely obtained per day from 15-20 rat livers. Method B yields about 2.2 nmol of 60% pure receptor with an overall yield of congruent to 60%. The quality of these purifications has been controlled by affinity labeling. In each case, more than 95% of purified binding activity represented the intact 92,000 +/- 400-Da glucocorticoid receptor polypeptide as shown by sodium dodecyl sulfate-gel electrophoresis and fluorography. No difference in the labeling pattern was observed using either [3H]triamcinolone acetonide (photoaffinity labeling) or [3H]dexamethasone 21-mesylate (electrophilic labeling). The electrophilic labeling step was performed in the cytosol prior to purification by method A to compare the labeled components thus purified with those obtained when the photoaffinity labeling was performed after the purification. Using this approach, distinct breakdown products of the glucocorticoid receptor were revealed, co-purifying during DNA affinity chromatography. Cross-linked receptor obtained by method A has been further purified to homogeneity by preparative sodium dodecyl sulfate-gel electrophoresis and successfully used as immunogen to raise glucocorticoid receptor antibodies in rabbits. These antibodies raised against glucocorticoid receptor, as well as those previously obtained using affinity chromatography-purified receptor, react with the receptor molecules irrespective of their method of purification. Glucocorticoid receptors purified by methods A and B have been analyzed for specific DNA-binding properties by the nitrocellulose filter binding assay.

Synthesis of 21-diazo-9 alpha-fluoro-16 alpha-methyl-21-deoxy-[1,2(n)-3H]prednisolone.

The preparation of 21-diazo-9 alpha-fluoro-16 alpha-methyl-21-deoxy-[1,2(n)-3H]prednisolone is described. [1,2(n)-3H]Dexamethasone 26 Ci/mmol, was oxidized with cupric acetate/air to give the 21-aldehyde. Treatment of the aldehyde with hydroxylamine gave the corresponding 21-oxime which was reacted with chloroamine to give a 25--35% yield of 21-diazo-9 alpha-fluoro-16 alpha-methyl-21-[1,2(n)-3H]deoxyprednisolone with a specific activity of 25--26 Ci/mmol.

Three-step purification of glucocorticoid receptors from rat liver.

The 90 000-Mr glucocorticoid receptor was purified to homogeneity according to sodium dodecylsulfate/polyacrylamide gel electrophoresis. An affinity column containing either dexamethasone-17 beta-carboxylic acid or dexamethasone-21-methanesulfonate bound to the matrix with the help of a disulfide bond is used in this study. Using this affinity matrix, in a single step, 8700-fold purification of glucocorticoid receptor from rat liver cytosol could be achieved. Following the method of activation and DNA-cellulose chromatography the 90 000-Mr receptor subunit was purified to homogeneity.