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1. Exchange techniques were developed for measurement of total progesterone receptor binding sites concentration in the cytosol and nuclei of chicken oviduct. 2. The level of progesterone receptor in the cytosol was under both oestrogen and progesterone control. Primary stimulation by oestradiol benzoate increased the receptor concentration from approximately 10000 sites/cell to approximately 40000 sites/cell in the magnum cytosol, subsequent withdrawal from oestrogen treatment led to a decrease to approximately 14000 sites/cell after 6 weeks After progesterone administration (3 mg/kg) to oestrogen-stimulated, withdrawn chicken, the receptor concentration decreased to approximately 40 % of the initial level within the first 4 h; afterwards the receptor level rose again and by 40 h exceeded slightly the initial one. 3. The nuclear levels of the receptor reached a maximum at 1 h after the progesterone injection; however, the gain of the binding sites by the nuclei (≈ 900/nucleus) did not account for their loss from the cytosol. The maximum nuclear receptor level was not influenced by the dose of progesterone within range of 1-10 mg/kg. 4 The extractibility of the nuclear progesterone receptor by 0.5 M NaCl was strongly influenced by preincubation of the nuclei at 30°C; irrespective of this preincubation, approximately 12 % of the nuclear receptor resisted extraction with 2 M NaCl/5 M urea. [ABSTRACT FROM AUTHOR]

[3H]Oestradiol exchange techniques were developed for the determination of specific oestrogen receptor site concentrations in the cytoplasm and nuclei of chick oviduct cells. Non-labelled, receptor-bound oestrogens were exchanged with [3H]oestradiol during a 24-h incubation at 20 °C, 2 h at 30 °C or 45 min at 37 °C. Both ‘soluble’ and ‘insoluble’ nuclear receptors were stable for at least 6 h at 30 °C and 37 °C but a proportion (approx. 30 %) of cytoplasmic sites from withdrawn chickens were inactivated after 2 h at 20 °C. The magnum of 4-week-old immature chickens (weight = 15 mg) contained 0.20 pmol of oestrogen receptor which corresponds to 4275 receptor sites/cell, when it is assumed that all magnum cells have equal concentrations of receptor. In primarily stimulated chickens of similar age which had received 10 × 1 mg of oestradiol benzoate/day, the magnum weighed approximately 800 mg and contained 8.65 pmol of oestrogen receptor (4610 sites/cell). Withdrawal from primary oestrogenic stimulation for 3–6 weeks resulted in a 110 mg magnum which contained 1.20 pmol of receptor (2225 sites/cell). Oviducts from immature and withdrawn chickens had the majority (73–77%) of their oestrogen receptor sites in the cytoplasmic fraction, while in primary stimulated chicken oviducts the majority (82 %) of receptor sites were located in the nucleus. A single secondary injection of oestradiol, to oestrogen-withdrawn chickens, resulted in apparent translocation of cytoplasmic receptors to the nucleus during the first hour after injection. The magnitude of the decline in cytoplasmic receptor, and the concurrent increase in nuclear receptor concentration, was dose-dependent between 2 and 100 μg oestradiol/kg body weight. Larger doses of oestradiol up to 1 mg/kg did not increase the concentration of nuclear receptor above the maximum level seen at 100 μg oestradiol/kg. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. By 15 h after an injection of 100 μg oestradiol/kg, the concentration of nuclear sites had reached pre-injection levels. During the same time period, the depleted cytoplasmic receptor levels were replenished such that they reached control values by 12 h and were about 150% of the pre-injection level at 24 h. [ABSTRACT FROM AUTHOR]

FKBP59, a 59-kDa FK506 binding protein, was discovered in heterooligomeric complexes containing nontransformed, non-DNA binding, steroid receptors. Sequence similarity search and secondary structure prediction suggested that the protein has a multi-domain organization, the N-terminal domain having a great similarity to human FKBP12 (12-kDa FK506-binding protein). FKBP59 binds immunosuppressant FK506 and has peptidylprolyl cis-trans-isomerase activity, both properties being localized in the N-terminal domain (FKBP59-I). In order to characterize its conformational features and to better understand its biological significance, we overexpressed and 15N-labeled this domain (149 amino acids) in Escherichia coli and initiated an NMR structural study in solution. Almost complete sequence-specific assignment of the 1H and 15N resonances was achieved using two-dimensional and three-dimensional homonuclear and heteronuclear experiments. Localization of the secondary structure elements was derived essentially from CαH chemical shift distribution along the sequence, the short-range and medium-range NOE connectivities and exchange kinetics of amide protons. The domain has a structured part comprising six β-strands and a three-turn (α-helix between K87 and M96. The first 17 residues are highly flexible and show no regular secondary structure. The β-sheet structure, derived from long-range connectivities between backbone protons, consists of six β-strands defined as follows: B1, V22–I24; B2, V32–K37; B3, D50–L61; B4, T64–S68 and F76–L80; B5, E100–K107; B6, L127–F137. They are organized in an antiparallel β-sheet with the connecting topology +1, +3, +1, -3, +1. The α-helix connects strand B4 to strand B5. Globally, the structure of FKBP59-I, derived from the present work, is similar to the NMR-derived structures of uncomplexed FKBP12. However, several conformational differences were noted at this level of structural analysis. The b-sheet of the FKBP59 domain has an additional strand at the N-terminal and the α-helix is longer by about one helical turn. In addition strand B4 has two components, separated by a large bulge (seven residues); the first component was observed in the X-ray or NMR structures of complexed FKBP12 but not in the NMR-derived, uncomplexed structure. [ABSTRACT FROM AUTHOR]

The glucocorticosteroid receptor (GR) has been studied in oviduct cytosol prepared from estrogen-primed, 4-week-withdrawn chicken. The equilibrium dissociation constant was 6 nM for dexamethasone, and 18300 receptor sites/cell were measured assuming that all ceils contain identical concentrations of GR. Dexamethasone, used in most studies investigating glucocorticosteroid action, was found not to be the best GR ligand. The affinities of several natural and synthetic glucocorticosteroids for GR increased in the following order: corti- sol < deoxycorticosterone < dexamethasone < corticosterone < triamcinolone acetonide. The synthetic steroid RU 486 was the most specific ligand of GR (its affinity was ≈ 10-fold higher than that of triamcinolone acetonide), while it did not bind either to plasma transcortin (which binds dexamethasone nor, surprisingly, to progesterone receptor (PR), contrary to what occurs in mammalian species. The molybdate-stabilized, 8-S form of GR was prepared from withdrawn chick oviduct, whole chick embryo or cultured chick embryo fibroblasts (which do not contain PR), and was labeled with either [3H]dexamethasone or [3H]RU 486. The sedimentation coefficient of radioactive ligand-8-S GR complexes was shifted towards heavier forms after incubation with polyclonal (IgG-G3) or monoclonal (BF4) antibodies generated against the molybdate-stabilized, 8-S form of the chick oviduct PR. Since neither IgG-G3 nor BF4 interacted with the steroid binding 4-S form of GR, it is suggested that these antibodies recognized a non-steroid binding protein common to molybdate-stabilized, 8-S forms of GR and PR. [ABSTRACT FROM AUTHOR]

The highly purified, molybdate-stabilized '8-9-S' form of the chick oviduct progesterone receptor complexed with [³H]progesterone (Specific activity ≈ 10-12 nmol bound steroid/mg protein) was prepared according to the three-step method [Renoir et al. (1982) Eur. J. Biochem. 127., 71-79] and 50-90 µg samples were injected into a goat and a rabbit. Specific antibodies were observed as early as 2 weeks following the first booster injection and increased considerably 12 weeks later after further booster injections. Results were similar in the two animals, with higher titers observed in the goat. Antibodies cross-reacted with the 'transformed' 4-S form of the chick oviduct progesterone receptor. They also cross-reacted with molybdate-stabilized 8-9-S progesterone receptors of mammalian species (obtained from MCF-7 human breast cancer cells and from rabbit and mouse uteri), suggesting some evolutionary conservation of steroid hormone receptors. No cross-reaction was observed with chick oviduct oestradiol receptor and with chick plasma transcortin (a non-receptor progesterone-binding protein). [ABSTRACT FROM AUTHOR]

A molydate-stabilized. 'non-activated' form of the progesterone receptor from the cytosol of oestrogen-stimulated chick oviduct has been purified to homogeneity by a three-step procedure. The first step, affinity chromatography using a N-(12-amino-dodecyl)-3-oxo-4-androsten-17β-carboxamide-substituted Sepharose gel, purified the receptor 1500-2700-fold with ≈ 50% recovery. In the second step, ion-exchange chromatography through a DEAE-cellulose column, progesterone receptor was eluted as a single peak at 0.1 M KCl. Purification after this step was >6700-fold. The third step was filtration through Ultrogel AcA 34, resulting in overall purification ≈ 7400-fold with overall recovery ≈ 25% of pure receptor on the basis of 1 binding site/molecule of Mr 85 000. The purified molybdate-stabilized receptor had a sedimentation coefficient ≈ 7.9 S ± 0.1 (n = 4) in 0.15 M or 0.4 M KCl containing sucrose 5-20% gradient and ≈ 8.9 S ± 0.2 (n = 6) in 0.15 M KCl containing glycerol 10-35% gradient, and its Stokes radius was 7.05 ± 0.10 nm (n = 3) (calculated Mr between 240 000 and 280 000). Binding specificity of the purified receptor was the same as that found in crude cytosol. SDS-PAGE revealed a single band migrating as a polypeptide of Mr ≈ 85 000 ± 2300 (n = 9). PAGE under nondenaturing conditions at total acrylamide concentrations 5%, 7% and 9% showed a single [³H]ORG 2058-protein band (ORG 2058 is a high-affinity analogue more suitable than progesterone for electrophoretic studies). The data suggest that the high molecular weight molybdate-stabilized progesterone receptor purified from oestrogen-primed chick oviduct is composed of only ≈ 85 000-Mr polypeptide chains. [ABSTRACT FROM AUTHOR]

Tamoxifen, a non-steroidal antioestrogen, did not display any oestrogenic effects biochemically or histologically in the chick oviduct when 10 mg/kg were administered to oestrogen-withdrawn animals each day and effects were monitored during periods ranging from a few hours to 10 days. After a single injection of oestradiol benzoate. 1 mg/kg, to oestrogen-withdrawn animals, the relative rate of ovalbumin synthesis was significantly elevated by 3 h, and reached a maximum by &approx; 16 h. Conalbumin synthesis increased immediately after oestrogen administration, and attained a maximum by &approx; 16 h. Between 16 and 24 h there was a decrease of &approx; 50% in the rate of synthesis of the two proteins and in the levels of nuclear oestrogen receptor. Administration of tamoxifen together with oestradiol benzoate inhibited the oestrogen effect on ovalbumin and conalbumin synthesis. This effect of tamoxifen was dose-dependent; 50% inhibition of the maximum induction of both proteins was obtained with 1 mg of tamoxifen/kg. A series of experiments indicated that tamoxifen given after oestradiol benzoate could rapidly inhibit the oestrogenic effect, and oestradiol benzoate administered subsequent to tamoxifen could overcome the antioestrogenic effect; that is each ligand could produce its own characteristic activity, although in both cases, at the time of administration of the second drug, the level of cytoplasmic oestrogen receptor was low. When tamoxifen was administered during the lag period of ovalbumin induction, it decreased the entire pattern of ovalbumin synthesis, but when given later, the inhibitory effect was retarded. On the other hand, the inhibition by tamoxifen of oestradiol-dependent conalbumin synthesis was virtually immediate. The basal level of conalbumin synthesis, already significant in withdrawn chickens (1%), was unaffected by tamoxifen, suggesting that endogenous oestrogens are not required to maintain this level of synthesis. [ABSTRACT FROM AUTHOR]

After a single injection of oestradiol benzoate (1.5 mg/kg) to oestrogen-withdrawn chickens, there was an increase in magnum wet weight, DNA polymerase α activity, adenosine-3′,5′-mono-phosphate-dependent protein-kinase activity and estrogen-receptor concentration, as measured over 36 h. Besides these intracellular proteins, the secretory proteins ovalbumin and conalbumin were also augmented, and detailed time-course studies were performed. Early induction kinetics for ovalbumin and conalbumin synthesis, which differed for each protein, were independent of the dose of oestradiol benzoate injected if it exceeded 0.1 mg/kg. After 6 h for ovalbumin and 2 h for conalbumin, the induction curves diverged according to the dose of hormone administered and in correlation with the persistence of elevated nuclear oestrogen-receptor concentrations, a result confirmed with 11Β-methoxy-17α-ethynyloestradiol (R 2858), a powerful synthetic oestrogen. When oestradiol benzoate (1 mg/kg) and progesterone (3 mg/kg) were injected simultaneously, the rate of conalbumin synthesis, during the first 6–8 h, was lower than that observed in animals injected with oestradiol benzoate alone. However at later times conalbumin synthesis was greater in animals receiving both hormones than with oestradiol alone. In contrast, the rate of ovalbumin synthesis after the combined injection was higher than that induced by either hormone alone throughout the entire experimental period. In order to study further the synergistic and antagonistic activities of these two hormones, a single injection of progesterone (3 mg/kg) was administered 6, 12 or 18 h after 1.5 mg/kg oestradiol benzoate. Progesterone administration resulted in a reduction in cytoplasmic, nuclear and total oestrogen receptor concentration for at least 6 h when compared with the values in birds treated with oestrogen alone. DNA polymerase and protein kinase activities were also reduced during this period. [ABSTRACT FROM AUTHOR]

The properties of three types of adsorbents obtained by coupling oestradiol 7α-derivatives to agarose were compared. The adsorbents examined were: oestradiol 7α-decamethylene-agarose, oestradiol 7α-decamethylene-poly(alanyl-lysine)-agarose and oestradiol 7α-trimethylene-poly(alanyl- lysine)-agarose. The following results were obtained. (1) All these adsorbents are stable at 0 C for at least a year when stored in water. In the presence of cytosol they are stable for several hours and are reusable after a simple wash. (2) A new method allowing the calculation of the maximal receptor binding capacity of an absorbent was developed. (3) The geometry of the column and the dynamics of the loading have no influence on the binding capacity of the adsorbents. (4) Binding of the cytosol receptor to the adsorbent depends on whether the receptor had previously been partially purified by heparin-Ultrogel chromatography or treated with low or high salt concentration or trypsin. It was demonstrated that aggregation decreases the binding of the receptor to the adsorbents. (5) A satisfactory recovery of receptor upon elution is pos- sible only with biospecific adsorbents containing low concentrations of coupled steroid (≤ 0.2 mg/ml). The use of these adsorbents for the purification of the trypsin-treated receptor directly from cytosol allowed a 2500-fold purification corresponding to 5% pure protein (assuming one oestradiol binding site per molecule of Mr 60000). When starting from a low salt preparation containing the native 8-S receptor, partially purified by heparin-Ultrogel chromatography, preliminary experiments using affinity chromatography gave a further purification of 250-500-fold and led to a 50-90% pure protein (assuming one oestradiol binding site per molecule of Mr 70000). [ABSTRACT FROM AUTHOR]

An early 'induced protein', after exposure of the rat uterus to estradiol, is detected among the soluble proteins with a double-labelling technique and electrophoretic fractionation. Efforts have been directed to establish the subcellular distribution of the induced protein, since such a protein, observable 1 h after hormone administration, may play an important role in the subsequent amplified responses, especially in terms of RNA synthesis. Moreover such an early discrete induced protein was sought in a comparable system responding to another hormone, namely prostate and seminal vesicles under androgens. The induced protein was not found in uterine nuclei of 21-day-old rats after 1 h of estradiol action in vivo and 1 h of tissue incubation with labeled leucine. This negative result summarizes a search among different nuclear protein fractions using various procedures; nor was induced protein observed in mitochondrial and microsomal pellets. Contrary to these negative findings, slight changes of histone labelling were observed under the experimental conditions used to demonstrate induced protein. In addition histone acetylation was increased after 1 h of estradiol action in vivo and 15 min tissue labeling in vitro with radioactive acetate. Furthermore, an increase in total protein synthesis between 0 and 2 h after estradiol action was observed, the relative increase of incorporation of radioactive leucine into protein of estradiol-treated vs non-stimulated uteri being corrected for variations of the acid-soluble radioactive leucine pool. Attempts to obtain an early and discrete induced protein with androgens in prostate and seminal vesicles of immature or castrated rats after different times of exposure to testosterone, androstanolone and estradiol have been unsuccessful. The contribution of both negative and positive findings is steroid-induced early protein synthesis is discussed in the context of the current knowledge of hormone action. [ABSTRACT FROM AUTHOR]

Macromolecular components with properties of oestrogen receptors have been identified in the 0.5 M KCl nuclear soluble, the nuclear insoluble and the cytosol fractions of laying hen and immature (2-4 weeks, untreated by hormone) chicken oviduct. In the 0.5 M KCl extract of laying hen oviduct nuclei, a receptor, of protein nature according to the effects of enzymic treatments, has been identified. It exhibits high affinity for oestradiol with an apparent equilibrium association constant KA = 4 · 109 M-1 at 4 °C. The binding of [³H] oestradiol is abolished by 1 µM oestriol, oestrone and diethylstilboestrol, but not by the same concentration of progesterone, testosterone, and cortisol. Sucrose gradient ultracentrifugation studies in the presence of 0.5 M KCl indicate a sedimentation coefficient of 4.3 S, and there is partial aggregation in low-ionic-strength medium. The estimated number of binding sites per nucleus is about 5 000, as calculated from DNA content of chick diploid genome. Most of the binding sites were found to be occupied by endogenous oestrogen(s). Oestradiol dissociates from the receptor according to an apparent two-step mechanism. The half-life time for the faster dissociation step is 18 h at 0 °C, 25 min at 20 °C and 10 min at 30 °C, and for the slower one is 180 h, 115 min and 60 min, respectively. In the 0.5 M KCl extract of immature chicken oviduct nuclei, there are approximately 500 receptor sites per nucleus; their affinity for oestradiol is the same as in the case of laying hen soluble nuclear receptor. After repeated extractions of nuclei with 0.5 M KCl medium, a substantial quantity of oestrogen binding sites remains in the residual fraction. Binding characteristics of this insoluble nuclear receptor resemble those of the soluble nuclear receptor: high affinity for oestradiol (KA = 7 · 108 M-1 at 37 °C) and specificity for oestrogens. The estimated number of binding sites are approximately 2000/cell for laying hen, and approximately 1000/cell for immature chicken. In the high-speed supernatant fraction of laying hen oviduct homogenates, an oestrogen receptor is also present, but its concentration is low (≤ 100 sites/cell) and at the limits of sensitivity of the methods used. In the cytosol of immature chicken oviduct, there are approximately 2500 oestradiol receptor sites per cell. [ABSTRACT FROM AUTHOR]