Transformation of the rat uterine estrogen receptor after partial purification

Warming crude rat uterine cytosol after the addition of [³H] estradiol accelerates the association of the 4-S estrogen-binding protein with a second macromolecule, resulting in the formation of the 5-S estrogen-binding protein. To determine whether the 5-S estrogen-binding protein consists of two similar or dissimilar subunits, uterine cytosol was subjected to a number of fractionation procedures that separate macromolecules by solubility, molecular gel sieving, sedimentation rate, ionic charge, and heat lability. Following each of these methods, the fraction containing the 4-S estrogen-binding protein was incubated at 28°C; each of these 4-S estrogen-binding protein-containing fractions retained its capacity to completely transform to the 5-S estrogen-binding protein. In samples subjected to partial purification procedures, it was necessary that the buffer contain 40 mM Tris, 60 mM KCI, 1–10 mM dithiothreitol, and 1 M urea at pH 7.4, in order to accomplish the 4-S to 5-S estrogen-binding protein transformation at 28°C. Formation of the 5-S estrogen-binding protein requires association of the 4-S estrogen-binding protein with a molecule identical to or very similar to itself.     

Characterization of juvenile hormone-binding proteins of hemolymph of Locusta migratoria

The binding of juvenile hormone (JH) by components from hemolymph of adult female Locusta migratoria was characterized to establish whether hemolymph JH-binding proteins could be distinguished from a protein of fat body (BP-1) that may be a JH receptor. Hemolymph was analyzed by the hydroxyapatite assay, gel separation chromatography, polyacrylamide gel electrophoresis, and density gradient centrifugation. Three fractions that bound JH were separated from whole hemolymph by DEAE cellulose column chromatography, and these differed from all three cytosol-binding components. The major hemolymph component (H-A) showed relatively stable binding of JH, a slight loss of binding capacity after delipidation, and a K_d for JH-I of 16 nM. The K_ds for JH-l and JH-lll with unfractionated hemolymph were 26 and 42 nM respectively. The order of effectiveness of competitors for binding of [³H]JH-l was JH-lll > JH-l ? methoprene > hydroprene ? acids of methoprene and hydroprene. The data indicated that unlabeled JH-lll was bound more effectively than its radioactive counterpart. The sedimentation values determined by sucrose density gradient ultracentrifugation were 13-14 S for hemolymph, and the sedimentation value was not altered by the inclusion of 0.4 M KCl throughout the gradient. The data indicated that H-A resembled the specific JH carriers and differed from the putative receptor of fat body cytosol by several criteria.     

Characterization of estrogen receptor in estrogen-dependent transplantable rat pituitary tumor MtT/F84

Properties of nuclear and cytosolic estrogen receptors (ERs) were examined in a new transplantable rat pituitary tumor designated as MtT/F84, of which growth is stimulated by estrogen. The optimal incubation conditions of both nuclear and cytosolic exchange were found to be at 37 degrees C for 15 min and at 25 degrees C for 2 hr, respectively. Molybdate increased a specific binding of estradiol (E2) as determined by [3H]E2-binding assay. Sucrose density gradient analyses of crude cytosol revealed specific peaks of radioactivity in both 4-5S and 8-10S areas. However, only a single 5S peak was present in 0.4M KCl-extractable nuclear ER. Molybdate also enhanced the stability of cytosolic 8-10S receptor in density gradient sedimentation behavior. Scatchard plot analysis for nuclear ER yielded a single class of binding sites with a dissociation constant (Kd) of 0.317 nM and the maximum number of binding sites (NBSmax) of 25.4 fmol/mg protein. Saturation analysis of [3H]estrogen binding to cytosolic ER also yielded a straight line with a Kd of 0.146 nM and NBSmax of 58.5 fmol/mg protein. The effect of E2 administration on the intracellular distribution of ER was also examined. A marked disappearance in the ER binding in cytosol with a concomitant increase in binding in nuclear fraction was found after the administration of the unlabeled E2 in vivo, whereas the total number of ER did not change. Thus, it is concluded that properties of ER in the MtT/F84 were very similar to those in other target organs such as uterus and pituitary gland.     

Hepatic estrogen-binding proteins in male and female vervet monkeys

Hepatic cytosols from male and female vervet monkeys (Cercopithecus pygerythrus) were found to contain the same levels of high affinity estrogen-binding proteins. Multipoint saturation analyses revealed that male liver cytosols contain two distinctly different binding components: a high affinity (HAEB) and a low affinity estrogen binder (LAEB). Female livers appeared to contain only the HAEB. Sucrose density gradient (SDG) analyses, however, clearly established the presence of a 3.8 S as well as an 8.1 S estrogen-binding component in the hepatic cytosols of both sexes. The 3.8 S binding component appeared to be more prominent in male SDG profiles. Cytosols, prepared in the presence of sodium molybdate (cyt +) exhibited significantly lower (50%) levels of specific estrogen-binding than cytosols prepared in the absence of the oxyanion (cyt-). SDG analyses, however, indicated that in cyt+ the 8.1 S binding component was stabilized at the cost of the 3.8 S binder. This phenomenon was observed in both sexes. Large excess levels of cortisol did not have any effect on specific estrogen binding by hepatic cytosols. The hepatic estrogen-binding proteins displayed a lower relative binding affinity for diethylstilbestrol than for its native ligand and higher affinities for estriol and estrone than expected.     

Transformation of the rat uterine estrogen receptor after partial purification.

Warming crude ratuterine cytosol after the addition of [3H] estradiol accelerates the association of the 4-S estrogen-binding protein with a second macromolecule, resulting in the formation of the 5-S estrogen-binding protein. To determine whether the 5-S estrogen-binding protein consists of two similar or dissimilar subunits, uterine cytosol was subjected to a number of fractionation procedures that separate macromolecules by solubility, molecular gel sieving, sedimentation rate, ionic charge, and heat lability. Following each of these methods, the fraction containing the 4-S estrogen-binding protein was incubated at 28 degrees C; each of the these 4-S estrogen-binding protein-containing fractions retained its capacity to completely transform to the 5-S estrogen-binding protein. In samples subjected to partial purification procedures, it was necessary that the buffer contain 40 mM Tris, 60 mM Tris, 60 mM KC1, 1-10 MM dithiothreitol, and 1 M urea at pH 7.4, in order to accomplish the 4-S to 5-S estrogen-binding protein transformation at 25 degrees C. Formation of the 5-S estrogen-binding protein requires association of the 4-Estrogen-binding protein with a molecule identical to or very similar to itself.     

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

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

Estradiol down-regulation of the rat uterine estrogen receptor

We have previously shown that neonatal exposure of rats to pharmacologic doses of diethylstilbestrol via daily injections resulted in a significant decrease in the estrogen-binding capacity of the uterine estrogen receptor (ER). In this study, we examined the effects of physiologic and pharmacologic doses of estradiol (E2) administered to adult ovariectomized rats via Silastic implants. Two days after implantation, uteri were removed, weighted, and homogenized, and ER levels were determined in the supernatant (hydroxylapatite assay) and low-speed pellet (nuclear exchange assay). Implants containing E2 concentrations of 0.005 or 0.05 mg/ml increased cytosolic but not total ER-binding capacity, whereas 0.5 or 5.0 mg of E2/ml implants decreased the binding capacity of cytosol ER to 40% and total ER to 50% of control values. The 0.005-mg/ml dose increased cytosol ER without increasing uterine weight; all higher doses significantly increased uterine weight. Determination of ER protein by an ER radioimmunoassay showed the same extent of reduction of ER concentration as the binding assays, demonstrating that the loss in E2 binding capacity is homologous down-regulation. The down-regulation of ER was maximal at 24 hr and was completely reversible after implant removal, although the time required to recover from down-regulation was dose dependent. Uterine weight also returned to control levels slowly after implant removal. Neither the sedimentation rate of the down-regulated ER nor the Kd of the cytosolic ER changed following long-term implantation; however, the Kd of the nuclear ER decreased significantly. This is the first demonstration of in vivo homologous down-regulation of uterine ER. ER down-regulation may play a role in several biologic processes.     

Characterization of an estrogen-binding protein in the yeast Saccharomyces cerevisiae

This paper further characterizes the estrogen-binding protein we have described in the cytosol of the yeast Saccharomyces cerevisiae. [3H]Estradiol was used as the radioprobe, and specific binding of cytosol fractions was measured by chromatography on Sephadex minicolumns. Other 3H-steroids did not exhibit specific binding. [3H]Estradiol binding was destroyed by treatment with trypsin, but not RNase, DNase, or phospholipase; N-ethylmaleimide substantially decreased the binding. The yeast did not metabolize estradiol added to the medium, and extraction and chromatography of the bound moiety showed it to be unmetabolized estradiol. Scatchard analysis of cytosol from both a and alpha mating types as well as the a/alpha diploid cell revealed similar binding properties: an apparent dissociation constant or Kd(25 degrees) for [3H]estradiol of 1.6-1.8 nM and a maximal binding capacity or Nmax of approximately 2000-2800 fmol/mg of cytosol protein. Gel exclusion chromatography on Sephacryl S-200 and high performance liquid chromatography suggested a Stokes radius of approximately 30 A. Sucrose gradient centrifugation showed a sedimentation coefficient of approximately 5 S, and the complex did not exhibit ionic dependent aggregation. The estrogen binder in S. cerevisiae differed in its steroidal specificities from classical mammalian estrogen receptors in rat uterus. 17 beta-Estradiol was the best competitor, 17 alpha-estradiol had about 5% the activity, and diethylstilbestrol exhibited negligible binding affinity as did tamoxifen, nafoxidine, and the zearalenones. In summary, a high affinity, stereospecific, steroid-selective binding protein has been demonstrated in the cytosol of the simple yeast S. cerevisiae. We speculate that this molecule may represent a primitive hormone receptor system, possibly for an estrogen-like message molecule.     

Immunological demonstration of alpha-fetoprotein in uterine cytosol from immature rats

An immunological demonstration of alpha-fetoprotein (AFP) in uterine cytosol from immature rats is reported. The identity between serum AFP and the estrogen binding 4.5 S macromolecular complex of uterine cytosol was demonstrated by the use of a specific immunoadsorbent to AFP. Analysis of the sedimentation profile in glycerol gradients of uterine cytosol incubated with tritiated estrone or estradiol suggests that the total estrogen binding capacity of the 4.5 S complex is provided by AFP. Changes of AFP content in rat uterus with the age of the animals suggest that this protein is probably present in the cytosol as a serum contaminant.     

Properties of estradiol binding components in hamster uterine nuclei

Only one estrogen-binding component (Type I) was observed in salt (0.5 M KCl) extracts of proestrous hamster uterine nuclei. In addition to the classical estrogen receptor (Type I), a second binding component (Type II) was detected by [³H]estradiol exchange assay performed with hamster uterine nuclear suspensions. Although this Type II binder was not detected in salt extracts, a similar binding component was found in the nuclear debris remaining after salt extraction. The Type II binding component in the nuclear debris did not posess estrogen-binding specificity. Lack of specificity for estrogens, resistance to KCl extraction, and high capacity differentiated this Type II binder from the classical estrogen receptor. Preparation of nuclear fractions in buffer containing glycerol and monothioglycerol resulted in greater recovery of nuclear estrogen receptor (Type I) as compared to buffer lacking these constituents.     

Characterization of a progestin-binding component in lactating rat mammary glands

Experiments were carried out to identify progestin-binding receptors in the mammary gland where casein synthesis is known to be inhibited by this hormone. A progestin-binding component with high affinity, low capacity and a sedimentation coefficient of 8.8 S was isolated from the cytosol of lactating rat mammary glands. This component strongly bound [³H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) with a dissociation constant of 3.9 · 10^?9 M under low-salt conditions and with that of 8.2 · 10^?10 M in the presence of 0.3 M KCl. Specificity studies showed a higher degree of progestin specificity under high salt conditions. In the absence of KCl, binding of [³H]-R5020 was inhibited by unlabeled glucocorticoid in the same degree as unlabeled progestin, but the inhibition by glucocorticoid was greatly diminished by the presence of 0.3 M KCl. These observations suggest that the [³H]R5020-binding-component is the progestin receptor and that its function may be regulated by the concentration of glucocorticoid and salt.     

Immunoprecipitation Assay of Alpha-fetoprotein Synthesis by Cultured Mouse Hepatoma Cells Treated with Estrogens and Glucocorticords

This investigation was to study the biosynthesis of ³H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. The secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticords and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17β indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties in the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticords, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticord receptors revealed binding components in the 7S and 3–4S regions of the gradient. The ³H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17β, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticord receptors in normal liver and other hepatomas.     

Analysis of Competition Binding Assays: Assessment of the Range of Validity of a Commonly Invoked Assumption

Abstract

A common assumption invoked in the analysis of competition binding assays is that the fractional saturation of sites with the unlabeled ligand is given by 1 - (the concentration of bound labeled ligand in the presence of unlabeled ligand)/(the concentration of bound labeled ligand in the absence of unlabeled ligand). This assumption is critically evaluated in the context of several binding models: (a) binding of univalent ligands to multiple classes of equivalent and independent sites, with and without nonspecific binding; (b) cooperative binding of univalent ligands; and (c) binding of multivalent ligands to a single class of univalent acceptors. We show that the conventional assumption is only valid when the labeled ligand is mainly in the free form, occupies a small fraction of the total sites and binds univalently to all sites in an equivalent and independent manner, and when the unlabeled ligand forms l : l complexes with the acceptor sites. When these conditions are satisfied, the conventional assumption is valid even if the unlabeled ligand binds to nonequivalent sites or exhibits cooperativity. Finally, we apply the theory derived for case (a) above to the binding of fluoresceinated epidermal growth factor to A431 cells and demonstrate that the analysis of data obtained from both conventional and competition assays provides information which is difficult, if not impossible, to obtain from either assay alone.     

Photoaffinity labeling of three renal cyclic 3′,5′-adenosine monophosphate-binding proteins

Evidence is presented for the presence of multiple cyclic AMP binding components in the plasma membrane and cytosol fractions of porcine renal cortex and medulla. N⁶-(Ethyl-2-diazomalonyl)-3′,5′-adenosine monophosphate, a photoaffinity label for cyclic AMP binding sites, exhibits non-covalent binding characteristics similar to cyclic AMP in membrane and soluble fractions. Binding data for either compound to the plasma membrane fraction yields biphasic Scatchard plots while triphasic plots are obtained with the dialyzed cytosol. When covalently labeled fractions are separated on SDS-polyacrylamide gel electrophoresis, the cyclic AMP photoaffinity label is found on 49 000 and 130 000 dalton components in each kidney fraction. DEAE-cellulose and gel filtration chromatography of the labeled cortical cytosol fraction establishes that the three components suggested by the binding data correspond to two 49 000 dalton species and a 130 000 component. The 49 000 species have higher affinities for cyclic AMP than the 130 000 component (K_a(1) = 2.0 · 10⁹, K_a(2) = 1.7 · 10⁸, K_a(3) = 1.0 · 10⁷). The 49 000 components are associated with protein kinase activity while the 130 000 component does not exhibit protein kinase, adenosine deaminase, or cyclic nucleotide phosphodiesterase activity. Immunologic results and effects of phosphorylation and cyclic GMP on cyclic AMP binding further suggest that the 49 000 components are regulatory subunits of cyclic AMP-dependent protein kinases. Cyclic AMP binding to the 130 000 component is markedly inhibited by adenosine and adenine nucleotides, but not cyclic GMP. Thus, this component may reflect an aspect of adenosine control or metabolism which may or may not be a cyclic AMP-related cellular function.     

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

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

Immunoprecipitation assay of alpha-fetoprotein synthesis by cultured mouse hepatoma cells treated with estrogens and glucocorticoids

This investigation was to study the biosynthesis of 3H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. the secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticoids and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17 beta indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties on the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticoids, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticoid receptors revealed binding components in the 7S and 3-4S regions of the gradient. The 3H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17 beta, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticoid receptors in normal liver and other hepatomas.     

Effect of Quinoline-Type Antimalarial Drugs on the Binding of Oestradiol-17β and Progesterone by Rabbit and Human Uterine Cytosols

Abstract

Rabbit and human uterine cytosol, prepared and tested in phosphate buffer, bound less oestradiol-17β or progesterone than cytosol from the same source prepared and tested in Tris-HCl buffer. Dissociation constants were the same in both buffer systems, and the difference in binding was due to a difference in the number of binding sites. Three quinoline-type antimalarial drugs, chloroquine, quinine and mefloquine, and the quinoline derivative, 4-(4'-hydroxy-l'-methylbutylamino)-7-chloroquinoline, increased the steroid binding capacity of phosphate-buffered cytosol to that of Tris-buffered cytosol, the optimal concentration of quinoline derivative being 1.4–1.6 mM. Tris (50 mM) increased the binding capacity of phosphate-buffered cytosol to that of Tris-buffered cytosol. The effects of Tris and quinoline derivatives were not additive. By gel chromatography and sucrose density gradient centrifugation it was shown that the molecular size and sedimentation behaviour of the oestradiol and progesterone receptors were not affected by the quinoline derivatives. Two types of binding site are proposed, one requiring the presence of low molecular weight, basic compounds. The uterine levels of chloroquine attained by normal pharmacological doses of the drug are potentially capable of influencing the binding of oestradiol-17β and progesterone in the uterine cytosol.     

Transformation of the 8-9 S molybdate-stabilized estrogen receptor from low-affinity to high-affinity state without dissociation into subunits

The rate of dissociation of labeled estradiol from [3H] estradiol-8-9 S receptor complexes ([3H]E2-8-9 S ER) molybdate-stabilized was determined in the presence of either an excess of unlabeled hormone ("chase") or of charcoal/dextran suspension ("stripping"). Biphasic dissociation of the hormone was observed in both cases, but the fraction of the fast-dissociating component was dramatically reduced (5% instead of 60%) when stripping was used. As the dissociation patterns were independent of the degree of saturation of the receptor, the results do not favor the possibility of cooperative effects between binding sites in the 8-9 S ER. After pretreatment of cytosol by charcoal at 28 degrees C for 15 min, the dissociation studied by chase displayed only the slowly dissociating component (t1/2 approximately 65 min). This effect was dependent on temperature and influenced by the ligand bound to 8-9 S ER, being pronounced with estradiol (E2) and absent with [3H]4-hydroxytamoxifen. The slow-dissociating component obtained after charcoal treatment was reconverted to fast-dissociating state by adding dithiothreitol or by incubation with cytosol at 20 degrees C. The charcoal treatment did not change the sedimentation coefficient (approximately 9 S) and the Stokes radius (approximately 7 nm) of the [3H]E2-8-9 S ER, and the slow-dissociating form obtained did not bind to DNA-cellulose either in the presence or absence of molybdate ions. Thus there are likely small but functionally significant changes of structure in the 8-9 S ER which remain in a non-DNA-binding form, whereas the rate of estradiol dissociation is modified.     

Multiple species of estrogen binding sites in the nuclear fraction of the rat prostate

Specific estrogen-binding sites have been demonstrated in purified nuclear fractions of prostates from intact rats. Saturation analysis of nuclei over a wide range of [³H]-estradiol concentrations (0.15 to 90 nM) has shown two different types of binding sites: a) one with high affinity (Kd of 0.5–0.8 nM) and low capacity for estradiol (approximately 162 fmole/mg DNA); b) a second with a lower affinity (Kd of 30–40 nM), which shows a higher capacity (approximately 860 fmole/mg DNA), and displays a saturation curve that is sigmoidal and that appears to be similar to those for Type II estrogen-binding sites in rat uterus. These results suggest that the actions of estradiol in the prostate are mediated by specific nuclear binding sites.