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1.
Biochem Biophys Res Commun ; 199(2): 826-33, 1994 Mar 15.
Article in English | MEDLINE | ID: mdl-8135829

ABSTRACT

A non-covalent complex of a 17 and a 7 kDa peptide was isolated from a Lys-C digest of the C-terminal 32 kDa half of the estradiol receptor by immunoadsorption. The 17 kDa part extends from K303 to K467, the 7 kDa part from S468 to K529 (or K531). The components are held together by hydrophobic interactions and can be separated by SDS/PAGE. They react on Western blots with MAB 13H2 (17 kDa) and MAB H222 (7 kDa), respectively. The native complex binds estradiol with high affinity and is recognized both by MAB 13H2 and H222, indicating that both peptides contribute to the ligand-binding niche.


Subject(s)
Receptors, Estradiol/chemistry , Receptors, Estradiol/metabolism , Uterus/metabolism , Amino Acid Sequence , Animals , Binding Sites , Blotting, Western , Centrifugation, Density Gradient , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Female , Humans , Ligands , Molecular Sequence Data , Molecular Weight , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Receptors, Estradiol/isolation & purification , Sequence Homology, Amino Acid , Swine
2.
Biochem Biophys Res Commun ; 194(3): 1248-55, 1993 Aug 16.
Article in English | MEDLINE | ID: mdl-8352781

ABSTRACT

A 32 kDa fragment of the porcine estradiol receptor, which contains the entire hormone-binding domain E and parts of the adjacent domains D and F, binds 65Zn with high affinity. Nonradioactive Zn and Cu are equally potent competitors, Co and Ni are less effective. The capacity for Zn-binding is pH-dependent, it is abolished by the ethoxycarboxylation of imidazole, but remains unimpaired after the modification of sulfhydryls by vinylpyridine. The presence of a HDXXH motif in domain E of the receptor and the significance of its similarity with the HEXXH zinc-binding motif of metallo-proteases are discussed.


Subject(s)
Histidine/metabolism , Peptide Fragments/metabolism , Receptors, Estradiol/metabolism , Uterus/metabolism , Zinc/metabolism , Amino Acid Sequence , Animals , Binding, Competitive , Copper/metabolism , Female , Hydrogen-Ion Concentration , Metalloendopeptidases/genetics , Molecular Sequence Data , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Receptors, Estradiol/genetics , Receptors, Estradiol/isolation & purification , Sequence Homology, Amino Acid , Structure-Activity Relationship , Swine , Uterus/chemistry
3.
Folia Biol (Praha) ; 39(1): 58-61, 1993.
Article in English | MEDLINE | ID: mdl-8348987

ABSTRACT

A three-step procedure to enrich estradiol receptor protein from calf uterus nearly 30,000-fold has been described. Over-all yield is 12%. Control of the single steps has been performed by sodium dodecyl sulfate-electrophoresis, sucrose density gradient centrifugation, gel filtration and determination of receptor quantity. Immunological properties of the preparation obtained have not been controlled.


Subject(s)
Receptors, Estradiol/isolation & purification , Uterus/chemistry , Ammonium Sulfate , Animals , Cattle , Centrifugation, Density Gradient , Chemical Precipitation , Chromatography, Affinity , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Female , Receptors, Estradiol/analysis
4.
Rev. Inst. Nac. Cancerol. (Méx.) ; 38(3): 1611-8, jul.-sept. 1992. tab
Article in Spanish | LILACS | ID: lil-117838

ABSTRACT

La cuantificación de los receptores hormonales empleando hormona radiactiva en neoplasias hormonodependientes regularmente se realiza únicamente en el citosol celular. Esta determinacon es incompleta, ya que la presencia de los receptores localizados en el núcleo no se efectúa y, por lo tanto, no se determina la totalidad de los receptores en un tumor. En el presente estudio se otimizó la cuantificación de los receptores hormonales para estradiol localizados en el núcleo celular, empleando diferentes temperaturas de incubación con la hormona radiactiva (4,22 y 37 Grados C). Los receptores nucleares se extrajeron de la cromatina con soluciones amortiguadoras de KC1 0.4 M. se emplearon como modelo experimental úteros de ratas Wistar, adrenalectomizadas y ooforectomizadas, las cuales fueron posteriormente estimuladas con estradiol, progesterona y vehículo de administración. Se pudo observar que la cuantificación de los receptores nucleares para estradiol a 22 Grados C detecta un 13 por ciento más de estas proteínas, sin que se vea afectada por la temperatura la afinidad del receptor por su hormona. El receptor de progesterona no se pudo detectar en el núcleo, ya que al parecer es altamente sensible al KC1. En conclusión, es factible determinar los receptores nucleares para estradiol, empleando incubaciones a 22 Grados C como temperatura óptima en el núcleo.


Subject(s)
Animals , Female , Rats , Cytosol/chemistry , Estradiol , Neoplasms, Hormone-Dependent/diagnosis , Cell Nucleus/chemistry , Progesterone , Rats, Inbred Strains , Receptors, Estradiol/analysis , Receptors, Estradiol/isolation & purification , Receptors, Progesterone/analysis , Receptors, Progesterone/isolation & purification , Uterus/physiopathology
5.
Receptor ; 2(1): 17-28, 1992.
Article in English | MEDLINE | ID: mdl-1477677

ABSTRACT

Nuclei were isolated from porcine endometrium of castrated pigs either unexposed or exposed to estradiol in vivo by two techniques, one of which included a hypotonic step. Aliquots were analyzed for estradiol content. Receptor was extracted from buffered, Surfynol-stabilized suspensions by either (a) KCl alone, (b) in combination with dithiothreitol, or (c) by dithiothreitol with polypentosanesulfate and addition of KCl. The yields rose from a-->c. The same proportional gains with increasing extractant efficacies were obtained from nuclei of unstimulated and estradiol-treated cells. Receptor recovery with extractant "c" rose linearly over the range of 9-80 x 10(6) nuclei/mL and was independent of the technique used for isolation. Nuclear fractions isolated using steroid-free solutions contained more estrogen receptor than estradiol; the numerical excess in control nuclei persisted in the nuclei of stimulated cells featuring a stoichiometric rise of ligand and receptor contents. The increase of receptor contents in nuclei isolated from hormone-stimulated cells coincided with a decline in the cytoplasmic fractions. An excess of hormone over receptor was seen only when nuclei were isolated from untreated cells with media containing 10 nM estradiol. Our data strengthen earlier notions of an estradiol-promoted receptor translocation into the nucleus and are not compatible with the ligand-filling hypothesis of preexisting nuclear binding sites.


Subject(s)
Cell Nucleus/drug effects , Endometrium/drug effects , Estradiol/pharmacology , Receptors, Estradiol/drug effects , Subcellular Fractions/metabolism , Animals , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Endometrium/cytology , Endometrium/metabolism , Estradiol/analysis , Female , Microscopy, Electron , Receptors, Estradiol/isolation & purification , Receptors, Estradiol/metabolism , Reference Values , Scintillation Counting , Swine
6.
Biochem J ; 276 ( Pt 3): 709-14, 1991 Jun 15.
Article in English | MEDLINE | ID: mdl-2064608

ABSTRACT

Homodimers of the porcine oestradiol receptor dissociated at pH 6.4. The monomers reassociate after neutralization. This property is retained in a 32 kDa receptor fragment generated by co-adsorbed endopeptidases from cytosolic receptor bound to heparin-Sepharose. The fragment was purified by successive gel filtrations in the dimer and monomer states. Precipitations with ethanol and (NH4)2SO4 respectively served as concentrating steps. In all, 10-15 nmol of the homogeneous fragment were recovered from 8 kg batches of porcine uteri with a approximately 10(5)-fold enrichment and in approximately 20% yields. Its oestradiol-binding capacity was identical with that of the intact receptor. The N-terminus was blocked. Two decapeptides from a tryptic digest were sequenced. One of them corresponded to amino acids 353-362 of the human receptor, a sequence fully conserved in all species investigated. The second peptide differed in positions 553, 554 and 557 from the 549-558 sequence of the human protein.


Subject(s)
Receptors, Estradiol/isolation & purification , Uterus/chemistry , Amino Acid Sequence , Animals , Cattle , Chickens , Female , Humans , Hydrogen-Ion Concentration , Mice , Molecular Sequence Data , Molecular Weight , Rats , Receptors, Estradiol/chemistry , Sequence Alignment , Swine , Uterus/metabolism , Xenopus
7.
J Biol Chem ; 265(33): 20686-91, 1990 Nov 25.
Article in English | MEDLINE | ID: mdl-2243115

ABSTRACT

Several mutants of the human estrogen receptor (ER) were transiently expressed in Cos 7 cells in order to determine the regions involved in the formation of complexes with the heat shock protein Mr approximately 90,000 (hsp 90). The formation of the cytosol non-DNA binding 8-9 S complexes (8-9 S ER) was monitored by glycerol gradient ultracentrifugation. It was established that the N-terminal region of the receptor, including the two zinc fingers of the DNA binding domain (DBD), is not required for the formation of the 8-9 S ER complexes. Conversely, deletion of the entire ligand binding domain (LBD) produced truncated receptor mutants that are constitutive transcriptional activators and did not form 8-9 S ER complexes, confirming results obtained previously with the glucocorticosteroid receptor (Pratt, W.B., Jolly, D.J., Pratt, D.V., Hollenberg, S.M., Giguerre, V., Cadepond, F., Schweizer-Groyer, G., Catelli, M.G., Evans, R.M., and Baulieu, E.E. (1988) J. Biol. Chem. 263, 267-273). However, no limited subregion of the LBD was found to be uniquely involved in hsp 90 binding. A highly positively charged region situated at the C-terminal extremity of the DBD (between amino acids 251 and 271) also appeared to be implicated. Although not sufficient, this sequence is necessary for the formation of the 8-9 S ER; it also corresponds to the NL1 nuclear localization domain of steroid receptors. Taken together, these results suggest that the formation of complexes with hsp 90 involves several receptor regions, and they are consistent with the proposal that hsp 90 inhibits receptor function and can be released by hormone binding to the LBD.


Subject(s)
DNA-Binding Proteins/metabolism , Heat-Shock Proteins/metabolism , Receptors, Estradiol/metabolism , Amino Acid Sequence , Animals , Cell Line , Cytosol/metabolism , Estradiol/metabolism , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Receptors, Estradiol/genetics , Receptors, Estradiol/isolation & purification , Transfection
8.
J Steroid Biochem ; 36(6): 617-29, 1990 Aug 28.
Article in English | MEDLINE | ID: mdl-2214779

ABSTRACT

Binding of [3H]estradiol, [3H]testosterone and [3H]progesterone to purified NADP-dependent estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase (EHSD) from rabbit liver cytosol has been examined. The three steroids bind to the enzyme with moderate [corrected] affinity (Ka congruent to 10(7) [corrected] M-1 at 4 degrees C) and equal binding capacity. High-rates were shown for both association and dissociation processes. The steroids competitively inhibited the binding of each other to EHSD. At the same time, their relative binding affinities (RBA) were dependent on the nature of [3H]ligand. The results of RBA determinations for 72 steroids and their analogues by inhibition of [3H]progesterone binding to EHSD suggest that androgens and gestagens bind preferentially to the same site on EHSD molecule, while estrogens (at least by their D-ring) bind to another site. The assumption that EHSD molecule has more than one binding site for steroids is corroborated by (i) substrate inhibition revealed for a number of steroids; (ii) the estrogen ability to potentiate 20 alpha-reduction of progesterone; (iii) stimulatory effect of 5 alpha (beta)-androstane-3 alpha (beta), 17 beta-diols on [3H]testosterone and progesterone binding; and (iv) reciprocal effect of NADP on [3H]estradiol and [3H]testosterone binding to EHSD. Significant differences in sensitivity to pH and changes in NaCl concentration upon metabolism and binding of various steroids have been found. At concentrations of 16 mM dithiothreitol potentiated catalytic conversion of some steroids and had no effect on metabolism of others. Both the affinity for steroids and binding capacity of EHSD are found to be cofactor-dependent. It is speculated that EHSD has a complex active center including at least two mutually influencing steroid-binding sites tightly related with cofactor-binding site. The polyfunctionality of EHSD may be due to both the excess of functional protein groups that form individual constellations upon binding of any steroid and also to conformational lability of EHSD molecule implying alternative orientations of steroids at the binding site.


Subject(s)
Hydroxysteroid Dehydrogenases/isolation & purification , Liver/enzymology , Receptors, Estrogen/isolation & purification , 17-Hydroxysteroid Dehydrogenases/isolation & purification , 20-Hydroxysteroid Dehydrogenases/isolation & purification , 3-Hydroxysteroid Dehydrogenases/isolation & purification , Animals , Binding Sites/drug effects , Binding, Competitive , Estradiol/pharmacology , Hydrogen-Ion Concentration , Hydroxysteroid Dehydrogenases/drug effects , Hydroxysteroid Dehydrogenases/metabolism , Kinetics , Models, Molecular , NADP/pharmacology , Progesterone/antagonists & inhibitors , Progesterone/pharmacology , Rabbits , Receptors, Androgen/isolation & purification , Receptors, Estradiol/isolation & purification , Receptors, Estrogen/drug effects , Receptors, Estrogen/metabolism , Receptors, Progesterone/isolation & purification , Testosterone/pharmacology
9.
Comp Biochem Physiol B ; 96(2): 375-80, 1990.
Article in English | MEDLINE | ID: mdl-2361366

ABSTRACT

1. Specific [3H]estradiol binding activity with characteristics of estrogen receptors was found in the cytosols and nuclear extracts of the adrenal cortex proper and special zone of the brushtail possum (Trichosurus vulpecula). 2. The specific estradiol receptor had a sedimentation coefficient on sucrose gradients of approximately 9S and a molecular weight on gel filtration of more than 200,000. The adrenal cortex cytosol binds [3H]estradiol with high affinity (Ka 5.5 X 10(9) M-1), and limited capacity (Bmax 62.7 fmol/mg cytosol prot). In competition experiments with different steroids the receptor showed a high affinity for four estrogens and a very low affinity to androgens, progesterone and cortisol. 3. There was no difference in the affinity and maximum binding capacity of the cytosols from cortex proper in male and female animals, but the binding capacity of the special zone of females was half that of cortex proper. Estradiol receptors were found in the kidney, liver, lung, testis and muscle but only in the adrenal and prostate was the binding capacity relatively high compared with the uterus. 4. The specific binding capacity of [3H]estradiol to cytosols of adrenal cortex at different stages of the estrus cycle and pregnancy was unrelated to that of the uterus. In the adrenal the receptor concentration was lowest at estrus, when uterine concentration was high, while in late pregnancy the binding of adrenal cortex and uterus cytosols was almost the same. 5. The possible physiological significance of the presence of a specific estrogen receptor in male and female possums is discussed.


Subject(s)
Adrenal Cortex/metabolism , Receptors, Estradiol/metabolism , Animals , Binding, Competitive , Chromatography, Gel , Cytosol/metabolism , Estradiol/metabolism , Female , Kinetics , Male , Opossums , Organ Specificity , Receptors, Estradiol/isolation & purification , Sex Factors
10.
J Biol Chem ; 263(15): 7203-10, 1988 May 25.
Article in English | MEDLINE | ID: mdl-3366774

ABSTRACT

We previously described and partially characterized endogenous ligands for nuclear type II sites in normal and malignant tissues. Chromatography of these ligands on Sephadex LH-20 revealed that two peaks with binding activity (alpha and beta) could be resolved. The beta-peak component was present in all normal tissues that we examined, but not in malignant tissues, and it inhibited the growth of MCF-7 human breast cancer cells in vitro. Conversely, the alpha-peak component was found to be present in both normal and malignant tissues, and did not inhibit MCF-7 cell growth. The present studies describe the purification and identification of the alpha-peak and beta-peak components in bovine serum and an assessment of the effects of these compounds on normal and malignant cell growth. Gas chromatography-mass spectroscopy analysis of the purified beta-peak component demonstrated that the compound was methyl p-hydroxyphenyllactate (MeHPLA). Competition analysis revealed that MeHPLA binds to nuclear type II sites with a high binding affinity, while physiological levels of this compound blocked estradiol stimulation of uterine growth in vivo and inhibited the growth of MCF-7 human breast cancer cells in vitro. The alpha-peak component was found to be the corresponding acid, p-hydroxyphenyllactic acid (HPLA). This compound interacted with nuclear type II sites with a relatively low affinity and did not block uterotropic response to estradiol or inhibit MCF-7 cell growth. These studies demonstrate that HPLA and MeHPLA are ligands for nuclear type II sites and that MeHPLA may be a very important regulator of normal and malignant cell growth.


Subject(s)
Cell Division/drug effects , Cell Nucleus/metabolism , Lactates/pharmacology , Receptors, Estradiol/metabolism , Receptors, Estrogen/metabolism , Animals , Breast Neoplasms , Cell Line , Chromatography, Gel , Chromatography, High Pressure Liquid , Female , Humans , Lactates/metabolism , Mass Spectrometry , Ovariectomy , Rats , Rats, Inbred Strains , Receptors, Estradiol/isolation & purification
11.
Biochem Biophys Res Commun ; 150(3): 1131-7, 1988 Feb 15.
Article in English | MEDLINE | ID: mdl-3342061

ABSTRACT

A component of human breast tumors, isolated by chromatographing the cytosol on hydroxylapatite, increased specific estradiol binding by the cytosolic proteins (23/24) while a similarly isolated component of normal sections of the cancerous breasts inhibited sharply the binding (17/17). Using the same procedures, a component isolated from rat uteri excised at proestrus acted like the one from human breast tumors and a component isolated from uteri at the other phases of the cycle acted like the one from normal breast tissue. The described hormone binding differences are revealed in incubations at 37 degrees C with divalent copper present in the incubation mixture.


Subject(s)
Breast Neoplasms/analysis , Breast/analysis , Estradiol/metabolism , Receptors, Estradiol/metabolism , Receptors, Estrogen/metabolism , Animals , Breast/metabolism , Breast Neoplasms/metabolism , Chromatography , Copper/pharmacology , Cytosol/analysis , Cytosol/metabolism , Estrus , Female , Humans , Proestrus , Rats , Rats, Inbred Strains , Receptors, Estradiol/drug effects , Receptors, Estradiol/isolation & purification , Uterus/analysis
12.
J Steroid Biochem ; 29(1): 41-5, 1988 Jan.
Article in English | MEDLINE | ID: mdl-3347049

ABSTRACT

Specific estradiol binding proteins (EBP) that have been described in the pancreatic tissues of a number of species are thought to be important in maintaining the structural and functional integrity of the pancreas. However, possible sex-related differences in the presence and characteristics of EBP have not been examined. In the present study, we have analyzed the pancreatic tissues of male and female Syrian golden hamsters for the presence of EBP and progesterone binding protein (PBP), and further characterized these sites. Our results indicate the presence of only one class of EBP with a high capacity (greater than 500 fmol/mg protein) and low affinity (Kd greater than 1.0 nM) in the pancreatic cytosol of female hamsters. On the other hand, there appeared to be two distinct classes of EBP in the male pancreas. One class of EBP in the male pancreas had a high binding affinity (Kd = less than 0.05 nM) and low capacity (less than 10 fmol/mg protein); we have arbitrarily called these Type I EBP. The second class of EBP in the male pancreas which resembled EBP in the female pancreas had a high capacity (greater than 100 fmol/mg protein) and a low binding affinity (Kd = greater than 1.0 nM); we have called these Type II EBP. The sucrose-density gradient profile of EBP for male and female hamster pancreas demonstrated the presence of both an 8S binding protein and a 4S binding protein in the male pancreas; the female pancreas had only a 4S binding protein. PBP were not detected in pancreas of either male or female hamsters. We conclude that significant sex-related differences are present in the EBP populations of the hamster pancreas.


Subject(s)
Pancreas/metabolism , Receptors, Estrogen/isolation & purification , Animals , Cricetinae , Cytosol/metabolism , Female , Male , Mesocricetus , Receptors, Estradiol/isolation & purification , Receptors, Estrogen/classification , Sex Characteristics , Species Specificity
13.
EMBO J ; 6(10): 2923-9, 1987 Oct.
Article in English | MEDLINE | ID: mdl-3691476

ABSTRACT

Recent experiments have shown that calf uterus oestrogen receptor exists in a tyrosine-phosphorylated hormone binding form and in non-phosphorylated, non-hormone binding form. We report here that physiological concentrations of oestradiol in complex with the receptor stimulate the calf uterus receptor kinase that converts the non-hormone binding receptor into hormone binding receptor through phosphorylation of the receptor on tyrosine. The activity of this enzyme has been followed by reactivation of hormone binding sites and phosphorylation on tyrosine of calf uterus phosphatase-inactivated receptor. Phosphorylation of the receptor has been demonstrated by interaction of kinase 32P-phosphorylated proteins with anti-receptor antibody followed either by sucrose gradient centrifugation or SDS-PAGE of the immunoprecipitated proteins. Hormone stimulation of the kinase is inhibited by receptor occupancy of the anti-oestrogen tamoxifen. Oestradiol-receptor complex increases the affinity of the kinase for the dephosphorylated receptor. Findings of this report are consistent with the observation that several protein tyrosine kinases that are associated with peptide hormone receptors are stimulated by the binding of the hormone to the receptor. This is the first report on the activation of a tyrosine kinase by a steroid hormone. The finding that hormones can regulate their own receptor binding activity through a tyrosine kinase is also new.


Subject(s)
Estradiol/pharmacology , Protein-Tyrosine Kinases/metabolism , Receptors, Estradiol/metabolism , Receptors, Estrogen/metabolism , Uterus/metabolism , Animals , Cattle , Cell-Free System , Cytosol/metabolism , Estradiol/metabolism , Female , Phosphorylation , Receptors, Estradiol/isolation & purification , Tyrosine
14.
J Biol Chem ; 262(15): 6969-75, 1987 May 25.
Article in English | MEDLINE | ID: mdl-3584104

ABSTRACT

The structure of the calf uterus nontransformed molybdate-stabilized estradiol receptor (ER) has been investigated using affinity labeling with tamoxifen aziridine and several monoclonal antibodies directed either against the steroid binding protein (Mr approximately 65,000) or against the heat shock protein of Mr approximately 90,000 (hsp 90). The purification was performed using affinity chromatography and a DEAE-Sephacel column. The [3H] estradiol-ER complex was obtained as a well-defined radioactive peak, the specific activity varying between 1,600 and 3,400 pmol/mg of protein. The purified ER sediments in glycerol gradients at 9.4 S +/- 0.2 (n = 5) and at 8.1 S +/- 0.2 (n = 15) in a 0.15 M KCl containing gradient ("8-9 S" ER). From a measured Stokes radius of 7.4 +/- 0.2 nm (n = 12), an Mr of approximately 300,000 has been calculated. Studies of the purified 8-9 S ER by glycerol gradient centrifugation and by "twin antibody" assay with the JS34/32 anti-ER monoclonal antibody suggest the presence of two binding subunits in the nontransformed molecular complex. Results of immunological analysis with polyclonal and several monoclonal antibodies against hsp 90 suggest the association of two molecules of this protein to the two steroid binding subunits. In high salt medium (0.4 M KCl), the purified ER sediments at 5.2 +/- 0.3 (n = 8), has a Stokes radius of 5.7 nm +/- 0.1 (n = 2) and the Mr is approximately 129,000, values expected for a homodimer consisting of two hormone-binding subunits (Mr approximately 65,000), a result confirmed by glycerol gradient centrifugation experiments, using the monoclonal antibody JS34/32. The relationship between the nontransformed 8-9 S ER and the transformed 5 S-ER forms are discussed, the simplest possibility being the release of the already formed homodimeric ER from 8-9 S ER during transformation.


Subject(s)
Molybdenum , Receptors, Estradiol , Receptors, Estrogen , Uterus/analysis , Affinity Labels , Animals , Antibodies, Monoclonal , Cattle , Centrifugation, Density Gradient , Chemical Phenomena , Chemistry, Physical , Chromatography , Electrophoresis, Polyacrylamide Gel , Female , Heat-Shock Proteins/immunology , Immunologic Tests , Macromolecular Substances , Molecular Weight , Receptors, Estradiol/immunology , Receptors, Estradiol/isolation & purification , Receptors, Estrogen/immunology , Receptors, Estrogen/isolation & purification , Tamoxifen/analogs & derivatives
15.
J Biol Chem ; 262(12): 5530-5, 1987 Apr 25.
Article in English | MEDLINE | ID: mdl-3571221

ABSTRACT

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.


Subject(s)
Molybdenum/pharmacology , Receptors, Estradiol/metabolism , Receptors, Estrogen/metabolism , Uterus/metabolism , Animals , Cattle , Drug Stability , Estradiol/metabolism , Female , Kinetics , Macromolecular Substances , Osmolar Concentration , Receptors, Estradiol/drug effects , Receptors, Estradiol/isolation & purification , Thermodynamics
16.
Biochem Biophys Res Commun ; 143(1): 218-24, 1987 Feb 27.
Article in English | MEDLINE | ID: mdl-3827917

ABSTRACT

The first six N-terminal amino acid residues of the 85-90K non-estrogen binding component of the calf uterine, molybdate-stabilized estradiol receptor have been determined by Edman degradation. After affinity chromatography of the stabilized receptor oligomer, the 85-90K unit was purified to homogeneity by preparative gel electrophoresis using electroelution for protein recovery. Inverse-gradient high performance liquid chromatography provided the 85-90K protein suitable for amino-terminal sequence analysis.


Subject(s)
Molybdenum/pharmacology , Receptors, Estradiol/metabolism , Receptors, Estrogen/metabolism , Uterus/metabolism , Amino Acid Sequence , Animals , Cattle , Female , Molecular Weight , Protein Binding , Receptors, Estradiol/drug effects , Receptors, Estradiol/isolation & purification
18.
EMBO J ; 5(11): 2867-72, 1986 Nov.
Article in English | MEDLINE | ID: mdl-2431901

ABSTRACT

Estradiol receptor from rat uteri incubated with [32P] orthophosphate has been purified by diethylstilbestrol--Sepharose followed by heparin--Sepharose chromatography. The purified receptor, analyzed by centrifugation through sucrose gradients after incubation with monoclonal antibodies against purified estradiol receptor, appears to be labeled with 32P. The receptor preparation has been further purified by immunoaffinity chromatography and submitted to SDS--poly-acrylamide gel electrophoresis. A heavily 32P-labeled 68 kd protein and a very lightly 32P-labeled 48 kd protein, probably a proteolytic product of the 68 kd protein, were detected. Phosphoamino acid analysis of the receptor eluted from the immunoaffinity column shows that its 32P-labeling occurs exclusively on tyrosine. This is the first report on phosphorylation on tyrosine of a steroid receptor in tissue. It is consistent with our previous finding that a uterus estradiol receptor-kinase, which confers hormone binding ability to the estradiol receptor, in vitro phosphorylates this receptor exclusively on tyrosine. Calf uterus receptor binds with high specificity and affinity to monoclonal anti-phosphotyrosine antibodies covalently bound to Sepharose (Kd = 0.28 nM). Dephosphorylation of the receptor by nuclei containing the calf uterus nuclear phosphatase abolishes the interaction with antibodies. These results suggest that also in calf uterus, estradiol receptor is phosphorylated on tyrosine. Anti-phosphotyrosine antibodies bound to Sepharose have been used to partially purify the estradiol receptor from calf uterus.


Subject(s)
Receptors, Estradiol/metabolism , Receptors, Estrogen/metabolism , Tyrosine , Tyrosine/analogs & derivatives , Uterus/metabolism , Animals , Antibodies, Monoclonal , Cytosol/metabolism , Estradiol/metabolism , Female , Phosphates/metabolism , Phosphorylation , Phosphotyrosine , Rats , Rats, Inbred Strains , Receptors, Estradiol/isolation & purification , Tyrosine/analysis
19.
Exp Gerontol ; 21(4-5): 255-65, 1986.
Article in English | MEDLINE | ID: mdl-3817038

ABSTRACT

We have examined the effects of aging on the capacity of rat uterine estradiol receptors to be transformed from 8S to 4S and 5S species. Cytosol receptors from mature (6-month-old) rats or senescent (24-month-old) rats have been exposed to various KCl concentrations, ammonium sulfate precipitation and 25 degrees C heating. Estradiol receptors of both the mature and senescent age groups exist in an 8S form on linear 5-20% sucrose gradients in the absence of KCl and are converted to a 4S molecule in the presence of 0.4 M KCl. At intermediate salt concentrations a greater portion of mature receptors was converted to the 4S species. At 0.15 M KCl 62.3% +/- 2.8 of the mature receptors are converted to 4S versus 41% +/- 1.9 of the senescent receptors, and at 0.2 M KCl 79.6% +/- 3.2 of the mature receptors are converted to the 4S versus 58.2% +/- 2.1 of the senescent. Ammonium sulfate treatment in the presence of 0.3 M KCl converted about 80% of the receptors from the 4S to the 5S form, while only about half of the old receptors are affected. When ammonium sulfate precipitates were heated to 25 degrees C all to mature receptors were converted to the 5S species, while only two thirds of the senescent receptors were sedimented at 5S under the same conditions. Inclusion of 20 mM molybdate during preparation blocks conversion of about 15% of the senescent receptors from the 8S to the 4S form but does not affect the mature preparations. Similarly, molybdate treatment does not affect the conversion of the mature estradiol receptors to the 5S form but increases the percentage of senescent receptors remaining in the 4S form from 30 to 45%. Such qualitative differences in receptor conversion may be related to age associated deterioration of estradiol stimulated uterine responsiveness.


Subject(s)
Aging/physiology , Receptors, Estradiol/metabolism , Receptors, Estrogen/metabolism , Uterus/ultrastructure , Ammonium Sulfate/pharmacology , Animals , Centrifugation, Density Gradient , Cytosol/metabolism , Female , Hot Temperature , Molecular Weight , Molybdenum/pharmacology , Potassium Chloride/pharmacology , Rats , Rats, Inbred Strains , Receptors, Estradiol/drug effects , Receptors, Estradiol/isolation & purification
20.
J Biol Chem ; 260(10): 5936-41, 1985 May 25.
Article in English | MEDLINE | ID: mdl-3997805

ABSTRACT

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.


Subject(s)
Receptors, Estradiol/isolation & purification , Receptors, Estrogen/isolation & purification , Uterus/metabolism , Animals , Cattle , Chromatography, Affinity , Chromatography, High Pressure Liquid , Cytosol/metabolism , Estradiol/metabolism , Female , In Vitro Techniques , Molecular Weight , Molybdenum , Protein Conformation , Receptors, Estradiol/metabolism
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