RESUMO
The characteristic ability of rat uteri to take up tritiated estradiol in vitro or to retain estradiol previously incorporated either in vivo or in vitro is destroyed by treating the tissue with various sulfhydryl-blocking reagents. The two radioactive estradiol-receptor complexes, observed in uterine homogenates in the supernatant fraction and in an extract of the nuclear fraction, respectively, are disrupted by brief exposure to organic mercurials in the cold. Sulfhydryl groups of uterine receptor substances apparently play a vital role in estradiol binding, perhaps indirectly through contribution to receptor conformation.
Assuntos
Amidas/farmacologia , Benzoatos/farmacologia , Estradiol/farmacologia , Etilmaleimida/farmacologia , Receptores de Droga , Útero/metabolismo , Animais , Centrifugação com Gradiente de Concentração , Estradiol/metabolismo , Feminino , Técnicas In Vitro , RatosRESUMO
The lysosomes present in homogenates of porcine endometrium epithelium equilibrate in two density regions of Percoll gradients. Patterns with varying proportions between high and low density peaks are observed, when aliquots of a tissue sample are processed with different all-glass Potter-Elvejhem homogenizers. The described constant-tolerance shearing device (CTSD), in contrast, provides homogenate fractions with higher latencies and steady distribution patterns. They are characteristic for each of the six lysosomal markers and the six other structure-bound enzymes measured in gradient fractions of the particulate matter harvested between 600g and 17,000g. The 17,000g sediments of CTSD homogenates contain more than 40% of the total lysosomal enzymatic activities. Recoveries from Percoll gradients are between 93 and 101%. Enrichments in the high density region range from 35-fold (beta-glucosidase) to 82-fold (acid ribonuclease). Both lysosomal populations exhibit latencies between 89 and 94%. Our results indicate that light lysosomes can be artificially generated by inappropriate homogenization, which should be considered in experiments on the formation and maturation of lysosomes.
Assuntos
Endométrio/ultraestrutura , Lisossomos/análise , Animais , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Endométrio/análise , Endométrio/enzimologia , Feminino , Lisossomos/enzimologia , Microscopia Eletrônica , SuínosRESUMO
Porcine 17 beta-estradiol dehydrogenase was recently purified and cloned. It catalyzes the NAD(+)-dependent oxidation of estradiol to estrone 360-fold more efficiently than the reverse reaction with NADPH. Immunogold electron microscopy localizes 17 beta-estradiol dehydrogenase in organelles of 120 to 500 nm with moderate electron-dense matrices bounded by single membranes. Antibodies against the peroxisomal markers catalase and acyl-CoA oxidase recognize the same organelles in double-labeling studies. This is the first report on the participation of peroxisomes in the metabolism of estradiol.
Assuntos
Estradiol Desidrogenases/análise , Microcorpos/enzimologia , Acil-CoA Oxidase , Animais , Catalase/análise , Endométrio/enzimologia , Feminino , Córtex Renal/enzimologia , Oxirredutases/análise , SuínosRESUMO
The steroid complexes of (plasma) corticosteroid-binding globulin can be distinguished from intracellular steroid-receptor complexes by agar electrophoresis at low temperature in neuraminidase-treated tissue extracts. With this method, the presence of progesterone receptor has been demonstrated in heavily plasma-protein-contaminated human uterus "cytosol", but not in human mammary carcinoma extracts. SHBG and "basic" receptors for estradiol and dihydrotestosterone in human uterus cytosol could also be assayed simultaneously.
Assuntos
Corticosteroides/metabolismo , Neoplasias da Mama/metabolismo , Di-Hidrotestosterona/metabolismo , Hidrocortisona/metabolismo , Progesterona/metabolismo , Receptores de Superfície Celular , Útero/metabolismo , Adulto , Idoso , Animais , Sítios de Ligação , Ligação Competitiva , Bovinos , Citosol/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Neuraminidase , Gravidez , Ligação Proteica , Ácidos Siálicos/metabolismoRESUMO
The C-terminal part of ligand filled porcine estradiol receptor extending from H267 to I595 was isolated by adsorption to the monoclonal antibody 13H2, subjected to cleavage by CNBr, o-iodosobenzoic acid and endopeptidase Lys-C as well as other proteases, both in the native and the denatured state. The overlapping peptides produced were separated by reverse phase HPLC and sequenced by Edman degradation, lacking T570-M581 in domain F. We found no evidence of post-translational modification; the native fragment is not glycosylated and the tyrosyl residues in domain E (aa 328, 331, 459, 526, 537) and F (aa 582, 583) are not phosphorylated. In addition, all serine and threonine PTH derivatives were obtained in normal yields. The amino acid sequence of the fragment corresponds in full with that derived from the cDNA. The complete cDNA-derived sequence codes for a polypeptide of 595 amino acids with a calculated mass of 66,357 Da. The high degree of homology between species in domains C and E is shared by the porcine receptor.
Assuntos
Processamento de Proteína Pós-Traducional , Receptores de Estradiol/química , Análise de Sequência , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Brometo de Cianogênio , DNA Complementar/química , Endopeptidases/metabolismo , Feminino , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Receptores de Estradiol/genética , Homologia de Sequência , Especificidade da Espécie , SuínosRESUMO
Three types of periodic fluctuation in tissue concentrations of estradiol receptor protein have been observed. A seasonal variation is described in the uteri of 12-16-week-old calves and of ovariectomized pigs, and in mammary tumor tissue obtained from postmenopausal women. A circadian rhythm has been demonstrated in uteri of ovariectomized rats. An irregular periodic fluctuation has been found in uteri of ovariectomized and of ovariectomized/hypophysectomized rats, with the period varying from 9 to 15 days. These observations establish that a substantial turnover of receptor occurs in the absence of hormone and that 'normal' baseline values of receptor concentration do not exist.
Assuntos
Estradiol/metabolismo , Ovário/fisiologia , Receptores de Estrogênio/metabolismo , Útero/metabolismo , Animais , Neoplasias da Mama/metabolismo , Castração , Bovinos , Ritmo Circadiano , Feminino , Humanos , Hipofisectomia , Menopausa , Ratos , Estações do Ano , Especificidade da Espécie , SuínosRESUMO
PIP: Theoretically, all estrogen-receptor positive (ER+) tumors of the breast should respond to hormonal therapy; however, only 50-60% do. Therefore, this study collected information on hormonal variations which affect the cytoplasmic receptor levels and offers other parameters besides estrogenicity for biochemically characterizing breast cancers. One reason ER+ does not insure hormonal responsiveness is that cytoplasmic receptor levels are influenced by endogenous hormones as well as by exogenous factors, specifically rhythmic fluctuations of unknown origin (circadian and seasonal). Because of these various influences, it is obvious that no normal baseline level of receptors in target tissue exists on which to place the decision for prognosis of hormonal therapy. In addition to quantitative assessment of ER by assay, determination of nuclear estradiol content and measurement of cytoplasmic progestin receptor must be employed. Experiments outlined have shown that the receptor can enter the nucleus of target tissue without the assistance of hormone. Hence, complete hormone withdrawal (as in ovariectomized and adrenalectomized subjects) is not a totally effective treatment for hormone-responsive cancers. Instead, this withdrawal must be supplemented by an antihormonal treatment that incapacitates the receptor. In this article, the virtue of tamoxifen for performing this blockade is pointed out. Since tamoxifen competes with estradiol at the receptor-binding site (according to pig uterus experiments outlined here), its biological effect might be explained by a blockade of the nuclear ER transfer, the parameter that hormonal withdrawal alone cannot affect.^ieng
Assuntos
Neoplasias da Mama/metabolismo , Receptores de Estrogênio/metabolismo , Idoso , Animais , Neoplasias da Mama/terapia , Bovinos , Núcleo Celular/metabolismo , Estradiol/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Periodicidade , Suínos , Tamoxifeno/metabolismo , Útero/metabolismoRESUMO
The technique and the equipment necessary for the transcervical intrauterine injection in trained, ovariectomized pigs is detailed. Experimental groups of 14-16 week-old animals are recruited from German landrace litters with large numbers (4-9) of females. A silastic tubing containing a crystalline suspension of estradiol in propylene glycol is subcutaneously implanted behind the right ear. Ovariectomy and the detachment of the left uterine horn from the cervix are performed 7-10 days later. The animals are then kept for 6-7 weeks in ventilated stables, heated to 20 degrees C in wintertime, with 12 hr light dark cycle, free access to water and semi-ad libitum supplies of pelleted standard feed. The silastic implant gives rise to plasma levels of 8-12 pg estradiol/ml and still contains crystalline sediments when removed before the experiment. The manipulations for the transcervical, intrauterine instillation of solutions, imitating the reproductive action of the boar, are practiced daily for at least one week. The animals learn quickly to enter a restraining box, which is facilitated by the simultaneous offering of pellets drenched with root beer. The intrauterine injections proceed thereafter without any signs of stress from the animals. Stress is also avoided during slaughter by unexpected electric stunning on leaving the box, followed by exsanguination. Uteri are quickly excised and chilled in crushed ice until processed. One to two grams of endometrium cells can be harvested by curettage from each, treated and untreated (detached) horn. The Mariensee model allows for defined kinetic analyses of treated cells and provides nontreated controls from the same animal. It combines the advantages of in-vivo and in-vitro experiments.
Assuntos
Injeções/veterinária , Inseminação Artificial/veterinária , Modelos Biológicos , Suínos/fisiologia , Útero/fisiologia , Animais , Implantes de Medicamento , Estradiol/administração & dosagem , Feminino , Inseminação Artificial/métodos , Ovariectomia/veterináriaAssuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Isoenzimas/metabolismo , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Humanos , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Cinética , Dados de Sequência Molecular , Suínos , Distribuição TecidualRESUMO
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.
Assuntos
Receptores de Estradiol/química , Receptores de Estradiol/metabolismo , Útero/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Ligantes , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Receptores de Estradiol/isolamento & purificação , Homologia de Sequência de Aminoácidos , SuínosRESUMO
Estradiol is released from the binding niche of the receptor and covalently arrested in the molecular vicinity by the Mannich reaction during target fixation in acetic acid/formaldehyde. The exposed steroid is freely accessible for appropriate antibodies. It can be visualized in sections by the second antibody/enzyme technique in high resolution and without enhancements.
Assuntos
Estradiol/metabolismo , Técnicas Imunoenzimáticas , Animais , Mama/metabolismo , Endométrio/metabolismo , Estradiol/administração & dosagem , Tubas Uterinas/metabolismo , Feminino , Cabras , Humanos , Microtomia , Inclusão em Parafina , Pós-Menopausa , Ratos , Ratos Sprague-Dawley , Suínos , Tíbia/metabolismoRESUMO
The peptide A569-Y582 of the porcine estradiol receptor containing the missing sequence T570-M581 (1) was isolated and sequenced. The 4 seryl- and 2 threonyl-PTH amino acids were recovered in normal yields, excluding their posttranslational modification and reconfirming the absence of O-glycosylation and O-phosphorylation in H267-I595.
Assuntos
Fragmentos de Peptídeos/química , Receptores de Estradiol/química , Sequência de Aminoácidos , Animais , Carboidratos/análise , Cromatografia de Afinidade , Feminino , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Mapeamento de Peptídeos , Fosfatos/análise , Maturidade Sexual , Suínos , Útero/químicaRESUMO
Human mammary gland and tumours derived from it contain individual receptors for oestradiol) and for dihydrotestosterone. Analysis of 97 mammary cancers by agargel electrophoresis revealed a widely varying pattern of simultaneous occurrence in different concentration ratios (46), sole presence of oestradiol receptor (23) or dihydrotestosterone receptor (8) and absence of both receptors (18). The relevance of the results is discussed and the need for an extensive cooperative study is emphasized.
Assuntos
Neoplasias da Mama/metabolismo , Mama/metabolismo , Di-Hidrotestosterona/metabolismo , Estradiol/metabolismo , Receptores de Superfície Celular , Adulto , Proteínas de Transporte , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Ágar , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
In this paper, standards for steroid hormone receptor determinations in cancer tissue have been outlined. Standardization of methods for receptor extraction and receptor assay are equally important; and it is a condition sine qua non, if comparable receptor values between different centers for multilocal studies are required. Immediate and proper cooling of the tissue samples after removal, homogenization at the temperatures of the liquid nitrogen (as well as the presence of a sulfhydryl-protecting agent in the extraction medium), and the use of a simple, reproducible assay procedure are most important. As a standard procedure for ER- and PR-assays, the charcoal adsorption method combined with a scatchard-plot has been suggested. Reference procedures, like protein with DNA-assays, must also be standardized. Quality control experiments can be performed with lyophylized calf uterus homogenates. Additional determination of the nuclear estradiol in the high-speed sediments by radioimmunoassay is highly recommended.
Assuntos
Neoplasias da Mama/análise , Receptores de Esteroides/análise , Adulto , Idoso , Animais , Bovinos , Feminino , Humanos , Pessoa de Meia-Idade , Controle de Qualidade , Padrões de ReferênciaRESUMO
Immunoglobulin G preparations align unspecifically with all naturally occurring forms of the estradiol receptor, giving rise to soluble complexes of moderately increased sedimentation velocities. This interaction is not shown by the tryptic receptor fragment, which still contains the high affinity steroid binding site and the dimerization links of the native receptor.
Assuntos
Imunoglobulina G/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de Estrogênio/metabolismo , Tripsina/metabolismo , Animais , Centrifugação com Gradiente de Concentração , Feminino , Cabras , SuínosRESUMO
The steroid-binding core of estradiol receptor was purified from pig uterus cytosol by a protocol consisting of (1) adsorption to heparin-sepharose, (2) enzymatic release of the receptor core, (3) DEAE-chromatography, (4) Sephadex G-150 filtration and (5) chromatography on heparin-sepharose. The final product was approximately 18000-fold enriched over the starting material. It consisted of at least 18% core protein resembling dimeric microsomal receptor with a molecular mass of 75 kDa and an isoelectric point of 5.8 (microheterogeneity). A goat antiserum raised against the preparation contains immunoglobulins G precipitating estradiol-receptor complexes, and antibodies releasing the steroid from its binding site.
Assuntos
Receptores de Estrogênio/isolamento & purificação , Animais , Cromatografia DEAE-Celulose , Citosol/análise , Estradiol/metabolismo , Feminino , Cabras/imunologia , Focalização Isoelétrica , Microssomos/metabolismo , Suínos , Útero/metabolismoRESUMO
The lysosomal population in endometrial cells of prepuberal pigs varies with the seasons. In summertime, lysosomes equilibrate in sucrose density gradients over a broad, domeshaped area from p = 1.21 g/cm3 to p = 1.15 g/cm3. The winter pattern is characterized by a major sharp peak of aggregated lysosomes at p = 1.20 g/cm3, visible as a "snowflurry"-like band. The two extremes are linked by transition patterns in spring and fall. The possible correlation of this phenomenon to the seasonal fluctuations in the level of soluble cytoplasmic estrogen receptor is discussed.
Assuntos
Endométrio/ultraestrutura , Lisossomos/ultraestrutura , Maturidade Sexual , Animais , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Feminino , Microscopia Eletrônica , Estações do Ano , SuínosRESUMO
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.