Sulfhydryl groups and estradiol-receptor interaction.

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.

Harvesting and separation of two populations of lysosomes from porcine endometrium.

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.

The organelles containing porcine 17 beta-estradiol dehydrogenase are peroxisomes.

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.

Differentiation between steroid hormone receptors CBG and SHBG in human target organ extracts by a single-step assay.

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.

The C-terminal half of the porcine estradiol receptor contains no post-translational modification: determination of the primary structure.

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.

Ovarian-independent fluctuations of estradiol receptor levels in mammalian tissues.

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.

Improved biochemical characterization of breast cancer as a guide to hormonal treatment.

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

[Transcervical injection into the uterine lumen of castrated swine as a cell and organ biological research design. The Mariensee model]. / Die transcervicale Injektion in das Uteruslumen von kastrierten Schweinen als zell- und organbiologische Versuchsanordnung. Das Mariensee Modell.

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.

Completion of the amino acid sequence of the C-terminal half of the porcine estradiol receptor by Edman degradation: reconfirmation of the absence of O-linked sugars and phosphates.

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.

Purification of the steroid-binding core of porcine estrogen receptor.

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.

Unspecific interactions of estrogen receptors with immunoglobulins and their absence in the tryptic receptor fragment.

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.

quality control in steroid hormone receptor analyses.

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.

Identification of target cells by immunohistochemical detection of covalently rearranged estradiol in rehydrated paraffin sections.

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.

Oestradiol- and dihydrotestosterone receptors in normal and neoplastic human mammary tissue.

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.

The ligand-binding site of the estradiol receptor resides in a non-covalent complex of two consecutive peptides of 17 and 7 kDa.

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.

The proton-driven dissociation of oestradiol-receptor dimers as a preparative tool. Isolation of a 32 kDa fragment from porcine uteri and assignment of C-terminal origin by partial sequencing.

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.

Purification and properties of oestradiol 17 beta-dehydrogenase extracted from cytoplasmic vesicles of porcine endometrial cells.

Porcine endometrial oestradiol-17 beta dehydrogenase was solubilized from the particulate fraction of homogenates sedimenting between 1200 g and 10,000 g by treatment with 0.4% Brij 35 in neutral buffers. The extracts were processed by successive passage through DEAE-Sepharose, Amberlyte XAD-2 and Blue-Sepharose, and the enzyme was collected from the washed affinity matrix at 0.8 M of a 0-2 M-KCl gradient. A genuine oestrone reductase was eluted at 1.9 M-KCl. The dehydrogenase pool was resolved by butyl-Sepharose chromatography into a major (80%) peak (EDHM) eluted at 0.8 M-(NH4)2SO4 and a very hydrophobic fraction (VHF) recovered at 0.1 M. EDHM was further purified by filtration through Sephadex G-200 and cation-exchange chromatography on Mono S. Sephacryl 300 was used for VHF followed by Mono S. Enrichments from the homogenate amounted to 1074-fold for EDHM and 632-fold for VHF. A single silver-stained band at 32 kDa is seen on SDS/PAGE of EDHM, and VHF contains additional bands at 45 and 80 kDa. Polyclonal antibodies (G436) raised against EDHM and the monoclonal antibody F1 raised against VHF recognize the single 32 kDa band in EDHM and both the 32 kDa and 80 kDa bands in composite VHF. The 45 kDa band of VHF reacts with neither. Monoclonal antibody W1 raised against EDHM only recognizes the 32 kDa peptide of EDHM and VHF. The specific activity for oestradiol oxidation amounts to 4081 mu-units/mg for EDHM and to 2402 mu-units/mg for VHF. Both possess a minimal (1/260) endogenous reductase activity and are devoid of 3 beta, 3 alpha- and 20 alpha-dehydrogenases. We consider EDHM to be authentic oestradiol-17 beta dehydrogenase of porcine endometrium. The composite VHF could reflect the situation of the enzyme in vivo or result from aggregations occurring during processing.