Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purified rat liver glucocorticoid receptor was covalently charged with [3H]glucocorticoid by photoaffinity labeling (UV irradiation of [3H]triamcinolone acetonide-glucocorticoid receptor) or affinity labeling (incubation with [3H]dexamethasone mesylate). After labeling, separate samples of the denatured receptor were cleaved with trypsin (directly or after prior succinylation), chymotrypsin, and cyanogen bromide. Labeled residues in the peptides obtained were identified by radiosequence analysis. The peaks of radioactivity corresponded to Met-622 and Cys-754 after photoaffinity labeling with [3H]triamcinolone acetonide and Cys-656 after affinity labeling with [3H]dexamethasone mesylate. The labeled residues are all positioned within hydrophobic segments of the steroid-binding domain. The patterns of hydropathy and secondary structure for the glucocorticoid receptor are highly similar to those for the progestin receptor and similar but less so to those for the estrogen receptor and to those for c-erb A.
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PMID:Identification of hormone-interacting amino acid residues within the steroid-binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors. 336 Aug 9

A high resolution quantitative method for estrogen receptor analysis has been elaborated using isoelectric focusing in 0.5% agarose gel, without any prior trypsin digestion. The 23 cytosols analyzed were stabilized by molybdate and prepared from human mammary tumors with progesterone receptors (PR + cytosols) or without (PR - cytosols). Progesterone receptor was used as a tumoral hormonal sensitivity marker. The estrogen receptors usually resolved as 4 isoform peaks of close isoelectric points. In PR - cytosols, the mean pI values were 4.7, 5.5, 6 and 6.5. Significant differences between the two cytosol populations were observed concerning pI 4.7 and 6.5 isoforms. In PR - cytosols, the pI 4.7 isoform occurred in greater proportions than in PR + cytosols, whereas lower proportions of pI 6.5 isoform were seen. The comparison between high performance size exclusion chromatography profiles and isoelectric focusing patterns, before and after cytosol incubation at 28 degrees C with KCl (0.4 M), confirmed an oligomer structure for the pI 4.7 isoform and suggested a monomer structure (Stokes radius 2.9 mm) for the pI 6.5 estrogen receptor isoform. The results indicated that isoelectric focusing analysis of estrogen receptors could be useful in the prediction of breast cancer hormonal sensitivity.
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PMID:Charge heterogeneity of human mammary tumor estrogen receptors. Relationship with a hormonal sensitivity tumor marker. 338 82

High-performance liquid chromatography was performed to separate the various isoforms of estrogen receptor from human breast cancer, based on size (high-performance size-exclusion chromatography) and surface charge (high-performance ion-exchange chromatography) properties. The ability of these isoforms to interact with the monoclonal antibodies was assessed. All isoforms exhibited similar immunodeterminant sites, but when they are bound to [125I]iodoestradiol-17 beta (IE), only 30% binding of the radioactive complex to the immobilized monoclonal antibodies was observed. However, the mass of the receptor recognized by the antibody bead, via the estrogen receptor-enzyme immunoassay (ER-EIA), was always significantly higher. This was true for both fractionated and non-fractionated cytosols, suggesting that non-ligand binding forms, such as precursors and products of the estrogen receptor, were also recognized; or the ligand was only selecting for a particular conformer(s); or the monoclonal antibody on the bead recognized other proteins associated with estrogen receptor. Ion-exchange fractionation of unlabeled receptor showed loss of immunodeterminant sites. However, size-exclusion fractionation did not show this effect. Diethylstilbestrol, a competitor of IE binding, showed marked stability of receptor recognized by ER-EIA during both size-exclusion and ion-exchange chromatography. Limited trypsin treatment of the receptor caused the loss of immunodeterminant sites without altering the ligand binding sites. Thus, proteolysis of estrogen receptors in cytosols of human breast cancer could easily lead to underestimation by ER-EIA. Although the components with immunodeterminant sites recognized by ER-EIA were always eluted with the ligand-binding isoforms of the estrogen receptor, our data suggest that the concentration of the protein having the epitope associated with the monoclonal antibody is unequal to that recognized by the steroid ligand. We conclude that application of ER-EIA to clinical assays of estrogen receptors clearly needs further clarification.
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PMID:Interaction of estrogen receptor isoforms with immobilized monoclonal antibodies. 352 85

Employing a hydroxylapatite batch assay, estrogen-binding activities (EBAs) were demonstrated in the cytosol and nuclear extract of the testis of the anadromous sea lamprey, Petromyzon marinus. The lamprey testicular EBAs are sensitive to trypsin digestion and bind [3H]estradiol-17 beta with high affinities (cytosolic Kd = 0.52 nM; nuclear Kd = 0.39 nM) and limited capacities (cytosolic: 56.2 fmol/g tissue; nuclear: 68.2 fmol/g tissue). Androgens, progesterone, and corticosterone displayed little affinities for lamprey EBAs. Thus, lamprey testicular EBA possessed many definitive properties of an estrogen receptor as described in amphibian, reptilian, and mammalian studies. No specific binding to androgens was detected in either testicular subcellular fraction. The presence of a putative estrogen receptor in lamprey testis suggests a functional role of estrogen in testicular regulation in this ancient vertebrate.
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PMID:Identification of an estrogen receptor in the testis of the sea lamprey, Petromyzon marinus. 362 65

The assumption that a different conformational form was induced in the nuclear estrogen receptor following binding by antiestrogens compared to estrogens was studied by analysing the proteolytic fragments of the receptor following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from MCF-7 cells previously exposed to [3H] 4-OHTAM. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients (S) determined on a sucrose gradient and from the Stokes radii (Rs) estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 155,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 63,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. A similar receptor molecule was released by chymotrypsin from intact nuclei. Digestion of the micrococcal nuclease hydrolysate with trypsin degraded the receptor to a form of a Mr = 67,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. Digestion of intact nuclei with trypsin followed by micrococcal nuclease, solubilized a receptor form of Mr = 80,000 which could be further dissociated with 0.4 M KCl and 3 M urea to a receptor form of Mr = 67,000. This trypsin degraded receptor form seems to be similar in Mr to the chymotrypsin degraded form. On the other hand different receptor fragments of Mr = 33,000 and Mr = 60,000 were excised by chymotrypsin and trypsin respectively from the estradiol ligated estrogen receptor. (Geier et al., J. steroid Biochem. 26 [1987] 35-40.) These results support the assumption of a different conformational form for the antiestrogen ligated receptor, compared to the estrogen ligated receptor since they were differentially susceptible to proteolytic degradation by chymotrypsin.
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PMID:Analysis of the 4-hydroxytamoxifen (4-OHTAM) bound nuclear estrogen receptor from MCF-7 cells by limited proteolysis. 368 14

The proteolytic fragments of the nuclear estrogen receptor in the MCF-7 cell line were characterized following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients determined on a sucrose gradient and from the Stokes radii estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 151,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 33,000 and by trypsin to a receptor of Mr = 60,000. Digestion of intact nuclei with chymotrypsin solubilized a receptor form of Mr = 62,000 which dissociated in 0.4 M KCl to a receptor of Mr = 32,000. Digestion of intact nuclei with trypsin followed by micrococcal nuclease solubilized a receptor form of Mr = 75,000 which was further dissociated by 0.4 M KCl to a receptor form of Mr = 60,000. The ability of the receptor forms to bind DNA was tested using DNA-cellulose column chromatography. About 40% of the micrococcal nuclease solubilized receptor form, compared to about 7% of the chymotrypsin degraded receptor and to about 13% of the trypsin degraded receptor forms, all bound to the column and could be eluted by high salt concentrated buffer. We conclude that the nuclear estrogen receptor in the MCF-7 cell line can be partially degraded either in the micrococcal nuclease hydrolysate or in intact nuclei by chymotrypsin or trypsin generating protein moieties, probably receptor fragments of Mr = 33,000 and 60,000 respectively. Both fragments retain their estradiol binding domain and it may be hypothesized that the heavier fragment retains its chromatin binding domain.
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PMID:Analysis of the nuclear estrogen receptor from MCF-7 cells by limited proteolysis. 382 Nov 6

Fusion of splenic lymphocytes from Lewis rats, immunized with affinity-purified estrogen receptor from the cytosol of MCF-7 human breast cancer cells, with two different mouse myeloma lines, has provided 13 monoclonal hybridoma lines secreting antiestrophilin antibodies, each of which (with one possible exception) recognizes a different antigenic determinant in the human receptor molecule. Of this library of monoclonal antibodies, some react with estrophilin from all sources tested, some react with mammalian but not avian receptors, whereas one preparation appears specific for estrophilin from primate sources. By proteolytic digestion under controlled conditions with mercury-deactivated papain, chymotrypsin, and trypsin, respectively, it is possible to remove sequentially the determinants recognized by one, two or three of the monoclonal antibodies, leaving the epitopes for the six remaining antibodies investigated on the steroid-binding portion of the receptor. The proteolytic fragment containing the epitope most readily removed (by mercuripapain) also contains the DNA-binding domain of the activated receptor molecule. Immunocytochemical staining, using the peroxidase procedure with various monoclonal antibody preparations, of frozen sections of human breast cancer tissue, fixed in ethanol or in picric acid-formaldehyde reagent, shows clearly that the majority of the native receptor, which appears in the cytosol after tissue homogenization, is actually localized within the nuclear compartment in the intact cell. The immunocytochemical technique also permits the identification of mixed populations of receptor-containing and non-containing cells in human breast cancers.
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PMID:Immunochemical studies of estrogen receptors. 620 Jul

The effects of ionizing irradiation on the sedimentation coefficients of both estrogen receptor (ER) and progesterone receptor (PgR) have been examined in comparison to the effects of proteolysis. DMBA-induced rat mammary tumors were subjected to a treatment of 20 Gy and the ER and PgR concentrations were determined at different time intervals after irradiation. On a 5-20% sucrose gradient the ER sedimented as 9-11 and 4-5 S molecular forms, while PgR sedimented as a small 8-9 S peak and a major 4-5 S peak. Radiotherapy particularly reduced the 4-5 S sedimentation peaks of both receptors but did not initiate any new sedimentation forms. Although the 4-5 S ER receptor concentrations remained low, both progesterone receptor forms appeared to recover by 60 days after treatment. As these effects could be due to the release of proteolytic enzymes following irradiation of tumors, the receptors from untreated tumors were exposed to different concentrations of trypsin. The effects of trypsin were identical for ER and for PgR, and proved to be dependent on the trypsin concentration. Only concentrations of trypsin up to 30 micrograms/ml resulted in a reduction of 9-11 S ER or 8-9 S PgR forms which was accompanied by a simultaneous increase in the 4-5 S peaks, resulting in no change in total binding sites. Still higher trypsinization (300-3000 micrograms/ml) also reduced the 4-5 S ER and PgR fractions. In the presence or the absence of sodium molybdate, a stabilizer of the faster sedimenting forms of the receptor, no alterations were observed in the position of, or the total number of binding sites of, the sucrose gradient fractions from control or irradiated tumors. The irradiation effects appear to be due either to damage of the cytosolic ER receptor, thereby preventing its participation in the induction of de novo synthesis of ER and PgR, or to the non-specific damage of transcription and/or translation systems.
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PMID:The effects of ionizing irradiation on the sedimentation coefficient of cytoplasmic steroid receptors in rat mammary tumors. 641 77

The in vitro nuclear binding of rat uterine estrogen-receptor complexes has been studied. Heating cytosol from mature rat uterus at 25 C for various times in the presence of 0.15 M KCl resulted in a transient increase in nuclear binding activity, followed by irreversible loss of this activity. The molecular state of these complexes heated at 25 C in the presence of 0.15 M KCl was determined using Sephadex G-200 chromatography and sucrose density centrifugation at high ionic strength (0.4 M KCl). Gel filtration resulted in steroid-binding activity in the void volume. Sucrose density gradient analysis revealed a broad peak, ranging from approximately 5-20S. When cytosol was heated at 25 C in the presence of 10 mM molybdate to block the temperature-induced activation of receptor, nuclear binding ability was easily recovered by dialysis, while heating already activated estrogen receptor in the presence of 0.15 M KCl and 10 mM molybdate caused irreversible loss of nuclear binding ability. When cytosols prepared from immature rats (19-23 days old) were heated at 25 C in the presence of 0.15 M KCl, only a minimum loss of nuclear binding ability was shown. The radioactive peak in a high salt sucrose density gradient appeared almost exclusively in the 5S region. However, the addition of receptor-free mature uterine cytosol to estrogen-receptor complexes from immature rat uterus caused a marked loss of nuclear binding utility, with a resultant receptor aggregation, whereas rat liver cytosol had no effect on this reaction. Furthermore, heating liver glucocorticoid receptor did not cause a loss of nuclear binding ability even in the presence of receptor-free adult rat uterine cytosol. These observations suggest that there is a factor(s) in rat uterus which recognizes only activated estrogen receptor and induces receptor aggregation and a rapid loss of the nuclear binding ability of receptor in a KCl concentration- and temperature-dependent manner. Preliminary characterization indicates that this factor is macromolecular in nature and resistant to RNase and trypsin treatment, but labile at 100 C.
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PMID:A modulator which converts activated estrogen receptor to a biologically inactive aggregated form. 679 27

An estrogen-responsive mouse Leydig cell tumor line (Tumor 124958) has been shown to contain only a low-affinity binder for estradiol in the cytosol fraction. This differed from the putative estrogen receptor in terms of its hormone-binding specificity as well as affinity. In addition, the possibility that an estrogen receptor-like molecule exists in the nuclei even without hormonal stimuli was examined using purified nuclei. Scatchard plot analyses showed that these nuclei possessed a large amount of estrogen binder having a high affinity for estradiol and diethylstilbestrol. The content of this nuclear binding component was not diminished by using molybdate, a potent inhibitor for receptor activation, and in vitro incubation of collagenase-dispersed cells with estradiol did not cause significant increase in the number of nuclear binding sites when compared with the values obtained by direct incubation of isolated nuclei with estradiol. These results support the view that this nuclear estrogen binder is not due to artificial migration of the cytosol receptor into nuclei during homogenization. The characterization of this nuclear binding component under cell-free conditions revealed that its affinity for estradiol in Mg2+-containing buffer was temperature dependent (Kd 3 nM at 30 degrees and 12 nM at 0 degrees) without significant alteration in the number of maximum binding sites. Introduction of a chelating agent (ethylenediaminetetraacetate) into the buffer system abolished the temperature effect on the affinity, resulting in high affinity for estradiol at both low and high temperatures. These Mg2+ and temperature effects were reversible. In addition, when compared with putative nuclear estrogen receptors, this nuclear binding was observed to be relatively resistant to high salt or micrococcal nuclease treatments in relation to solubilization from nuclei. However, trypsin digestion was found to result in a marked decrease in the nuclear binding sites, indicating that this unique nuclear binding component contains a protein unit(s). These results suggest the possibility that this tumor line contains a unique unoccupied nuclear estrogen binder which might be able to transmit estrogen signals to tumor cell nuclei with regard to tumor growth.
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PMID:Characterization of a unique nuclear estrogen-binding component in an estrogen-responsive mouse Leydig cell tumor. 687 50


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