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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein disulfide isomerase (PDI) is a multifunctional microsomal enzyme that participates in the formation of protein disulfide bonds. PDI catalyzes the reduction of protein disulfide bonds in the presence of excess reduced glutathione and has been implicated in the reductive degradation of insulin; E. coli thioredoxin is homologous to two regions in PDI and can also degrade insulin. PDI activity, measured by 125I-insulin degradation or reactivation of randomly oxidized RNase in the presence of reduced glutathione, is non-competitively inhibited by estrogens; half-maximal inhibition was observed at approximately 100 nM estrogen. Other steroid hormones at 1 microM had little or no effect. PDI segment 120-163 (which corresponds to exon 3 of the PDI gene) and 182-230 have significant similarity with estrogen receptor segments 350-392 and 304-349, respectively, located in the estrogen binding domain but not with the steroid domains of the progesterone and glucocorticoid receptors or with thioredoxin, which is insensitive to estrogens. We propose the hypothesis that enzymes can acquire sensitivity to a hormone via exon shuffling to the enzyme gene from the DNA region coding for the hormone binding domain of the hormone's receptor.
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PMID:Selective inhibition of protein disulfide isomerase by estrogens. 266 79

Immunohistochemical and immunochemical analysis using Western blot techniques were carried out with estrogen receptor (ER) monoclonal antibody H-222 to 1) clarify the "nuclear translocation" phenomenon of ER, 2) elucidate the primary nuclear binding site of ER, and 3) to evaluate the binding force between ER and its nuclear binding site in the uterus of ovariectomized adult mice. Exclusive nuclear localization of ER was recognized in the epithelial cells, stroma cells, and smooth muscle cells. Uterine tissues prepared from animals injected with saline, 17 beta-estradiol (E2), estriol (E3), and diethylstilbestrol (DES) exhibited almost the same ER immunostaining when they were fixed prior to sectioning (prefixation method) and frozen sections were used. On the other hand, when fresh-frozen sections were fixed before or after incubation with various solutions (postfixation method) and then treated with various salt solutions, greater differences were seen in immunostaining of ER between saline-injected and hormone-treated animals. Immunostaining of ER in control animals was low after incubation with PBS (0.01 M phosphate buffer containing 0.16 M NaCl, pH 7.2), whereas uterine tissue from hormone-injected mice showed strong nuclear immunostaining after this treatment. After treatment with 0.4 M KCl or 0.5 M NaCl, immunostaining in the uterus of both hormone-injected and control animals was completely abolished. DNase treatment caused an almost complete loss of immunostaining of ER; however, RNase digestion slightly increased immunoreactivity in both E2-injected and control animals. Quantitative analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques showed that after incubation of tissue sections for 30 min with PBS, 0.4 M KCl, or DNase, 60%, 10%, and 30% of ER were present, respectively, compared to amount of ER present in unincubated sections. These findings suggest the following for the ER in uterine tissue; nuclear occupancy is a phenomenon that occurs due to a differential affinity between occupied and unoccupied receptors in the nucleus; after hormone treatment, the receptor levels do not fluctuate in the nucleus to the extent demonstrated by binding assays; and the properties of the ER detected in the immunohistochemical analysis are identical to those observed in biochemical studies.
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PMID:Immunological analysis of the biochemical properties of the uterine estrogen receptor. 277 19

It has been reported that the response of target cells to steroid hormone (SH) stimulation may depend on their position in the cell cycle. The DNA and RNA contents of malignant cells of the endometrium cultured in vitro were measured using flow cytometry (FCM). We also measured estrogen receptor (ER) and progesterone receptor (PR) levels of cells at different positions in the cell cycle. The G1 and S phases of the cell cycle were investigated using cells synchronized by sodium n-butyrate (G1 block), methotrexate (S block), and excess thymidine (S block). For DNA measurements, the cells were stained with propidium iodide following RNase treatment. For RNA measurements (double-stranded RNA) the cells were treated with DNase. We found that S phase synchronization by methotrexate was 136.2% of control (100%). Using the excess thymidine block and release procedure, the S phase fraction was 185.1% of control. G1 phase synchronization by sodium n-butyrate was 134% of control. The estrogen receptor level in G1 phase synchronized cells increased to 5.94 fmol/micrograms DNA in the cytosol and 12.35 fmol/micrograms DNA in the nuclear fraction. These levels represent a sevenfold total increase over that of the control estrogen receptor level. Cells in S phase showed no significant increase in estrogen receptor levels over control cells. Based on this study, the functional increase of the steroid receptor was most significant in the G1 phase.
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PMID:Relationship between changes of the steroid receptor and synchronization in human endometrial adenocarcinoma cells in vitro. 320 23

The role of estradiol in the regulation of its cognate receptor in MCF-7 cells was investigated in this study. After treatment with 10(-9) M estradiol, the level of receptor protein was measured using an enzymeimmunoassay. By 6 h, the receptor protein declined by about 60% from a level of approximately 3.6 to 1.2 fmol/micrograms DNA. The level of receptor remained suppressed for 24-48 h. Similar results were obtained with an estrogen receptor (ER) binding assay. The steady state level of ER mRNA was determined by an RNase protection assay. Estrogen treatment resulted in a maximum suppression of mRNA by 6 h. Receptor mRNA remained depressed for 48 h. Transcription run on experiments demonstrated a transient decrease of about 90% in ER transcription after 1 h. By 3-6 h transcription increased approximately 2-fold and remained elevated for at least 48 h. These data suggest that estrogen down-regulates ER mRNA by inhibition of ER gene transcription at early times and by a posttranscriptional effect on receptor mRNA at later times.
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PMID:Regulation of the estrogen receptor in MCF-7 cells by estradiol. 321 58

Epididymides from sexually mature rabbits contain a factor that induces a discrete reduction in the sedimentation coefficient of cytosolic estrogen receptors from various tissues (rabbit epididymis and accessory sex organs; rabbit, rat and mouse uterus) and of cytosolic progesterone receptors from the rabbit uterus. The factor is not species-specific since a similar activity was detected in extracts of mature rat epididymides. Although present in cytosol, the factor is obtained in much higher yield in hypertonic extracts of the nucleomyofibrillar fraction of mature rabbit epididymal tissue. Using rabbit uterine estrogen receptor as substrate, we have determined the following details about the rabbit epididymal factor: (1) it is tissue-specific (undetectable in extracts from rabbit accessory sex organs, testis, uterus, liver, lung, kidney and intestine); (2) it is age-dependent (undetectable in extracts from sexually immature rabbit epididymides); (3) its maintenance is testis-independent following its post-pubertal induction or activation; (4) it is primarily localized in the caput region of the epididymis; (5) it is inactivated by elevated temperature; (6) it is macromolecular in nature; (7) it is DNase- and RNase-resistant; (8) it is irreversibly inactivated by leupeptin, indicating that it is a protease; and (9) it is effective on unoccupied and occupied receptors.
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PMID:Structural conversion of cytosolic steroid receptors by an age-dependent epididymal protease. 391 13

Rat uterine and anterior pituitary microsomes each contain a population of specific estrogen-binding sites. Saturation binding of estradiol is demonstrable, with an affinity similar to that of the cytosol estrogen receptor (Ka = 1-2 X 10(10) M-1). Dissociation rate kinetic determinations, however, revealed that estrogen-microsomal complexes are 4 times as stable as cytosol estrogen-receptor complexes. Sedimentation properties in sucrose gradients were salt-dependent, yielding values of 10S in KCl-free buffer and 5.5S in the presence of 0.4 M KCl. The concentration of microsomal sites varies in proportion to the level of cytosol estrogen receptor, such that microsomal binding constitutes a consistent 20% of the total extranuclear binding capacity. Binding is sensitive to pronase, but not to ribonuclease or deoxyribonuclease; steroidal specificity differs from cytosol receptor only with respect to a greater extent of competition by progesterone. Microsomal binding sites are readily extractable with KCl-free hypotonic buffer or with 0.4 M KCl, but are resistant to extraction by 0.15 M KCl. The presence of estradiol lends stability to the microsomal binding sites, while high salt has a deleterious effect on their longevity. After exhaustive extraction of binding sites, microsomes are capable of accepting cytosol estradiol-receptor complexes to a level corresponding to the concentration of depleted binding sites; microsomes from nontarget tissue do not manifest such capability. However, the original microsomal estrogen-binding sites are not simply cytosol receptor contaminants, as evidenced by the observations that the microsomal binding site concentration is independent of the volume of tissue homogenate (indicating that a trapping phenomenon is not operative) and that nonextracted microsomes are not potential acceptor sites for cytosol estradiol-receptor complexes. In considering total cellular dynamics of estrogen and estrogen receptor turnover, it thus becomes important to explore the role of the microsomal compartment, since it functions as a repository of specific estrogen-binding sites and may have significant acceptor capability for the cytosol estrogen-receptor complex.
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PMID:Specific binding of estrogen and estrogen-receptor complex by microsomes from estrogen-responsive tissues of the rat. 402 80

Nuclear and cytoplasmic binding sites for estradiol (E2-17 beta) in granulosa cells of immature rats were characterized. These binding sites for estrogen were high affinity, low capacity with an affinity constant (Kd) of 1.9 X 10(-10)M (binding capacity, Ro = 80 pM) for nuclear sites and a Kd = 3.5 X 10(-10) M (Ro = 45 pM) for cytosol sites. Binding was specific for biologically active estrogens. The estrogen receptor in granulosa cells is a protein and heat-labile as treatment with protease or pre-incubation at 37 degrees C for 1 h significantly diminished binding. RNase and DNase had no effect on estrogen binding. Sedimentation coefficients for nuclear and cytosol binding components were 5S and 8S respectively, similar to values obtained with uteri. Finally, translocation was demonstrated after a s.c. injection of E2-17 beta. Forty-five minutes post-injection, cytosol binding sites for estradiol were depleted concomitant with accumulation of nuclear binding sites. We concluded that granulosa cells of immature rats have binding sites specific for estradiol which have characteristics similar to the classical estrogen receptor in uteri.
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PMID:Identification of estrogen receptors in granulosa cells of immature rats. 667 47

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

Incubation of crude estrogen receptor preparations from mammary tumor cytosol with RNase A increases the sedimentation coefficient of the receptor from 9.7 S to 10.4 S. The effect is not obtained with other low molecular weight basic proteins (lysozyme, cytochrome c, or histone H2B). Nonenzymically active RNase A derivatives such as performic acid oxidized RNase A, fully reductively methylated RNase A, carboxymethyl-His-119-RNase A, and RNase S-protein were ineffective. RNase T1, an acidic endoribonuclease, was also without effect. However, enzymically active RNase S', prepared from a mixture of RNase S-protein and S-peptide, shifted the sedimentation to 10.4 S. The increased sedimentation is not accompanied by a change in the Stokes radius of the receptor (74 A) or buoyant density in metrizamide (1.24 g/ml). The effect of RNase A on the sedimentation of the receptor can be reversed by subsequent incubation with human placental RNase inhibitor or with rabbit anti-RNase A antibodies. Direct interaction was shown by chromatography of the receptor on RNase A Sepharose. Thus, the shift in sedimentation results from binding of RNase A to the receptor and, although this requires that the enzyme active site be available, enzymic activity is not responsible for the effect. The interaction of RNase A with the receptor occurs at low ionic strength; it does not occur at elevated ionic strength or after activation of the receptor by precipitation with ammonium sulfate.
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PMID:Interaction of ribonuclease A with estrogen receptor from rat mammary tumor MTW9. 683 88

Cytosols of whole testicular homogenates from the Syrian golden hamster contained specific binding sites for [3H]triamcinolone acetonide that exhibited limited capacity and high affinity binding characteristic of glucocorticoid receptors in other target tissues. The receptor complex sedimented as an 8.6S binder in low salt 5-20% linear sucrose gradients and as 6.2S and 4.0S moieties in 0.15M and 0.4 M KCl, respectively. The Ka at equilibrium was 3.1-3.3 X 10(9) M-1 at 4 C in intact and adrenalectomized males. The testicular glucocorticoid binder was vulnerable to proteolytic degradation while being completely resistant to the action of RNase and DNase. In addition the binding protein exhibited the usual steroid specificities for type I glucocorticoid receptor: triamcinolone acetonide greater than dexamethasone greater than cortisol greater than corticosterone greater than progesterone greater than aldosterone greater than prednisone greater than 5 alpha-dihydrotestosterone greater than diethylstilbestrol. Unexpectedly, 17 beta-estradiol competed for receptor binding to the same extent as prednisone. A 3.2 S nuclear receptor was extracted from purified testicular nuclei after incubation of whole suspensions in culture media containing 5 nm radiolabeled triamcinolone acetonide at 32 C. Although the glucocorticoid receptor concentrations in prepubertal, adrenalectomized, and hypophysectomized animals were markedly higher in the testis compared to the concentration in the normal adult hamster (52 +/- 4 fmol/mg cytosol protein), the greatest total amount of receptor per testis was found in the mature intact animal. Moreover, under the conditions studied, the concentration of glucocorticoid receptor substantially exceeded the levels of either androgen or estrogen receptor when determined simultaneously. In contrast, no measurable cytoplasmic [3H]triamcinolone acetonide binding was detected in adjacent urogenital organs such as the epididymis and seminal vesicle. It is therefore unlikely that the testicular glucocorticoid receptor is associated with the spermatid or present as a secretory product in the seminiferous tubule lumen.
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PMID:Characterization of specific glucocorticoid receptor in the Syrian hamster testis. 729 80


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