Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.31.1 (micrococcal nuclease)
 2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Androgen receptors were quantified in nuclei from human prostate tissue and in nuclear fractions derived by exhaustive digestion with micrococcal nuclease. In nuclei from benign hypertrophic prostate (BPH), the population of androgen receptors solubilized during nucleolysis predominated whereas in carcinoma nuclei the nuclease-resistant population was in excess. This phenomenon was restricted to intranuclear deployment and could not be attributed to recompartmentalization within the cell. Receptor content could not be correlated to the expression of the cellular protooncogenes myc, H-ras, K-ras or sis, in either BPH or carcinoma. However, in both BPH and carcinoma, significant correlation was observed between nuclear androgen receptor content and expression of c-fos. Expression of c-fos was not elevated in carcinoma compared to BPH, whereas expression of c-myc was elevated in carcinoma specimens of all grades of glandular differentiation, and expression of H-ras became increasingly elevated as differentiation was lost.
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PMID:Intranuclear androgen receptor deployment and protooncogene expression in human diseased prostate. 244 4

Nuclear androgen-receptor binding sites in chromatin regions of mouse submandibular gland were studied. Cytosol androgen receptors prelabeled with [3H]androgen interacted with the crude nuclei in mouse submandibular gland and activation of the receptor was a prerequisite for this interaction. After in vivo administration of [3H]androgen to female mice, radioactivities were found in the nuclei purified from submandibular gland tissues and the [3H]androgen-labeled purified nuclei were further digested with micrococcal nuclease. The androgen receptor was found in solubilized, active chromatin fractions which contained mono- and dinucleosomes. By an in vitro exchange assay, endogenous androgen-receptor complexes associated with chromatin binding sites in intact males were found in the solubilized fraction after micrococcal nuclear digestion, whereas such complexes were not found in females. These results suggest that the androgen receptors translocated to the nuclei and became associated with chromatin and that this association occurred in transcriptionally active chromatin regions that were preferentially sensitive to micrococcal nuclease.
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PMID:Intranuclear androgen-receptor complex binding sites of mouse submandibular gland. 251 84

With increasing purification of the androgen receptor from nuclei of rat ventral prostate, a receptor-like protein could be demonstrated by chemical staining with silver nitrate. After sonication and digestion of nuclei with micrococcal nuclease, the solubilized receptor was applied to a column of Matrex Gel Green A and eluted with a linear gradient of 0-2 M NaCl. Characterized by specific binding of dihydrotestosterone, this form of the receptor was also androgen dependent and yielded an apparent Mr of 33,000 when analyzed by polyacrylamide gel electrophoresis and silver nitrate staining. To facilitate recovery following chromatography, the receptor was precipitated with 0-40% ammonium sulfate. Analysis of the 15-fold enriched fraction by sucrose density-gradient centrifugation confirmed the presence of a 3S androgen-binding protein. About 200 ng of the precipitated protein was applied to a column of dihydrotestosterone-17 beta-succinyl agarose (ligand concentration, 0.25 mumol/ml). The fractions eluted with 50 microM dihydrotestosterone were electrophoresed and stained as before; again, the presence of a 33,000 Mr protein sensitive to castration was demonstrated. Alternatively, when the precipitated protein was fractionated by fast protein liquid chromatography utilizing a Superose 12 HR 10/30 column, the receptor coeluted with nuclear proteins in the 29,000-36,000 Mr range as determined both by retention time and electrophoresis. In combination, the above methods may be used to obtain a receptor protein purified to near homogeneity with a yield of 5-10%. The amount of receptor afforded by the purification sequence is small but nevertheless sufficient for chemical detection. We anticipate that with modification, the procedures may prove suitable for the recovery of nuclear androgen receptor on a preparative scale.
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PMID:Chemical demonstration of nuclear androgen receptor following affinity chromatography with immobilized ligands. 358 12

Controlled digestion of rat ventral prostate nuclei by careful adjustment of conditions of temperature, divalent cation concentration, ionic strength and micrococcal nuclease:DNA ratios yielded oligonucleosome fractions corresponding to less than 10% of the total genome which contain the majority of RNA polymerase B activity and androgen-receptor complexes of the nucleus. These parameters were affected acutely by androgen withdrawal and administration: furthermore, such manipulations affected the susceptibility to micrococcal nuclease release of prostate binding protein gene sequences. This transcriptionally-active androgen-influenced fraction was considered ideal for studies of interaction of chromatin components with androgen receptor protein. Androgen receptor was purified approximately 20 000-fold from rat prostate cytosol. The purified protein retained its ability to stimulate RNA polymerase B activity in prostate nuclei and chromatin fractions, and its properties of binding to chromatin and to DNA. However, although purified receptor protein showed tissue-specific binding to prostate chromatin and enhanced binding to fractions released by low nuclease digestion, no such specificity was indicated by binding to total DNA, DNA from specific fractions or cloned prostatic binding protein cDNA.
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PMID:Interaction of androgen receptors with chromatin and DNA. 653 19

The physical properties of two types of androgen-binding sites in prostatic nuclei were compared and found to be identical. The first type was released from chromatin by micrococcal nuclease digestion and solution in 0.6 M NaCl; the second resisted such treatment and remained associated with nuclear structures. After in vivo administration of [1,2-3H]testosterone to 24-h castrated rats and sonication of purified nuclei, 90% of the nuclear radioactivity was extracted with nuclease/salt treatment and was found by sucrose density gradient analysis to be associated with a 3 S androgen receptor. If sonication was omitted, 50 to 60% of the nuclear radioactivity was recovered in the nuclease/salt-resistant pellets or bound to nuclear matrices. Mild digestion of either of these particulate fractions with trypsin resulted in the release of a 3 S androgen receptor. After in vitro isotope-exchange labeling with [1,2-3H]dihydrotestosterone, the sedimentation coefficient, steroid specificity, and dissociation constant of the androgen receptors released by trypsin digestion of nuclease/salt-resistant pellets or nuclear matrices were similar to those of the receptors extracted by nuclease/salt treatment. These results indicate first, that all androgen-binding sites in prostatic nuclei can be released, either with nuclease/salt or trypsin digestion procedures to yield a 3 S androgen receptor with uniform binding characteristics, and second, that the androgen receptors are distributed between two intra-nuclear pools--one containing about 10,000 molecules/nucleus sensitive to micrococcal nuclease digestion and salt and the other containing about 8,000 to 13,000 androgen receptors tightly bound to the nuclear matrix.
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PMID:Isolation of 3 S androgen receptors from salt-resistant fractions and nuclear matrices of prostatic nuclei after mild trypsin digestion. 686 57

Nuclei from rat ventral prostate were disrupted by sonication and treated with micrococcal nuclease to precipitate nuclear proteins including the androgen receptor. The precipitate was dissolved in 0.6--1.2 M ammonium bicarbonate buffer, pH 7.6, with no loss of receptor when compared to the conventional Tes buffer, pH 7.0 containing 0.6--1.2 M NaCl. Lyophilisation of the solubilised protein did not produce any qualitative or quantitative differences in the recovery of receptor relative to a non-lyophilised control preparation, both of which were analysed for binding properties by Sephadex G-25/G-100 dual-column chromatography. Over longer periods of storage at -80 degrees C, the rate of inactivation of receptor was found to be 6% per week. The stability of the lyophilised receptor was improved by the inclusion of MgCl2 and SH-reducing agents in the ammonium bicarbonate buffer. Recovery improved also with increasing ionic strength of the buffer used to dissolve the lyophilised receptor.
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PMID:Concentration and preservation of nuclear androgen receptor by lyophilisation. 710 83

Extensive (20%) digestion of linker DNA of prostatic chromatin with micrococcal nuclease resulted in the precipitation of 95% of the nuclear androgen receptors. The receptor-enriched precipitate was dissolved in Tes buffer, pH 7.0, containing 0.6--1.2 M NaCl and analysed by hydrophobic interaction chromatography. The adsorption of receptor to omega-amino-alkyl derivatives of agarose increased with the length of the alkyl substituent indicating the presence of hydrophobic regions on the surface of the receptor molecule. Digestion of linker DNA followed by chromatography of precipitated chromatin proteins using 5-aminohexyl-agarose gave rise to a mean 93-fold purificaton of receptor with a recovery of 45%. This approach to the partial separation of nuclear androgen receptor may prove useful in conjunction with more selective purification techniques such as affinity chromatography.
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PMID:Partial purification of nuclear androgen receptor by micrococcal nuclease digestion of chromatin and hydrophobic interaction chromatography. 731 34

Gene regulation by steroid hormone receptors depends on the particular character of the DNA response element, the array of neighboring transcription factors, and recruitment of coactivators that interface with the transcriptional machinery. We are studying these complex interactions for the androgen-dependent enhancer of the mouse sex-limited protein (Slp) gene. This enhancer has, in addition to multiple androgen receptor (AR)-binding sites, a central region (FPIV) with a binding site for the ubiquitous transcription factor Oct-1 that appears crucial for hormonal regulation in vivo. To examine the role of Oct-1 in androgen-specific gene activation, we tested the interaction of Oct-1 with AR versus glucocorticoid receptor (GR) in vivo and in vitro. Oct-1 coimmunoprecipitated from cell lysates with both AR and GR, but significant association with AR required both proteins to be DNA-bound. This was confirmed by sensitivity of the protein association to treatment with ethidium bromide or micrococcal nuclease. Addition of DNA to micrococcal nuclease-treated samples restored interaction, even when binding sites were on separate DNA molecules, suggesting association was due to direct protein-protein interaction and not indirect tethering via the DNA. AR/GR chimeras revealed that interaction of the N and C termini of AR was required to communicate the DNA-bound state that enhances interaction with Oct-1. Protease digestion assays of hormone-bound receptors revealed further conformational changes in the ligand binding domain of AR, but not GR, upon DNA binding. Furthermore, these conformational changes led to increased interaction with the coactivator SRC-1, via the NID 4 domain, suggesting DNA binding facilitates recruitment of SRC-1 by the AR-Oct-1 complex. Altogether, these results suggest that the precise arrangement of binding sites in the Slp enhancer ensures proper hormonal response by imposing differential interactions between receptors and Oct-1, which in turn contributes to SRC-1 recruitment to the promoter.
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PMID:Oct-1 preferentially interacts with androgen receptor in a DNA-dependent manner that facilitates recruitment of SRC-1. 1109 94