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

In order to characterize the nuclear thyroid hormone receptors in human tissue, an improved method for isolation of nuclei from cultured human fibroblasts was developed. This method provided nuclei with a protein/DNA ratio of 2.8 and recovery of 42%. The purity of nuclei was verified by phase contrast and electron microscopy, which showed normal appearance of chromatin structure. Nuclear binding assay was performed by incubation of whole cells at 37 degrees C or isolated nuclei at 22 degrees C with L-triiodothyronine (T3). In both cases, an affinity constant (Ka) of 2.0-3.0 X 10(10) M-1 and an average binding capacity of 41 femtomoles of T3/100 micrograms DNA (3,100 binding sites/nucleus) were obtained. During incubation of the nuclei, 13% to 16% of receptors that had an identical Ka was released into the medium. Salt extraction recovered 85% to 90% of the receptors, which had a Ka of 4.5 X 10(10) M-1 and the capacity of 0.13 pmol of T3/mg protein. The Ka fo. L-thyroxine (T4) was seven to 18 times lower than that for T3, but the capacity was the same in isolated nuclei, receptors released during incubation of nuclei, and in salt-extracted receptors. Of the iodothyronines examined, affinity for triiodothyroacetic acid was the highest, followed by L-T3, D-T3, L-T4. Isokinetic glycerol gradient analysis revealed that salt-extracted receptors had a sedimentation coefficient of 3.4 S, whereas micrococcal nuclease digested receptors showed two major (6.0 to 6.5 and 12.5 S), and two minor (17 and 19 S) peaks. These results were virtually identical to those obtained with rat liver nuclei analyzed in parallel studies.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Nuclear thyroid hormone receptors in cultured human fibroblasts: improved method of isolation, partial characterization, and interaction with chromatin. 301 26

High affinity-low capacity binding sites for thyroid hormone have been identified in the nuclei of glial (C6) and neuronal (Neuro 2A) cultured cells. Equilibrium dissociation constants, determined by Scatchard analysis, were very similar in both types of cells (0.2-0.3 nM). The relative affinity of hormonal analogs was also similar: the affinity for T3 was lower than for triiodothyroacetic acid and higher than for T4 or tetraiodothyroacetic acid. The sedimentation coefficients obtained by gradient centrifugation of nuclear receptor extracted with 0.4 M KCl or excised by micrococcal nuclease digestion were 3.5 S and 6.5 S, respectively. These results suggest that the thyroid hormone receptor is not restricted to neuronal cells, but also appears in cells of glial origin.
 ...
PMID:Identification and characterization of L-triiodothyronine receptors in cells of glial and neuronal origin. 376 67

The thyroid hormone receptor is a chromatin-associated protein which appears to mediate the actions of the thyroid hormones in mammalian cells. Unlike steroid hormone receptors, a cytoplasmic form of the receptor has not been identified, and the factors which govern the nuclear concentrations of the receptor are poorly understood. Using cultured GH1 cells, a rat pituitary cell line, we having previously demonstrated that thyroid hormones reduces the concentration of its receptor by a mechanism which involves the association of the ligand with the receptor binding site (Samuels, H.H., Stanley, F., and Shapiro, L.E. (1977) J. Biol. Chem. 252, 6052-6060). In this study, we demonstrate that n-butyrate and other aliphatic carboxylic acids elicit a reduction of thyroid hormone nuclear receptor levels without altering total cell protein synthetic rates. In contrast, the nuclear association and total cell level of the glucocorticoid receptor is not altered by n-butyrate. Evidence is presented that the aliphatic carboxylic acid-mediated reduction of thyroid hormone nuclear receptor levels is secondary to the inhibitory effect of these compounds on chromatin-associated deacetylases which is reflected as an increase in the acetylation of the nucleosome core histones. Isokinetic gradient centrifugation of chromatin solubilized from GH1 cell nuclei by micrococcal nuclease indicates that the receptor exists as a form associated with high molecular weight chromatin, as a 12.5 S form that sediments slightly faster than the bulk of the mononucleosomes, and as a 6.5 S form which appears to remain associated with low molecular weight chromatin components. Exclusive of the receptor associated with the high molecular weight chromatin, the 6.5 S form represents 80% and the 12.5 S form 10% of the receptor resolved in the gradient. n-Butyrate decreases both forms to the same degree suggesting that they are generated from the same "entity" of chromatin structure. Studies on the reappearance of receptor after restoration of the chromatin to the "normal" acetylated state are consistent with a model in which the affinity of chromatin for newly synthesized receptor is diminished in the "hyperacetylated" state.
 ...
PMID:Thyroid hormone nuclear receptor levels are influenced by the acetylation of chromatin-associated proteins. 624 82

Nuclear thyroid hormone (T3) receptors are nonhistone proteins which are tightly bound to rat liver chromatin. The solubilization of the T3 receptors by micrococcal nuclease was studied using an assay which allows the delection of in vitro hormone binding and which is independent of the state of solubility of the chromatin. Nuclease digestion produces a receptor containing moiety which sediments at a rate of 5--6S. This form of the receptor is different than that released from chromatin at high ionic strength (3.8S) and potentially represents the stable association of the receptor which other elements of chromatin. Partial release of chromatin compaction by the use of dilute buffer solutions increases the rate of nuclease digestion, facilitates the release of the (5--6S) T3-receptor complex, and allows the isolation of sucrose gradient fractions which are enriched with receptor.
 ...
PMID:3,5,3'-triiodothyronine receptor-containing chromatin fragments: production by nuclease digestion. 625 Aug 2

A large body of circumstantial evidence indicates that receptors located in nuclei of T3 responsive tissues represent a site of initiation of thyroid hormone action at the cellular level. Partial characterization of T3 receptors indicates that these proteins are monomeric structures in nuclei and are chromatin-associated non-histone proteins. Treatment of rat liver nuclei with either pancreatic DNase I or micrococcal nuclease releases T3 receptors from nuclei in two forms: a predominant (95 400 Mr; 5.5-6.0S) and a minor (265 000-365 000 Mr; 12.5S) nucleoprotein complex. Similar structures are excised from rat kidney, brain, and heart nuclei and from GH1 pituitary cell nuclei by micrococcal nuclease digestion. These endonuclease-excised receptor-containing complexes are significantly larger than the salt-extracted receptor (50 000 Mr; 3.5S). The presence of DNA and other non-receptor proteins in these structures indicates that T3 receptors probably function within multimeric complexes in vivo. Although T3 receptors appear to be associated with DNA between nucleosomes, i.e. linker DNA, it is not entirely clear whether all or only a fraction of T3 receptors interact with nucleosomal components. The 12.5S receptor-containing nucleoprotein complex may represent T3 receptors in association with linker DNA and nucleosomal components. T3 receptors do not appear to be uniformly distributed to all chromatin fractions, but are associated with structures having characteristics of transcriptionally active chromatin. They are found in a region of chromatin which is enriched in RNA polymerase activity, rapidly labeled RNA and non-histone proteins, and depleted of histone Hl. This region is also highly sensitive to both micrococcal nuclease and pancreatic DNase I digestion. The association of receptors with transcriptionally active chromatin, however, must be considered provisional until additional details of the precise receptor-chromatin interaction have been established. The recent demonstration of a 20-fold increase in a specific hepatic mRNA four hours following administration of T3 to hypothyroid rats indicates that thyroid hormone potentially has very rapid effects on hepatic gene expression. However, significant changes in nuclear protein phosphorylation, nuclear protein composition, and chromatin structure have not been detected within this four-hour period. Thus, effects of T3 on hepatic gene expression are brought about by local and presumably subtle changes in nuclear function.
 ...
PMID:Association of thyroid hormone receptors with chromatin. 631 18

Rat liver nuclei were incubated with either thyroid hormone or angiotensin (AII) at varying concentrations or with buffer (control) prior to digestion with micrococcal nuclease. Concentrations of hormones greater than 10(-10)M were effective in increasing the solubilization of chromatin with physiological levels (10(-9)M) of AII showing an approximate 2.4 fold increase over control. Nuclei were also isolated from animals treated in-vivo with either AII or buffer (control) and chromatin solubility was increased in the AII treated nuclei even prior to the addition of exogenous nuclease, presumably from the action of endogenous nucleases. The data suggest that hormone-induced increases in solubility are a reflection of structural changes in chromatin which enhance the accessibility of DNA to endonuclease attack.
 ...
PMID:Nuclear-hormone mediated changes in chromatin solubility. 683 24

Transcription of the ratalpha1-acid glycoprotein (AGP) gene is activated by glucocorticoid, thyroid hormone (T3) and cytokines. Following these treatments, the chromatin structure of this gene was analyzed by means of digestion with DNase I or micrococcal nuclease. Four DNase I hypersensitive sites were observed in the 5'-upstream region of the rat AGP gene of liver cells. They were designated HS1, HS2, HS3 and HS4 (3'-->5'). After T3treatment the sensitivity of HS1 and HS2 increased and after dexamethasone (Dex) treatment that of all four sites did so. Three new sites appeared after turpentine oil treatment, while the sensitivities of HS3 and HS4 increased. We conclude that transcriptional activation of the gene by T3and Dex have very similar mechanisms, but that at the inflammation stage they become slightly different. The increase in sensitivity at HS1 and HS2 after T3treatment in vivo was successfully reproduced in a cell-free system by in vitro treatment with T3. HS1, HS2 and HS3 were also sensitive for micrococcal nuclease.
 ...
PMID:Analysis of chromatin structure of rat alpha1-acid glycoprotein gene; changes in DNase I hypersensitive sites after thyroid hormone, glucocorticoid hormone and turpentine oil treatment. 918 75

The Xenopus thyroid hormone receptor betaA (TRbetaA) gene contains an important thyroid hormone response element (TRE) that is assembled into a positioned nucleosome. We determine the translational position of the nucleosome containing the TRE and the rotational positioning of the double helix with respect to the histone surface. Histone H1 is incorporated into the nucleosome leading to an asymmetric protection to micrococcal nuclease cleavage of linker DNA relative to the nucleosome core. Histone H1 association is without significant consequence for the binding of the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR/RXR) to nucleosomal DNA in vitro, or for the regulation of TRbetaA gene transcription following microinjection into the oocyte nucleus. Small alterations of 3 and 6 bp in the translational positioning of the TRE in chromatin are also without effect on the transcriptional activity of the TRbetaA gene, whereas a small change in the rotational position of the TRE (3 bp) relative to the histone surface significantly reduces the binding of TR/RXR to the nucleosome and decreases transcriptional activation directed by TR/RXR. Our results indicate that the specific architecture of the nucleosome containing the TRE may have regulatory significance for expression of the TRbetaA gene.
 ...
PMID:Structural and functional features of a specific nucleosome containing a recognition element for the thyroid hormone receptor. 938 90

To investigate the role of chromatin structure in cardiac gene expression, we used the DNase I and micrococcal nuclease to probe the chromatin structure of the hamster cardiac beta-MyHC gene. Two cardiac-specific DNase I hypersensitive sites (DHS) were identified, one of which was mapped to the -2.3 kb (beta-2.3 kb) region and the other to the proximal promoter region of the beta-MyHC gene. The two sites were readily detectable using nuclei from neonatal hamster heart; however, the proximal promoter site disappeared when adult hamster heart nuclei were used, and the -2.3 kb site decreased in intensity. We were able to demonstrate the gradual disappearance of this proximal promoter DHS by comparing heart nuclei isolated from animals at late-gestation and 1-day-old stages. Furthermore, injecting thyroid hormone caused the disappearance of the proximal promoter DHS in late gestational fetal ventricular nuclei. Digestion of nuclei from various tissues by micrococcal nuclease revealed that the beta-MyHC gene proximal promoter exists in an array of three specifically-positioned nucleosomes only in fetal heart chromatin. The beta-MyHC gene proximal promoter is DNase I hypersensitive within one of the nucleosomal particles. Our data suggest that chromatin structure may participate actively in cardiac gene expression.
 ...
PMID:Chromatin remodelling of the cardiac beta-myosin heavy chain gene. 948 Sep 3