Analysis of CpG methylation and genomic footprinting at the tyrosine aminotransferase gene: DNA methylation alone is not sufficient to prevent protein binding in vivo.
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Specific DNA sequences from several DNase I hypersensitive sites located upstream of the tyrosine aminotransferase (TAT) gene are bound by ubiquitous nuclear factors in vitro. Genomic footprinting has shown, however, that proteins are excluded from their potential binding sites in cells where the gene is inactive and that the absence of in vivo footprints is correlated with CpG methylation and altered chromatin structures at these sites. In vitro, interactions of proteins with sequences of the TAT gene, including binding of the transcription factor CREB to the cAMP-responsive element (CRE), are prevented by a methylated CpG dinucleotide in the respective binding sites, suggesting that methylation of DNA might be sufficient to exclude proteins from their sites in vivo. To test directly whether the absence of in vivo footprints is the result of DNA methylation, we treated two different cell lines with 5-azacytidine to demethylate CpG dinucleotides. While genomic sequencing confirmed demethylation at two widely separated regions upstream of the TAT promoter, no footprints appeared in these cell lines, even though proteins capable of binding these sites in vitro were present in the nuclei. Thus, the simple model whereby protein exclusion in vivo is caused solely by DNA methylation is not appropriate in this case. The nucleosomal organization of the potential binding sites suggests that chromatin structure is a dominant determinant in maintaining the inactive state of these sites. (+info)
Role of coenzyme in aminotransferase turnover.
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The role of coenzyme in determining intracellular contnet of pyridoxal enzymes was assessed by analyzing effects of pyridoxine deficiency on the rapidly degraded, readily dissociable tyrosine aminotransferase (EC 2.6.1.5) and the slowly degraded, nondissociable alanine aminotransferase (EC 2.6.1.2) of rat liver. Synthesis of the tyrosine enzyme was reduced, leading to a decreased amount of this enzyme, much of which was present as active apoenzyme. Synthesis of alanine aminotransferase was unchanged but much of this enzyme was present as an inactive apoenzyme which retained immunological reactivity. Degradation rates of both enzymes (t1/2 about 1.5 h, tyrosine aminotransferase; about 3 days, alanine aminotransferase) were not changed in pyridoxine deficiency. Hence, interaction with coenzyme is not a significant determinant in intracellular degradation of these aminotransferases. Coenzymes dissociation and intracellular stability probably reflect structural features of the proteins which determine both properties. (+info)
Partial purification and properties of frog liver tyrosine aminotransferase.
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Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5'-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics. (+info)
Mechanism of liver tyrosine aminotransferase increase in ethanol-treated mice and its effect on serum tyrosine level.
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Liver tyrosine aminotransferase (TAT) activity is known to increase with ethanol treatment; however, the mechanism of this increase is unclear. Upon investigation we found that TAT activity and mRNA levels started to increase 2 h after ethanol administration and continued to increase until 6 h after ethanol administration. The increase in ethanol-induced TAT activity could not be explained by calorie loading after fasting, since ethanol loading increased TAT expression, while glucose loading decreased TAT expression. In addition, liver TAT activity was not related to serum tyrosine levels. TAT activity increased when an adenosine A2 agonist, 5'-N-ethylcarboxamide adenosine, was given. Since TAT activity is increased by cAMP, and ethanol increases cAMP production via an adenosine receptor-dependent mechanism, this increase in ethanol-induced TAT activity may occur via an adenosine receptor-dependent mechanism. (+info)
Differential recruitment of glucocorticoid receptor phospho-isoforms to glucocorticoid-induced genes.
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Effect of dietary threonine supplementation on tyrosine toxicity in the rat.
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The objective of this study was to determine the effect of threonine supplementation on tyrosine metabolism in rats fed a low protein diet with excess tyrosine. The growth retardation and the development of eye and paw lesions that occur in rats ingesting a basal plus 3% or 5% L-tyrosine diet could be alleviated partially by the addition of 0.5% or 1.0% L-threonine to the diet. An increased blood tyrosine level in rats fed excess tyrosine was also lowered by threonine supplementation. In tyrotoxic conditions, the activities of liver tyrosine transaminase (EC 2.6.1.5) and threonine dehydratase (EC 4.2.9.16) were elevated, but p-hydroxyphenyl pyruvic acid oxidase (EC 1.13.11.27) which is also intimately associated with tyrosine toxicity was found to be inactivated. Furthermore, biosynthesis of ascorbic acid in liver was significantly lowered in this condition. However, addition of L-threonine in the diet, not only could cure the signs developed due to excess tyrosine, but also could affect the levels of enzymes studied. (+info)
A glucocorticoid-resistant rat hepatoma cell variant contains functional glucocorticoid receptor.
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The mechanism of glucocorticoid resistance was studied in a rat hepatoma cell variant (6.10.2) which contains low levels of glucocorticoid receptor. These cells seem to have lost glucocorticoid-induced transcriptional responses as measured by the induction of expression of stably integrated mouse mammary tumor virus gene and the endogenous tyrosine aminotransferase gene, as well as the transcriptional suppression of glucocorticoid receptor gene expression. However, characterization of the glucocorticoid resistance in 6.10.2 cells revealed that the receptor is indistinguishable from the wild-type receptor with respect to hormone binding and affinity for both nonspecific and specific DNA sequences. The levels of the receptor mRNA and the total immunoreactive protein found in 6.10.2 cells were about 20% of those found in wild-type cells. Further analysis of 6.10.2 cells demonstrated that the receptor was indeed biologically functional. First, treatment of 6.10.2 cells with 8-bromo-cAMP elevated the endogenous glucocorticoid receptor levels 2-fold and restored responsiveness to glucocorticoids. Second, pretreatment of the cells with cycloheximide also led to acquisition of cellular responsiveness to glucocorticoids. We propose that there exists a "threshold" level of glucocorticoid receptor which is required for responsiveness and that under normal culture conditions, the level of glucocorticoid receptor in 6.10.2 cells is below this threshold. However, glucocorticoid responsiveness can be restored by raising the glucocorticoid receptor level above the threshold with 8-bromo-cAMP or, alternatively, by removing the threshold barrier with cycloheximide. (+info)
Chromatin structures of the rat tyrosine aminotransferase gene relate to the function of its cis-acting elements.
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The relationship between DNase I-hypersensitive sites (HSs) and transcriptional enhancers of the rat tyrosine aminotransferase (TAT) gene was examined by comparing HSs in and around the TAT gene with the activity of the corresponding DNA sequences in transient transfection assays. In this manner, we identified two HSs as liver-specific enhancers. Of three hepatoma cell lines examined, only one sustained TAT mRNA levels comparable to those of liver. In this cell line, both enhancers were strongly active, and strong hypersensitivity in chromatin over the enhancers was evident. The other two hepatoma cell lines had reduced levels of TAT mRNA and no or altered hypersensitivity over either the enhancers or the promoter. One of these lines carried a negative regulator of the TAT gene, the tissue specific extinguisher Tse-1. This cell line exhibited all HSs characteristic of the strongly active gene except at the promoter; however, one enhancer was inactive even though hypersensitive in chromatin. In a TAT-nonexpressing cell line, inactivity of both enhancers correlated with absence of the respective HSs. We conclude that although hypersensitivity in chromatin necessarily accompanies cell-type-specific enhancer activity, the occurrence of cell-type-specific HSs does not imply that the underlying sequences harbor enhancers active in transient transfection assays. (+info)