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:4.1.1.32 (phosphoenolpyruvate carboxykinase)
4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dexamethasone rapidly stimulated transcription of the tyrosine aminotransferase and metallothionein-I genes--but not of the phosphoenolpyruvate carboxykinase gene--in rat hepatocytes cultured in serum-free medium. This differential response was not observed for cyclic AMP. The results suggest that the phosphoenolpyruvate carboxykinase gene--but not the tyrosine aminotransferase and metallothionein-I genes--requires a factor which is permissive for stimulation of transcription by the glucocorticoid receptor.
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PMID:Differential induction of transcription for glucocorticoid-responsive genes in cultured rat hepatocytes. 196 35

We have examined the binding of factors in rat liver nuclear extracts to the phosphoenolpyruvate carboxykinase (PEPCK) gene cyclic AMP (cAMP) response element (CRE) and other CREs and have isolated a rat liver CRE-binding protein (CREBP) cDNA. In addition, we have examined the influence of altering the phosphorylation state of nuclear factors on both CRE binding and in vitro transcription. Specific binding to the PEPCK CRE was measured in a mobility shift assay. CRE sequences of the PEPCK, somatostatin, and glycoprotein hormone alpha subunit genes competed equally for binding of rat liver nuclear factors to the PEPCK CRE, whereas mutant PEPCK CRE sequences did not compete for binding. Oligonucleotides complementary to rat pheochromocytoma CREBP (Gonzalez et al., Nature [London] 337:749-752, 1989) were used to prime rat liver and brain cDNA in the polymerase chain reaction. The predominant CREBP molecule obtained was identical to the rat pheochromocytoma CREBP except for a 14-amino-acid deletion in the N-terminal half that was also present in a human placental cDNA (Hoeffler et al., Science 242:1430-1433, 1988). The regulation of transcription by cAMP was examined by coincubation of rat liver nuclear extract with the purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A). Although binding to the CRE was unaffected, in vitro transcription directed by the PEPCK promoter was stimulated by catalytic subunit, and this effect was blocked by protein kinase inhibitor peptide. In contrast, when nuclear extract was coincubated with phosphatase, there was substantial inhibition of in vitro transcription directed by the PEPCK promoter, but there was no effect on binding to the CRE. The major effects of catalytic subunit were exerted through the CRE, but residual stimulation was evident in promoter fragments containing only the TATA element. These data suggest that factors are bound to the CRE at constitutively high levels and that their capacity for transcriptional activation is regulated by phosphorylation.
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PMID:Cyclic AMP-dependent protein kinase regulates transcription of the phosphoenolpyruvate carboxykinase gene but not binding of nuclear factors to the cyclic AMP regulatory element. 214 84

Previous studies have identified a region in the promoter of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (positions -460 to +73) containing the regulatory elements which respond to cyclic AMP, glucocorticoids, and insulin and confer the tissue- and developmental stage-specific properties to the gene. We report that CCAAT/enhancer-binding protein (C/EBP) binds to the cyclic AMP-responsive element CRE-1 as well as to two regions which have been previously shown to bind proteins enriched in liver nuclei. The DNase I footprint pattern provided by the recombinant C/EBP was identical to that produced by a 43-kDa protein purified from rat liver nuclear extracts, using a CRE oligonucleotide affinity column, which was originally thought to be the CRE-binding protein CREB. Transient contransfection experiments using a C/EBP expression vector demonstrated that C/EBP could trans activate the PEPCK promoter. The trans activation occurred through both the upstream, liver-specific protein-binding domains and the CRE. The CRE-binding protein bound only to CRE-1 and not to the upstream C/EBP-binding sites. The results of this study, along with physiological properties of C/EBP, indicate an important role for this transcription factor in providing the PEPCK gene with several of its regulatory characteristics.
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PMID:The role of the CCAAT/enhancer-binding protein in the transcriptional regulation of the gene for phosphoenolpyruvate carboxykinase (GTP). 214 22

The mixed anhydride of oxalic and phosphoric acids, oxalyl phosphate, has been prepared by reaction of oxalyl chloride and inorganic phosphate in aqueous solution. The product was purified by anion exchange chromatography and characterized by 31P and 13C NMR. This acyl phosphate has a half-life of 51 h at pH 5.0 and 4 degrees C. Oxalyl phosphate, an analogue of phosphoenolpyruvate, is a slow substrate for pyruvate kinase, undergoing an enzyme-dependent phosphotransfer reaction to produce ATP from ADP. Oxalyl phosphate substitutes for phosphoenolpyruvate in the reaction catalyzed by pyruvate, phosphate dikinase. The acyl phosphate reacts with the free enzyme to give the phosphorylated form of the enzyme. Removal of the potent product inhibitor, oxalate, from the reaction mixtures by gel filtration chromatography permitted further reaction of the phosphorylated enzyme with pyrophosphate and AMP to give ATP and Pi in a single turnover assay. Oxalyl phosphate also served as a phospho group donor in a partial reaction catalyzed by phosphoenolpyruvate carboxykinase wherein GDP is phosphorylated at the expense of oxalyl phosphate.
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PMID:Synthesis of oxalyl phosphate and processing of the acyl phosphate by phosphoenolpyruvate-dependent enzymes. 216 54

Vanadate, at concentrations between 0.5 and 2 mM, rapidly decreased the basal level of P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA and blocked the dibutyryl cyclic AMP (Bt2cAMP)-induced increase in enzyme mRNA in both FTO-2B and H4IIE rat hepatoma cells. The concentration of vanadate necessary to inhibit the expression of this gene was similar to that required for the vanadate-mediated activation of the insulin receptor tyrosine kinase. To determine whether vanadate could inhibit PEPCK gene transcription, a series of chimeric genes containing several deletions in the P-enolypyruvate carboxykinase promoter between -550 and -68 was linked to the structural genes for either amino-3-glycosyl phosphotransferase (neo) or chloramphenicol acetyltransferase and introduced into hepatoma cells using three methods: (a) infection with a Moloney murine leukemia virus-based retrovirus, (b) transfection and stable selection for neo expression, or (c) transient expression of chloroamphenicol acetyltransferase. In FTO-2B hepatoma cells infected with retrovirus, vanadate rapidly (within 1 h) inhibited transcription of the PEPCK-neo gene and blocked induction of gene expression caused by the addition of either Bt2cAMP or dexamethasone to the cells. Vanadate was not a general transcription inhibitor since, it like insulin, stimulated the expression of the c-fos gene. Also, the inhibitory effect of vanadate was rapidly reversible in FTO-2B cells since PEPCK gene expression could be stimulated by Bt2cAMP and dexamethasone after removal of vanadate. A series of 5' deletions in the P-enolpyruvate carboxykinase promoter (-550 to +73) was ligated to the structural gene for neo and stably transfected into hepatoma cells. Sequences responsive to vanadate were detected between -109 and -68. This result was confirmed using H4IIE hepatoma cells transiently expressing the PEPCK-CAT gene. The most likely target for vanadate in that region of the P-enolpyruvate carboxykinase promoter is cAMP regulatory element 1 which maps from -91 to -84. A comparison of the inhibitory effects of insulin and vanadate in this system indicated a major difference in the site of action of these two compounds on PEPCK gene transcription.
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PMID:Vanadate inhibits expression of the gene for phosphoenolpyruvate carboxykinase (GTP) in rat hepatoma cells. 216 40

Genetics is a powerful tool for analyzing metabolic regulation in bacteria, permitting definitive identification of key regulatory enzymes, and assignment of cause and effect relationships between molecular mechanisms that control activity of regulatory enzymes and flow of metabolites through the pathways of intermediary metabolism. In homeothermic vertebrates, however, traditional genetic analysis of metabolic regulation is difficult or impossible. Despite an enormous amount of factual information about metabolic pathways in vertebrates, our understanding of regulatory mechanisms is based largely on correlations between regulation of the activity of a purified enzyme by an effector and variations in the intracellular concentration of that effector under conditions that modulate flow of metabolites through the relevant metabolic pathway. Newly developed molecular genetic and gene transfer methods can now be used to study metabolic regulation in vertebrates. Some of these methods will be described in short reviews of the mechanisms by which diet, hormones, and tissue-type regulate transcription of the genes for L-type pyruvate kinase and phosphoenolpyruvate carboxykinase. Application of these techniques to analyses of structure/function in intact cells and animals will be illustrated with recent work on the receptor for low density lipoproteins and cyclic AMP-dependent protein kinases.
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PMID:The new metabolism: molecular genetics in the analysis of metabolic regulation. 221 Jan 56

A cell-free system for the study of transcription from the promoter of the phosphoenolpyruvate carboxykinase (GTP) gene by using nuclear extracts from rat tissues was developed. The level of basal transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter between -490 and +73 was highest when extracts from liver nuclei, rather than kidney, spleen, and HeLa nuclear extracts, were used. A series of 5' deletions and block mutations were also tested for their effects on basal transcription in vitro. The promoter truncated to -355 had the highest rate of basal transcription, while subsequent deletion to -277 markedly decreased the rate of transcription. Further deletion of the promoter to -134 resulted in a twofold increase in the basal level of transcription compared with that of the promoter deleted to -277. However, subsequent deletion of the NF-1-CCAAT-binding transcription factor binding site or the proximal cyclic AMP (cAMP) regulatory element caused a decrease in basal transcription. Block mutations were inserted into nine specific protein-binding regions of the PEPCK promoter previously shown to be of functional significance or to bind nuclear proteins. Mutation of the TATA box resulted in a 94% decrease in the level of transcription noted with the intact promoter, while sequence substitutions within the proximal cAMP regulatory element decreased the transcription rate to 25%. The addition of the catalytic subunit of cAMP-dependent protein kinase to the in vitro system stimulated transcription from the intact promoter or from a promoter deletion to -109. However, a promoter deletion to -68, which removes the proximal cAMP regulatory element, was unresponsive to added protein kinase catalytic subunit. These findings indicate that the PEPCK promoter between -490 and +73 contains sequences responsive to hormonal and tissue-specific factors in nuclei from rat tissues. The sensitivity of this in vitro transcription system closely mimics the process regulating PEPCK transcription in rat tissues and should make it ideal for testing the function of purified transcription factors.
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PMID:In vitro analysis of promoter elements regulating transcription of the phosphoenolpyruvate carboxykinase (GTP) gene. 230 49

We have analyzed the chromatin structure of the phosphoenolpyruvate carboxykinase (PEPCK) gene in hepatoma x fibroblast hybrids with different extinction phenotypes. These hybrids included a karyotypically complete hybrid in which all liver gene activity was extinguished, a microcell hybrid that contained a single mouse chromosome 11 and in which PEPCK gene activity was decreased but inducible by cyclic AMP, and a segregant line that had lost all mouse chromosomes and in which the PEPCK gene was reexpressed. We found that only in the completely extinguished hybrid was PEPCK chromatin structure radically different from that in the parental hepatoma cells. In this hybrid, there was no evidence of any factors binding to the promoter or to the upstream hypersensitive site at -4800 base pairs. In the other cell lines, even when PEPCK gene transcription was low, the PEPCK chromatin showed characteristic structures typical of a transcriptionally competent gene, with hypersensitive sites at positions previously described. Loss of the upstream hypersensitive site was also shown to be correlated with the absence of a liver-specific protein factor that binds specifically to the upstream region.
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PMID:Extinction of phosphoenolpyruvate carboxykinase gene expression is associated with loss of a specific chromatin-binding protein from a far upstream domain. 235 23

Glucocorticosteroid, thyroid hormones and cyclic AMP can induce the synthesis of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of hepatocytes as soon as these cells differentiate from the embryonic foregut. The low levels of both enzymes that can accumulate in such still protodifferentiated hepatocytes are due to low levels of enzyme synthesis. In cultures, the rate of synthesis of both enzymes increases continually in the presence of hormones, showing that maturation of the capacity for synthesis towards the postnatal, fully differentiated situation is occurring in these cells. The turnover rate of both enzymes in embryonic hepatocytes is lower in the presence of hormones than in the absence, but does not change during the culture period. In the presence of hormones the turnover rate is comparable to that found in adult rat liver in vivo. The development of the capacity to accumulate organ-specific enzymes in vitro (and hence the rate of enzyme synthesis) is found to be comparable to that in utero.
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PMID:Synthesis, accumulation and turnover of carbamoylphosphate synthetase and phosphoenolpyruvate carboxykinase in cultures of embryonic rat hepatocytes. 243 10

Hepatocytes isolated from adult fasted rats and cultured in the presence of thyroid hormones, glucocorticoids, and in a serum-free medium conserve the essentials of their differentiated function and hormonal sensitivity for at least 1 week. In these cells, the gene for L-type pyruvate kinase is expressed only when glucose and insulin are present together, each of them being inactive by itself. Inhibition of the expression of the L-type pyruvate kinase gene which occurs when glucose and/or insulin are removed from the culture medium is not associated with accumulation of the phosphoenolpyruvate carboxykinase mRNA, which argues against the involvement of intracellular cyclic AMP in this phenomenon. Rather, a transcriptional activator, derived from carbohydrate metabolism and accumulating in the presence of insulin, seems to be needed to support the expression of the L-type pyruvate kinase gene. Glucagon, in vitro as in vivo, inhibits production of the L-type pyruvate kinase mRNAs. In addition to their roles on the production of these mRNAs, glucose and insulin on the one hand and glucagon on the other have profound effects on the stability of the L-type pyruvate kinase messengers: the half-life of the mRNA whose production has been blocked by actinomycin D is 1 h in the presence of glucagon and 24 h in the presence of glucose and insulin. Glucagon and glucose/insulin partially antagonize each other's effect on mRNA stability.
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PMID:Regulation of the expression of the L-type pyruvate kinase gene in adult rat hepatocytes in primary culture. 254 75


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