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

---

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

A primary rabbit kidney epithelial cell culture system has been developed which retains differentiated functions of the renal proximal tubule. In addition, the cells have a distinctive metabolism and spectrum of hormone responses. The primary cells were observed to retain in vitro a Na+-dependent sugar transport system (distinctive of the proximal segment of the nephron) and a Na+-dependent phosphate transport system. Both of these transport processes are localized on the apical membrane of proximal tubule cells in vivo. In addition, probenicid-sensitive p-aminohippurate (PAH) uptake was observed in basolateral membranes of the primary tubule cells, and the PAH uptake by these vesicles occurred at a rate that was very similar to that observed with membranes derived from the original tissue. Several other characteristics of the primary cells were examined, including hormone-sensitive cyclic AMP production and phosphoenolpyruvate carboxykinase (PEPCK) activity. Like the cells in vivo, the primary proximal tubule cells were observed to produce significant cyclic AMP in response to parathyroid hormone, but not in response to arginine vasopressin or salmon calcitonin. Significant PEPCK activity was observed in the particulate fraction derived from a homogenate of primary rabbit kidney proximal tubule cells.
...
PMID:Primary rabbit kidney proximal tubule cell cultures maintain differentiated functions when cultured in a hormonally defined serum-free medium. 255 82

The 5' flanking regions of the rat phosphoenolpyruvate carboxykinase gene were used to form chimeric gene constructs with the human growth hormone gene. These constructs were transfected into several renal and one liver cell line and the production of growth hormone (HGH) measured by immunoassay. Cyclic-AMP and glucocorticoid responsiveness of HGH production was observed in all cell lines. In two lines, the rat NRK52E renal epithelial line and the rat H4IIE hepatoma cell line, both capable of expressing PEPCK, lowering extracellular pH increased HGH production several fold. Comparison of hormone and pH effect on cells transfected with a thymidine kinase promoter-HGH chimera indicated that the PEPCK 5' flanking region effects were specific. Thus, part of the pH responsiveness of the PEPCK gene in vivo may be attributed to properties of the 5' flanking regions.
...
PMID:The 5'region of the rat phosphoenolpyruvate carboxykinase gene confers pH sensitivity to chimeric genes expressed in renal and liver cell lines capable of expressing PEPCK. 255 24

We used indirect end labeling to identify a series of five hypersensitive (HS) sites in the phosphoenolpyruvate carboxykinase (PEPCK) gene in H4IIE rat hepatoma cells. These sites were found at -4800 base pairs (bp) (site A), at -1300 bp (site B), over a broad domain between -400 and -30 bp (site C), at +4650 bp (site D), and at +6200 bp (site E). Sites A to D were detected only in cells capable of expressing the PEPCK gene, whereas site E was present in all of the cells examined thus far. The HS sites were present in H4IIE cells even when transcriptional activity was reduced to a minimum by treatment with insulin. Stimulation of transcription by a cyclic AMP analog to a 40-fold increase over the insulin-repressed level did not affect the main features of the HS sites. Furthermore, increased transcription did not disrupt the nucleosomal arrangement of the coding region of the gene, nor did it affect the immediate 5' region (site C), which is always nucleosome-free. In HTC cells, a rat hepatoma line that is hormonally responsive but unable to synthesize PEPCK mRNA, the four expression-specific HS sites were totally absent. Our experimental results also showed that, although there is a general correlation between lack of DNA methylation and transcriptional competence of the PEPCK gene, the role, if any, of methylation in the regulation of PEPCK gene activity is likely to be exerted at very specific sites.
...
PMID:Hormonal regulation of phosphoenolpyruvate carboxykinase gene expression is mediated through modulation of an already disrupted chromatin structure. 265 89

Previous work carried out in our laboratory (Burlini, N., Lamponi S., Radrizzani, M., Monti, E. and Tortora P. (1987) Biochim. Biophys. Acta 930, 220-229) led to the immunological identification of a yeast 65-kDa phosphoprotein as a modified form of phosphoenolpyruvate carboxykinase; moreover the appearance of this phospho form was proven to be independent of cAMP, whereas the glucose-induced inactivation of the native enzyme is cAMP-dependent. Here, we report further investigations on the mechanism of the glucose-triggered degradation of the enzyme which led to the following results: (a) the aforementioned phospho form displayed a binding pattern to 5 AMP-Sepharose 4B quite similar to that of native enzyme, although it did not retain its oligomeric structure, nor was it catalytically active; (b) its phosphate content was of about two residues per monomer; (c) its isoelectric point was slightly higher than that of native enzyme, this shows that the enzyme undergoes additional modifications besides phosphorylation; (d) it represented about 4% of the native enzyme in glucose-depressed cells; (e) other forms immunologically cross-reactive with the native enzyme were also isolated, whose molecular mass was in the range of 60-62 kDa, and they are probable candidates as degradation products of the phospho form; (f) time courses of the native and phospho forms in the presence and the absence of glucose provided data consistent with a kinetic model involving a strong stimulation of the decay of both forms effected by the sugar; (g) in the mutant ABYS1 (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343) which is devoid of the four major vacuolar proteinases, the decay pattern was essentially the same as in wild-type; (h) effectors lowering intracellular ATP also retarded the first step of enzyme degradation; this points to an ATP-dependence of this step. Based on these results we propose a degradation mechanism consisting of an initial cAMP- and ATP-dependent modification of the enzyme, followed by a cAMP-independent phosphorylation, which leads to the appearance of the aforementioned monomeric phospho form; this in turn seems to undergo limited proteolysis. These data strongly suggest the occurrence of an intermediate form arising from the native one and whose phosphorylation gives rise to the 65-kDa phosphoprotein described here.
...
PMID:Studies on the degradative mechanism of phosphoenolpyruvate carboxykinase from yeast Saccharomyces cerevisiae. 268 77

The effects of phosphate and several phosphate-containing compounds on the activity of purified phosphoenolpyruvate carboxylase (PEPC) from the crassulacean acid metabolism plant, Crassula argentea, were investigated. When assayed at subsaturating phosphoenolpyruvate (PEP) concentrations, low concentrations of most of the compounds tested were found to stimulate PEPC activity. This activation, variable in extent, was found in all cases to be competitive with glucose 6-phosphate (Glc-6-P) stimulation, suggesting that these effectors bind to the Glc-6-P site. At higher concentrations, depending upon the effector molecule studied, deactivation, inhibition, or no response was observed. More detailed studies were performed with Glc-6-P, AMP, phosphoglycolate, and phosphate. AMP had previously been shown to be a specific ligand for the Glc-6-P site. The main effect of Glc-6-P and AMP on the kinetic parameters was to decrease the apparent Km and increase Vmax/Km. AMP also caused a decrease in the Vmax of the reaction. In contrast, phosphoglycolate acted essentially as a competitive inhibitor increasing the apparent Km for PEP and decreasing Vmax/Km. Inorganic phosphate had a biphasic effect on the kinetic parameters, resulting in a transient decrease in Km followed by an increase of the apparent Km for PEP with increasing concentration of phosphate. The Vmax also was decreased with increasing phosphate concentrations. Further, the enzyme appeared to respond to the complex of phosphate with magnesium. In the presence of a saturating concentration of AMP, no activation but rather inhibition was observed with increasing phosphate concentration. This is consistent with the binding of phosphate to two separate sites--the Glc-6-P activation site and an inhibitory site, a phenomenon that may be occurring with other phosphate containing compounds. High concentrations of phosphate with magnesium were found to protect enzyme activity when PEPC, previously shown to contain an essential arginine at the active site, was incubated with the specific arginyl reagent 2,3-butanedione, consistent with the binding of phosphate at the active site. Data were successfully fitted to a rapid equilibrium model allowing for binding of the phosphate-magnesium complex to both the activation site and the active site which accounts for the activation/deactivation observed at low substrate concentrations. Effects on the Vmax of the reaction are also addressed. Factors controlling the differential affinity of various effectors to the active site or activation site appear to include charge distribution, size, and other steric factors.
...
PMID:A kinetic study of the effects of phosphate and organic phosphates on the activity of phosphoenolpyruvate carboxylase from Crassula argentea. 271 76

The aim of this work was to find by which mechanisms an increased availability of plasma free fatty acids (FFA) reduced carbohydrate utilization during exercise. Rats were fed high-protein medium-chain triglycerides (MCT), high-protein long-chain triglycerides (LCT), carbohydrate (CHO) or high-protein low-fat (HP) diets for 5 weeks, and liver and muscle glycogen, gluconeogenesis and FFA oxidation were studied in rested and trained runner rats. In the rested state the hepatic glycogen store was decreased by fat and protein feeding, whereas soleus muscle glycogen concentration was only affected by high-protein diets. The percentage decrease in liver and muscle glycogen stores, after running, was similar in fat-fed, high-protein and CHO-fed rats. The fact that plasma glucose did not drastically change during exercise could be explained by a stimulation of hepatic gluconeogenesis: the activity of phosphoenolpyruvate carboxykinase (PEPCK) and liver phosphoenolpyruvate (PEP) concentration increased as well as cyclic adenosine monophosphate (AMPc) while liver fructose 2,6-bisphosphate decreased and plasma FFA rose. In contrast, the stimulation of gluconeogenesis in rested HP-, MCT- and LCT-fed rats appears to be independent of cyclic AMP.
...
PMID:Effects of long-term feeding of high-protein or high-fat diets on the response to exercise in the rat. 273 29

Dexamethasone is necessary and sufficient to induce mRNA for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) by 19-fold in rat hepatocytes cultured in serum-free medium. However, the time required for maximum induction is 16 h. The slow induction suggested that glucocorticoids regulate the expression of an intermediate gene product(s) which is required for glucocorticoid stimulation of PEPCK-gene expression. Consistent with this notion was the finding that cycloheximide completely blocked the response to dexamethasone. In contrast, cycloheximide did not block the response to a cyclic AMP analogue.
...
PMID:Induction of mRNA for phosphoenolpyruvate carboxykinase (GTP) by dexamethasone in cultured rat hepatocytes requires on-going protein synthesis. 282 94

Changes in plasma glucose, hepatic cyclic AMP, glycogen and fructose 2,6-bisphosphate (F-2,6-P2), and liver 6-phosphofructo-2-kinase (6-PF-2kinase), fructose 2,6-bisphosphatase (F-2,6-P2ase) and phosphoenolpyruvate carboxykinase (PEPCK) activities were examined in rats fed a low protein, high carbohydrate (HC) diet during 3 d of either starvation or feeding a high protein, carbohydrate-free (HP) diet. Under both HP feeding or starvation, liver cyclic AMP increased after 1 d and remained constant thereafter. Whereas plasma glucose was low during starvation, it was unaffected by HP feeding. In both experimental groups, liver glycogen fell after 1 d; thereafter it remained low on starvation, but increased progressively on HP diet reaching 70% of the HC-fed rats value on day 3. Under both experimental conditions, F-2,6-P2 fell 85% after day 1 and was unchanged thereafter. One day after the start of starvation or consumption of the HP diet, 6-PF-2kinase decreased, F-2,6-P2ase increased and 6-PF-2kinase/F-2,6-P2ase ratio decreased, but changes were significantly more important with the HP diet than with starvation. PEPCK activity increased in both experimental conditions, but the increase was greater on the HP diet than on starvation. These findings suggest that during the first 3 d the adaptative response of hepatic gluconeogenesis is higher with a HP diet than upon starvation.
...
PMID:Changes in rat hepatic fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase activity during three days of consumption of a high protein diet or starvation. 282 29

It is now well established that cAMP induces the transcription rate of the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) and that this induction is dependent on a nucleotide domain located within the promoter-regulatory region of the gene (Short, J. M., Wynshaw-Boris, A., Short, H. P., and Hanson, R. W. (1986) J. Biol. Chem. 261, 9721-9726). We report here that cAMP also stabilizes phosphoenolpyruvate carboxykinase mRNA against degradation. Using two independent experimental approaches, we show that the half-life of the mRNA for phosphoenolpyruvate carboxykinase is extended when FTO-2B rat hepatoma cells are exposed to dibutyryl cyclic AMP (Bt2cAMP). In the first experiment, the rate of decay of phosphoenolpyruvate carboxykinase mRNA was determined in cells incubated in the presence of insulin, which has been shown to block the transcription rate of the gene for the enzyme. Under these conditions, the half-life of phosphoenolpyruvate carboxykinase mRNA was 30 min. However, in cells incubated in the presence of Bt2cAMP, the mRNA decayed with a half-life of 150 min. In the other experiment, mRNA stability was measured under steady state conditions, utilizing a "pulse-chase" approach. The apparent half-life of phosphoenolpyruvate carboxykinase mRNA increased from 40 min to over 250 min in Bt2cAMP-treated cells. No significant change in the stability of total cellular RNA was noted. Other experiments have shown that the transcription rate of the gene for phosphoenolpyruvate carboxykinase peaks within the first 20 min after exposing the cells to Bt2cAMP and then levels off, while the abundance of the mRNA reaches a maximum at about 90 min and remains at this level thereafter. Thus, the long term effect of cAMP on the expression of the gene coding for phosphoenolpyruvate carboxykinase occurs at least in part, through an alteration in the degradation rate of the mRNA for this enzyme.
...
PMID:Cyclic AMP stabilizes the mRNA for phosphoenolpyruvate carboxykinase (GTP) against degradation. 283 95

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.
...
PMID:Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene. 285 Apr 95


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---