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)

To investigate a possible chromosomal clustering of glycolytic enzyme genes in Corynebacterium glutamicum, a 6.4-kb DNA fragment located 5' adjacent to the structural phosphoenolpyruvate carboxylase (PEPCx) gene ppc was isolated. Sequence analysis of the ppc-proximal part of this fragment identified a cluster of three glycolytic genes, namely, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene gap, the 3-phosphoglycerate kinase (PGK) gene pgk, and the triosephosphate isomerase (TPI) gene tpi. The four genes are organized in the order gap-pgk-tpi-ppc and are separated by 215 bp (gap and pgk), 78 bp (pgk and tpi), and 185 bp (tpi and ppc). The predicted gene product of gap consists of 336 amino acids (M(r) of 36,204), that of pgk consists of 403 amino acids (M(r) of 42,654), and that of tpi consists of 259 amino acids (M(r) of 27,198). The amino acid sequences of the three enzymes show up to 62% (GAPDH), 48% (PGK), and 44% (TPI) identity in comparison with respective enzymes from other organisms. The gap, pgk, tpi, and ppc genes were cloned into the C. glutamicum-Escherichia coli shuttle vector pEK0 and introduced into C. glutamicum. Relative to the wild type, the recombinant strains showed up to 20-fold-higher specific activities of the respective enzymes. On the basis of codon usage analysis of gap, pgk, tpi, and previously sequenced genes from C. glutamicum, a codon preference profile for this organism which differs significantly from those of E. coli and Bacillus subtilis is presented.
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PMID:Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase. 140 Jan 58

Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose-degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo-D-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm-1 in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.
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PMID:Characterization of carbohydrate metabolism and demonstration of glycosomes in a Phytomonas sp. isolated from Euphorbia characias. 143 59

It has been widely believed that bloodstream forms of Trypanosoma brucei must be first transformed into intermediary and/or short-stumpy forms in the bloodstream of the mammalian host before differentiation to the procyclic culture form can occur. In our recent studies, the pleomorphic T. brucei strain TREU667 was found to differentiate directly from the long-slender bloodstream form to the procyclic form in Cunningham's medium at 26 degrees C [7]. In the present investigation, the same was found true for another pleomorphic strain of T. brucei, STIB366D. Four independent monomorphic strains of T. brucei were tested; two, #427 and EATRO164, were found capable of differentiating in vitro directly into procyclic forms, whereas the other two, TREU667/RP-56 and EATRO110, could not. There is thus no correlation between the capability of differentiating in vitro and the ability of being converted from long-slender to intermediary and short-stumpy bloodstream forms. Two additional markers for following differentiation, other than observing morphological changes, were tested. Assays for the emerging phosphoenolpyruvate carboxykinase (PEPCK) by immunoblottings worked well, with results agreeing closely with the morphological change. But immunoblottings of glycosomal phosphoglycerate kinase (gPGK) failed to demonstrate a significant decrease in the protein level upon completion of differentiation. Apparently, gPGK has a rather long half-life and is unsuitable as a marker of differentiation. When temperature was dropped from 37 degrees C to 26 degrees C at the starting point of in vitro differentiation, protein synthetic activity in the pleomorphic T. brucei TREU667 bloodstream form was decreased by 4-fold. When the activity was gradually brought back to and beyond the original level after a day's incubation, the profile of newly synthesized proteins was that of the procyclic form. A monomorphic variant of TREU667, RP-56, which is incapable of differentiating in vitro, has a much higher protein synthetic activity than its pleomorphic parent in the bloodstream form. This high activity and the bloodstream profile of proteins thus synthesized were unaffected by the decreased temperature in Cunningham's medium until cell death. We thus conclude that a general inhibition of protein synthesis in bloodstream forms caused by temperature drop may be among the early events triggering differentiation into the procyclic form.
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PMID:Transient inhibition of protein synthesis accompanies differentiation of Trypanosoma brucei from bloodstream to procyclic forms. 147 91

Optimal concentrations of the essential components for analyzing the activity of each enzyme associated with glycolysis and gluconeogenesis in rabbit periodontal ligament were examined, and enzyme assay systems for 15 enzymes including 22 reactions were established using triethanolamine buffer. Specific activities of all the enzymes, except for the gluconeogenic reaction of phosphoglycerate kinase, were systematically evaluated using the optimum buffer for each enzyme, since the activity of each enzyme varied depending on the buffer used. For glycolysis, the activity levels of hexokinase and 6-phosphofructokinase were very low, and consequently these enzyme reactions were inferred to be the rate-limiting steps. For gluconeogenesis, fructose 1,6-bisphosphatase and aldolase activities were extremely low, and the activities of glucose 6-phosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase were undetectable. These results suggest that the periodontal ligament may have no gluconeogenesis capability. With a rise in pH, the activities of the key enzymes of glycolysis gradually increased, and a specific "crossover" point was found between the activities of glyceraldehyde-phosphate dehydrogenase and phosphoglyceromutase. In addition, the activity of fructose 1,6-bisphosphatase, one of the key enzymes of gluconeogenesis, was markedly increased with a rise in pH, although pH changes had no effect on aldolase activity. Consequently, alkaline pH appeared to result in overall stimulation of glycolysis.
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PMID:Enzymatic regulation of glycolysis and gluconeogenesis in rabbit periodontal ligament under various physiological pH conditions. 165 53

A cell-free system prepared from rat liver containing cytosol and mitochondria as well as a number of cofactors and gluconeogenic intermediates at near-physiological concentrations was shown to form hexose 6-phosphates linearly from lactate + pyruvate + glutamate at a rate of 0.82 +/- 0.05 mumol/min per g of liver (mean +/- S.E.M., n = 8, 37 degrees C). The indicated rates were measured between 20 min and 60 min incubation time, when the system was near steady state. Experiments with either [1-14C]lactate or [U-14C]glutamate revealed that the incorporation of radioactive label into hexose 6-phosphates was proportional to the utilization of lactate + pyruvate and of glutamate during incubation and that both served as gluconeogenic substrates at a ratio of about 2:1. When the [ATP]/[ADP] ratio was lowered from 60 to 19 by addition of ATPase, the rate of hexose 6-phosphate formation fell to one-third. This decrease in gluconeogenic flux was mainly due to a decreased flow through the phosphoglycerate kinase step. Hexose 6-phosphate formation could also be decreased by increasing the ratio [NADH]/[NAD+], either by addition of ethanol or by increasing the initial concentration of lactate + pyruvate at a fixed ratio of 10:1. The observed inhibition was linked to a limitation in the availability of oxaloacetate for the phosphoenolpyruvate carboxykinase reaction and to an increased formation of sn-glycerol 3-phosphate. Finally, the rates of hexose 6-phosphate formation in incubations with cytosols from fed rats were only 50% of those observed with cytosols from animals starved for 48 h. One of the limiting steps was found to be the flow through the phosphoenolpyruvate carboxykinase step.
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PMID:Formation of hexose 6-phosphates from lactate + pyruvate + glutamate by a cell-free system from rat liver. 287 56

To evaluate published indications that about 25% of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), is located in mitochondria of adult rat liver, cell fractionations were conducted with hepatocytes isolated from rats that were fed ad libitum or starved for 2 days. Hepatocytes were exposed to digitonin for 10 s, and the released materials were separated from residual cell structures by centrifugation through a layer of brominated hydrocarbon. In addition to PEPCK, activities of 9 other enzymes were measured in the untreated cells and with good recovery in the two fractions obtained with digitonin treatment. By comparison with the release of marker enzymes for the cytosol and mitochondria, the subcellular distribution of PEPCK was determined. With cells from either fed or 2-day-starved rats, this enzyme was released exactly like lactate dehydrogenase and within 2-3% of phosphoglycerate kinase and pyruvate kinase. These results indicate that, even after induction by starvation, at least 97% of PEPCK activity is located in the cytosol of rat liver.
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PMID:Subcellular location of phosphoenolpyruvate carboxykinase in hepatocytes from fed and starved rats. 372 5

Evidence is presented for the occurrence of glycosomes (organelles resembling peroxisomes) in four major species of Leishmania (viz. L. major, L.m. mexicana, L. b. braziliensis and L. donovani), based on latency as well as differential and isopycnic centrifugation studies. The enzymes involved in glycolysis; (hexokinase, phosphoglucose isomerase, phosphofructokinase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase); glycerol metabolism (sn-glycerol-3-phosphate dehydrogenase and glycerol kinase); carbon dioxide fixation (phosphoenolpyruvate carboxykinase and possibly malate dehydrogenase); together with an enzyme involved in the beta-oxidation of fatty acids (3-beta-hydroxybutyryl coenzyme A dehydrogenase); a key enzyme in the synthesis of ether lipids (dihydroxyacetone phosphate acyltransferase) as well as the ADP utilising enzyme adenylate kinase, were all found associated, at least in part, with a subcellular organelle which had a buoyant density in sucrose gradients of 1.21 to 1.24 g cm-3. Little variance in enzyme composition was found between the different species of Leishmania or in comparison with other members of the Trypanosomatidae, supporting the unifying principle that glycosomes are a unique characteristic of this family. The occurrence of important catabolic, anabolic and anaplerotic pathways in the glycosomes of Leishmania renders them prime targets for chemotherapy.
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PMID:The occurrence of glycosomes (microbodies) in the promastigote stage of four major Leishmania species. 644 18

Highly purified glycosomes were isolated from Trypanosoma brucei bloodstream forms and cultured procyclic trypomastigotes. A comparison of the specific activities of glycosomal enzymes revealed that glycosomes from insect stages had decreased levels of hexokinase, phosphoglucose isomerase, phospho-fructokinase, fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, but contained increased levels of adenylate kinase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. Glycosomes from bloodstream forms were almost totally devoid of the latter two activities. Comparison of the two types of glycosomes by sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed that bloodstream form glycosomes contained 3 prominent polypeptides (64, 46 and 40 kDa) which were hardly detectable in insect stage glycosomes, whereas the latter contained 3 insect stage specific bands with molecular weight of 34 000, 61 000 and 77 000 and 4 additional bands with molecular weights between 94 000 and 110 000. Both types of glycosome contained the phospholipids phosphatidylcholine and phosphatidylethanolamine. Insect stage glycosomes contained in addition also phosphatidylinositol and some phosphatidylserine.
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PMID:A comparison of the glycosomes (microbodies) isolated from Trypanosoma brucei bloodstream form and cultured procyclic trypomastigotes. 674 87

Acyclovir diphosphate (acyclo-GDP) is a metabolite of the antiviral drug acyclovir [9-(2-hydroxyethoxymethyl)guanine]. Seven enzymes capable of catalyzing the phosphorylation of GDP and dGDP (nucleoside diphosphate kinase, pyruvate kinase, creatine kinase, phosphoglycerate kinase, succinyl-CoA synthetase, phosphoenolpyruvate carboxykinase and adenylosuccinate synthetase) also catalyzed the phosphorylation of acyclo-GDP. In general, acyclo-GDP had a lower V'max and a higher K'm than either GDP or dGDP. None of these enzymes showed significantly higher rates of phosphorylation with GDP or acyclo-GDP in herpes simplex virus-infected Vero cells as compared to uninfected Vero cells. The contribution of each enzyme to the phosphorylation of acyclo-GDP in vivo was estimated from the kinetic data from the partially purified enzymes, the level of each enzyme in Vero cells, and the physiological concentrations of both substrates and inhibitors. The relative order of estimated rates of acyclo-GDP phosphorylation in Vero cells was phosphoglycerate kinase much greater than pyruvate kinase greater than phosphoenolpyruvate carboxykinase greater than nucleoside diphosphate kinase greater than succinyl-CoA synthetase greater than creatine kinase greater than adenylosuccinate synthetase. The calculated potential for acyclo-GDP phosphorylation by these enzymes was adequate to account for the amounts of acyclo-GTP formed in cell culture.
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PMID:Phosphorylation of acyclovir diphosphate by cellular enzymes. 715 65

The in vitro differentiation of Trypanosoma brucei from bloodstream to procyclic (insect) forms is accompanied by diminishing variant surface glycoprotein (VSG) and increasing levels of procyclin and phosphoenolpyruvate carboxykinase (PEPCK). In this study, we examined the fate of several glycolytic enzymes of T. brucei during differentiation. We observed a down-regulation of glycosomal phosphoglycerate kinase (gPGK) during differentiation. In contrast, intracellular levels of glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH), aldolase (ALD), and phosphoglucoisomerase (PGI) remained unchanged during differentiation and apparently continued to be synthesized in the procyclic form. To determine the potential role of proteasomes and other proteases during the differentiation process, we tested the effect of lactacystin, a specific inhibitor of proteasome activity, and morpholinourea-Phe-homoPhe-benz-alpha-pyrone (P27), a selective inhibitor of cysteine proteases, on the in vitro differentiation of T. brucei. Cells differentiated normally in the presence of 1 microM lactacystin, which confirmed our previous observation that this differentiation does not require crossing any phase boundaries in the cell cycle (Mutomba and Wang, Mol Biochem Parasitol 1996;80:89-102). But the cells thus differentiated did not increase in number and retained gPGK. Cells differentiated under 2 microM P27 also proceeded at a normal rate but failed to multiply and retained gPGK. However, most of the differentiated cells under 2 microM P27 also retained VSG on the cell membrane surface and expressed higher levels of procyclin suggesting that a cysteine protease(s) may be involved in releasing VSG and partially reducing procyclin during differentiation. This cysteine protease(s) has been tentatively identified in the procyclic cells as a 48 kDa protein through labeling of cysteine protease(s) with a biotinylated P27 homolog K02 (morpholinourea-Phe-homoPhe-vinylsulfone).
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PMID:The role of proteolysis during differentiation of Trypanosoma brucei from the bloodstream to the procyclic form. 966 24


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