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Query: EC:4.1.1.49 (phosphoenolpyruvate carboxykinase)
 4,654 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An investigation was made of the respiratory properties and the role of the mitochondria isolated from one phosphoenolpyruvate carboxykinase (PCK)-CAM plant Ananas comosus (pineapple) in malate metabolism during CAM phase III. Pineapple mitochondria showed very high malate dehydrogenase (MDH), and low malic enzyme (ME) and glutamate-oxaloacetate transaminase (GOT) activities. The mitochondria readily oxidized succinate and NADH with high rates and coupling, while they only oxidized NADPH in the presence of Ca(2+). Pineapple mitochondria oxidized malate with low rates under most assay conditions, despite increasing malate concentrations, optimizing pH, providing cofactors such as coenzyme A, thiamine pyrophosphate, and NAD(+), and supplying individually external glutamate or GOT. However, providing glutamate and GOT simultaneously strongly increased the rates of malate oxidation. The OAA easily permeated the mitochondrial membranes to import into or export out of pineapple mitochondria during malate oxidation, but the mitochondria did not consume external Asp or alpha-KG. These results suggest that OAA played a significant role in the mitochondrial malate metabolism of pineapple, in which malate was mainly oxidized by active mMDH to produce OAA which could be exported outside the mitochondria via a malate-OAA shuttle. Cytosolic GOT then consumed OAA by transamination in the presence of glutamate, leading to a large increase in respiration rates. The malate-OAA shuttle might operate as a supporting system for decarboxylation in phase III of PCK-CAM pineapple. This shuttle system may be important in pineapple to provide a source of energy and substrate OAA for cytosolic PCK activity during the day when cytosolic OAA and ATP was limited for the overall decarboxylation process.
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PMID:Respiratory properties and malate metabolism in Percoll-purified mitochondria isolated from pineapple, Ananas comosus (L.) Merr. cv. smooth cayenne. 1536 38

Lactate and succinate were produced from glucose by Corynebacterium glutamicum under oxygen deprivation conditions without growth. Addition of bicarbonate to the reaction mixture led not only to a 3.6-fold increase in succinate production rate, but also to a 2.3- and 2.5-fold increase, respectively, of the rates of lactate production and glucose consumption, compared to the control. Furthermore, when small amounts of pyruvate were added to the reaction mixture, acid production rates and the glucose consumption rate were multiplied by a factor ranging from 2 to 3. These phenomena were paralleled by an increase in the NAD(+)/NADH ratio, thus corroborating the view that the efficient regeneration of NAD(+) could be triggered by the addition of either bicarbonate or pyruvate. To investigate the global metabolism of corynebacteria under oxygen deprivation conditions, we engineered several strains where the genes coding for key metabolic enzymes had been inactivated by gene disruption and replacement. A lactate dehydrogenase (LDH)-deficient mutant was not able to produce lactate, suggesting this enzyme has no other isozyme. Although a pyruvate carboxylase (pyc) mutant exhibited similar behavior to that of the wild type, phosphoenolpyruvate carboxylase (ppc) mutants were characterized by a dramatic decrease in succinate production, which was concomitant to decreased lactate production and glucose consumption rates. This set of observations corroborates the view that in coryneform bacteria under oxygen deprivation conditions the major anaplerotic reaction is driven by the ppc gene product rather than by the pyc gene product. Moreover, intracellular NADH concentrations in C. glutamicum were observed to correlate to oxygen-deprived metabolic flows.
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PMID:Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. 1538 16

Through a positional cloning approach, the thioredoxin-interacting protein gene (Txnip) was recently identified as causal for a form of combined hyperlipidemia in mice (Bodnar, J. S., A. Chatterjee, L. W. Castellani, D. A. Ross, J. Ohmen, J. Cavalcoli, C. Wu, K. M. Dains, J. Catanese, M. Chu, S. S. Sheth, K. Charugundla, P. Demant, D. B. West, P. de Jong, and A. J. Lusis. 2002. Positional cloning of the combined hyperlipidemia gene Hyplip1. Nat. Genet. 30: 110-116). We now show that Txnip-deficient mice in the fed state exhibit a metabolic profile similar to fasted mice, including increased levels of plasma ketone bodies and free fatty acids, decreased glucose, and increased hepatic expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and acyl-CoA oxidase. Dramatic differences in the expression of key metabolic enzymes were also observed in other tissues, and the fat-to-muscle ratio of Txnip-deficient mice was increased by approximately 40%. We demonstrate an effect of Txnip on the redox status, as the Txnip-deficient mice in the fed state had a significant increase in the ratio of NADH to NAD(+). Surprisingly, we observed that Txnip-deficient mice and wild-type mice had similar levels of thioredoxin activity, suggesting that the effects of Txnip deficiency may be mediated in part by other interactions. These results indicate a role for Txnip in the metabolic response to feeding and the maintenance of the redox status.
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PMID:Thioredoxin-interacting protein deficiency disrupts the fasting-feeding metabolic transition. 1552 Apr 47

The central metabolic pathway of Corynebacterium glutamicum was engineered to produce ethanol. A recombinant strain which expressed the Zymomonas mobilis genes coding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) was constructed. Both genes placed under the control of the C. glutamicum ldhA promoter were expressed at high levels in C. glutamicum, resulting, under oxygen-deprivation conditions, in a significant yield ofethanol from glucose in a process characterized by the absence of cellular growth. Addition of pyruvate in trace amounts to the reaction mixture induced a 2-fold increase in the ethanol production rate. A similar effect was observed when acetaldehyde was added. Disruption of the lactate dehydrogenase (ldhA) gene led to a 3-fold higher ethanol yield than wild type, with no lactate production. Moreover, inactivation of the phosphoenolpyruvate carboxylase (ppc) and ldhA genes revealed a significant amount of ethanol production and a dramatic decrease in succinate without any lactate production, when pyruvate was added. Since the reaction occurred in the absence of cell growth, the ethanol volumetric productivity increased in proportion to cell density of ethanologenic C. glutamicum in a process under oxygen-deprivation conditions. These observations corroborate the view that intracellular NADH concentrations in C. glutamicum are correlated to oxygen-deprived metabolic flows.
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PMID:Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions. 1617 1

Intercellular distribution of enzymes involved in amino nitrogen synthesis was studied in leaves of species representing three C(4) groups, i.e. Sorghum bicolor, Zea mays, Digitaria sanguinalis (NADP malic enzyme type); Panicum miliaceum (NAD malic enzyme type); and Panicum maximum (phosphoenolpyruvate carboxykinase type). Nitrate reductase, nitrite reductase, glutamine synthetase, and glutamate synthase were predominantly localized in mesophyll cells of all the species, except in P. maximum where nitrite reductase had similar activity on a chlorophyll basis, in both mesophyll and bundle sheath cells. NADH-glutamate dehydrogenase was concentrated in the bundle sheath cells, while NADPH-glutamate dehydrogenase was localized in both mesophyll and bundle sheath cells. The activities of nitrate-assimilating enzymes, except for nitrate reductase, were high enough to account for the proposed in vivo rates of nitrate assimilation.Based on the differential centrifugation of cell homogenates of P. miliaceum, mesophyll chloroplasts appear to be the major site of nitrate assimilation since nitrite reductase, glutamine synthetase, glutamate synthase, and NADPH-glutamate dehydrogenase were primarily localized in the chloroplast fraction. Both the glutamine synthetase-glutamate synthase and glutamate dehydrogenase pathways were considered as alternative routes of amino nitrogen synthesis.
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PMID:Distribution of Nitrate-assimilating Enzymes between Mesophyll Protoplasts and Bundle Sheath Cells in Leaves of Three Groups of C(4) Plants. 1665 90

Phosphoenolpyruvate partially inhibits the accumulation of Ca(2+) in isolated mung bean (Phaseolus aureus Roxb.) mitochondria. Succinate-supported Ca(2+) uptake is twice as sensitive to phosphoenolpyruvate inhibition as is NADH- or malate/pyruvate-supported Ca(2+) uptake. Pyruvate, atractylate, and ATP, but not ITP, reverse the phosphoenolpyruvate-induced inhibition. Oxaloacetic acid inhibits succinate-supported Ca(2+) uptake completely while partially inhibiting NADH-supported Ca(2+) uptake. The oxaloacetate inhibition of NADH-supported Ca(2+) uptake is greater than that produced by phosphoenolpyruvate. It is suggested that inhibition of Ca(2+) uptake is due to the conversion of phosphoenolpyruvate into oxaloacetate via phosphoenolpyruvate carboxykinase, with oxaloacetate responsible for the actual inhibition of Ca(2+) uptake.
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PMID:Effect of phosphoenolpyruvate and oxaloacetate on ca uptake by isolated mung bean mitochondria. 1665

Nitrogen assimilation in crabgrass Digitaria sanguinalis (L.) Scop., was studied by comparing leaf extracts with isolated mesophyll cell and bundle sheath strand extracts. The results show that both nitrate and nitrate reductase are localized in mesophyll cells; glutamine synthetase is nearly equally distributed in the mesophyll and bundle sheath; approximately 67% of the glutamate synthase activity is in the bundle sheath and 33% is in the mesophyll; and 80% of the glutamate dehydrogenase activity is in the bundle sheath, with the NADH-dependent form exhibiting a 2.5-fold higher activity than the NADPH-dependent form.Isolated crabgrass mesophyll cells reduce NO(2) (-) coupled to the photochemical production of O(2) but are inactive with NO(3) (-). The NO(2) (-) -dependent O(2) evolution is light-dependent; inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea; stimulated by photophosphorylation uncouplers; and exhibits a stoichiometry of O(2) evolved to NO(2) (-) reduced of 1.45 and 0.67 in coupled and uncoupled experiments, respectively. Isolated bundle sheath strands are inactive in O(2) evolution with NO(3) (-) or NO(2) (-).Based on these results, plus literature data, two schemes for crabgrass leaf nitrogen assimilation are presented, depending on whether the plant is using ammonium or nitrate as its nitrogen source. It is proposed that the increased nitrogen use efficiency in crabgrass and other C(4) plants is due partially to a "division of labor" between mesophyll and bundle sheath cells, where NO(3) (-) and NO(2) (-) reductase in mesophyll cells act as nitrogen reduction traps in an analogous fashion to phosphoenolpyruvate carboxylase acting as a CO(2) trap during C(4) photosynthesis.
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PMID:Nitrogen Assimilation Pathways in Leaf Mesophyll and Bundle Sheath Cells of C(4) Photosynthesis Plants Formulated from Comparative Studies with Digitaria sanguinalis (L.) Scop. 1666 Sep 55

A mechanical isolation procedure was developed to study the respiratory properties of mitochondria from the mesophyll and bundle sheath tissue of Panicum miliaceum, a NAD-malic enzyme C(4) plant. A mesophyll fraction and a bundle sheath fraction were obtained from young leaves by differential mechanical treatment. The purity of both fractions was about 80%, based on analysis of the cross-contamination of ribulose bisphosphate carboxylase activity and phosphoenolpyruvate carboxylase activity.Mitochondria were isolated from the two fractions by differential centrifugation and Percoll density gradient centrifugation. The enrichment of mitochondria relative to chloroplast material was about 75-fold in both preparations.Both types of mitochondria oxidized NADH and succinate with respiratory control. Malate oxidation in mesophyll mitochondria was sensitive to KCN and showed good respiratory control. In bundle sheath mitochondria, malate oxidation was largely insensitive to KCN and showed no respiratory control. The oxidation was strongly inhibited by salicylhydroxamic acid, showing that the alternative oxidase was involved. The bundle sheath mitochondria of this type of C(4) species contribute to C(4) photosynthesis through decarboxylation of malate. Malate oxidation linked to an uncoupled, alternative pathway may allow decarboxylation to proceed without the restraints which might occur via coupled electron flow through the cytochrome chain.
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PMID:Isolation of Mitochondria from Leaf Tissue of Panicum miliaceum, a NAD-Malic Enzyme Type C(4) Plant. 1666 92

The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V(max) with no apparent effect on K(m). The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation.
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PMID:A simple and accurate spectrophotometric assay for phosphoenolpyruvate carboxylase activity. 1666 4

The aim of this work was to discover the extent of interference by phosphoenolpyruvate (PEP) phosphatase in spectrophotometric assays of PEP carboxylase (EC 4.1.1.31) in crude extracts of plant organs. The presence of PEP phosphatase and lactate dehydrogenase (EC 1.1.1.27) in extracts leads to PEP-dependent NADH oxidation that is independent of PEP carboxylase activity, and hence to overestimation of PEP carboxylase activity. In extracts of three organs of pea (Pisum sativum L.: leaves, developing embryos, and Rhizobium nodules), two organs of wheat (Triticum aestivum L.: developing grain and endosperm), and leaves of Moricandia arvensis (L.) D.C., lactate dehydrogenase activity was at most only 16% of that of PEP carboxylase at the pH optimum for PEP carboxylase activity. Endogenous PEP phosphatase and lactate dehydrogenase are thus unlikely to interfere seriously with the assay for PEP carboxylase at its optimum pH. Addition of lactate dehydrogenase to PEP carboxylase assays- a proposed means of correcting for nonenzymic decarboxylation of oxaloacetate to pyruvate-resulted in increases in PEP-dependent NADH oxidation from zero (Rhizobium nodules) to 131% (wheat grains). There was no obvious relationship between the magnitude of this increase and conditions in the assay that might promote oxaloacetate decarboxylation. However, the magnitude of the increase was highly positively correlated with the activity of PEP phosphatase in the extract. Addition of lactate dehydrogenase to PEP carboxylase assays can thus result in very large overestimations of PEP carboxylase activity, and should only be used as a means of correction for oxaloacetate decarboxylation for extracts with negligible PEP phosphatase activity.
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PMID:Interference by phosphatases in the spectrophotometric assay for phosphoenolpyruvate carboxylase. 1666 52


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