Kinetic properties of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases from Corynebacterium glutamicum and their application for predicting pentose phosphate pathway flux in vivo.
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The glucose-6-phosphate (Glc6P) and 6-phosphogluconate (6PG) dehydrogenases of the amino-acid-producing bacterium Corynebacterium glutamicum were purified to homogeneity and kinetically characterized. The Glc6P dehydrogenase was a heteromultimeric complex, which consists of Zwf and OpcA subunits. The product inhibition pattern of the Glc6P dehydrogenase was consistent with an ordered bi-bi mechanism. The 6PG dehydrogenase was found to operate according to a Theorell-Chance ordered bi-ter mechanism. Both enzymes were inhibited by NADPH and the 6PG dehydrogenase additionally by ATP, fructose 1,6-bisphosphate (Fru1,6P2), D-glyceraldehyde 3-phosphate (Gra3P), erythrose 4-phosphate and ribulose 5-phosphate (Rib5P). The inhibition by NADPH was considered to be most important, with inhibition constants of around 25 microM for both enzymes. Intracellular metabolite concentrations were determined in two isogenic strains of C. glutamicum with plasmid-encoded NAD- and NADP-dependent glutamate dehydrogenases. NADP+ and NADPH levels were between 130 microM and 290 microM, which is very much higher than the respective Km and Ki values. The Glc6P concentration was around 500 microM in both strains. The in vivo fluxes through the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified enzymes determined in vitro were in agreement with the same fluxes determined by NMR after 13C-labelling. From the derived kinetic model thus validated, it is concluded that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH and NADP+ concentrations and the specific enzyme activities of both dehydrogenases. (+info)
Molecular characterization of the first two enzymes of the pentose-phosphate pathway of Trypanosoma brucei. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase.
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Trypanosomatids are parasitic protists that have part of their glycolytic pathway sequestered inside peroxisome-like organelles: the glycosomes. So far, at least one enzyme of the pentose-phosphate pathway has been found to be associated partially with glycosomes. Here, we describe how two genes from Trypanosoma brucei, coding for the first two enzymes of the pentose-phosphate pathway, i.e. glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase, were identified by in silico screening of trypanosome genome project data bases. These genes were cloned and sequenced. Analysis of the lactonase sequence revealed that it contained a C-terminal peroxisome targeting signal in agreement with its subcellular localization in the bloodstream form trypanosome (15% glycosomal and 85% cytosolic). However, the dehydrogenase sequence did not reveal any targeting signal, despite its localization inside glycosomes. The corresponding enzymes have been overexpressed in Escherichia coli and purified, and their biochemical characteristics have been determined. (+info)
Stimulation of the pentose phosphate pathway and glutathione levels by dehydroascorbate, the oxidized form of vitamin C.
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Ascorbic acid, or vitamin C, generally functions as an antioxidant by directly reacting with reactive oxygen intermediates and has a vital role in defenses against oxidative stress. However, ascorbic acid also has pro-oxidant properties and may cause apoptosis of lymphoid and myeloid cells. The present study shows that dehydroascorbate, the oxidized form of vitamin C, stimulates the antioxidant defenses of cells, preferentially importing dehydroascorbate over ascorbate. While 200-800 microM vitamin C caused apoptosis of Jurkat and H9 human T lymphocytes, pretreatment with 200-1000 microM dehydroascorbate stimulated activity of pentose phosphate pathway enzymes glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transaldolase, elevated intracellular glutathione levels, and inhibited H(2)O(2)-induced changes in mitochondrial transmembrane potential and cell death. A 3. 3-fold maximal glutathione elevation was observed after 48 h stimulation with 800 microM dehydroascorbate. In itself, dehydroascorbate did not affect cytosolic or mitochondrial reactive oxygen intermediate levels as monitored by flow cytometry using oxidation-sensitive fluorescent probes. The data reveal a novel mechanism for increasing glutathione levels through stimulation of the pentose phosphate pathway and identify dehydroascorbate as an antioxidant for cells susceptible to the pro-oxidant and proapoptotic properties of vitamin C. (+info)
Genomic analysis of a nutrient response in Arabidopsis reveals diverse expression patterns and novel metabolic and potential regulatory genes induced by nitrate.
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Microarray and RNA gel blot analyses were performed to identify Arabidopsis genes that responded to nitrate at both low (250 microM) and high (5 to 10 mM) nitrate concentrations. Genes involved directly or indirectly with nitrite reduction were the most highly induced by nitrate. Most of the known nitrate-regulated genes (including those encoding nitrate reductase, the nitrate transporter NRT1, and glutamate synthase) appeared in the 40 most strongly nitrate-induced genes/clones on at least one of the microarrays of the 5524 genes/clones investigated. Novel nitrate-induced genes were also found, including those encoding (1) possible regulatory proteins, including an MYB transcription factor, a calcium antiporter, and putative protein kinases; (2) metabolic enzymes, including transaldolase and transketolase of the nonoxidative pentose pathway, malate dehydrogenase, asparagine synthetase, and histidine decarboxylase; and (3) proteins with unknown functions, including nonsymbiotic hemoglobin, a senescence-associated protein, and two methyltransferases. The primary pattern of induction observed for many of these genes was a transient increase in mRNA at low nitrate concentrations and a sustained increase when treated with high nitrate concentrations. Other patterns of induction observed included transient inductions after both low and high nitrate treatments and sustained or increasing amounts of mRNA after either treatment. Two genes, AMT1;1 encoding an ammonium transporter and ANR1 encoding a MADS-box factor, were repressed by nitrate. These findings indicate that nitrate induces not just one but many diverse responses at the mRNA level in Arabidopsis. (+info)
Loss of [13C]glycerol carbon via the pentose cycle. Implications for gluconeogenesis measurement by mass isotoper distribution analysis.
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Whereas many reports substantiated the suitability of using [2-(13)C]glycerol and Mass Isotoper Distribution Analysis for gluconeogenesis, the use of [(13)C]glycerol had been shown to give lower estimates of gluconeogenesis (GNG). The reason for the underestimation has been attributed to asymmetric isotope incorporation during gluconeogenesis as well as zonation of gluconeogenic enzymes and a [(13)C]glycerol gradient across the liver. Since the cycling of glycerol carbons through the pentose cycle pathways can introduce asymmetry in glucose labeling pattern and tracer dilution, we present here a study of the role of the pentose cycle in gluconeogenesis in Fao cells. The metabolic regulation of glucose release and gluconeogenesis by insulin was also studied. Serum-starved cells were incubated for 24 h in Dulbecco's modified Eagle's media containing 1.5 mm [U-(13)C]glycerol. Mass isotopomers of whole glucose from medium or glycogen and those of the C-1-C-4 fragment were highly asymmetrical, typical of that resulting from the cycling of glucose carbon through the pentose cycle. Substantial exchange of tracer between hexose and pentose intermediates was observed. Our results offer an alternative mechanism for the asymmetrical labeling of glucose carbon from triose phosphate. The scrambling of (13)C in hexose phosphate via the pentose phosphate cycle prior to glucose release into the medium is indistinguishable from dilution of labeled glucose by glycogen using MIDA and probably accounts for the underestimation of GNG using (13)C tracer methods. (+info)
Cells overexpressing fructose-2,6-bisphosphatase showed enhanced pentose phosphate pathway flux and resistance to oxidative stress.
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Changes in the content of fructose-2,6-bisphosphate, a modulator of glycolytic flux, also affect other metabolic fluxes such as the non-oxidative pentose phosphate pathway. Since this is the main source of precursors for biosynthesis in proliferating cells, PFK-2/FBPase-2 has been proposed as a potential target for neoplastic treatments. Here we provide evidence that cells with a low content of fructose-2,6-bisphosphate have a lower energy status than controls, but they are also less sensitive to oxidative stress. This feature is related to the activation of the oxidative branch of the pentose phosphate pathway and the increased production of NADPH. (+info)
Exclusive expression of transketolase in the vanadocytes of the vanadium-rich ascidian, Ascidia sydneiensis samea.
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Ascidians, especially those belonging to the Ascidiidae, are known to accumulate extremely high levels of vanadium in vanadocytes, one type of blood (coelomic) cell. Vanadium, which exists in the +5 oxidation state in seawater, is accumulated in the vanadocytes and reduced to the +3 oxidation state. We have been trying to characterize all of the polypeptides specific to vanadocytes and to specify the proteins that participate in the accumulation and reduction of vanadium. To date, we have localized three enzymes in vanadocytes: 6-phosphogluconate dehydrogenase (6-PGDH: EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6PDH: EC 1.1.1.49), and glycogen phosphorylase (GP: EC 2.4.1.1), all of which are involved in the pentose phosphate pathway. In the current study, we cloned a cDNA for transketolase, an essential and rate-limiting enzyme in the non-oxidative part of the pentose phosphate pathway, from vanadocytes. The cDNA encoded a protein of 624 amino acids, which showed 61.8% identity to the human adult-type transketolase gene product. By immunocytochemistry and immunoblot analyses, the transketolase was revealed to be a protein that was expressed only in vanadocytes and not in any of the more than ten other types of blood cell. This finding, taken together with the localized expression of the other three enzymes, strongly supports the hypothesis that the pentose phosphate pathway functions exclusively in vanadocytes. (+info)
Oxidative stress and AP-1 activity in tamoxifen-resistant breast tumors in vivo.
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BACKGROUND: Most breast cancers, even those that are initially responsive to tamoxifen, ultimately become resistant. The molecular basis for this resistance, which in some patients is thought to involve stimulation of tumor growth by tamoxifen, is unclear. Tamoxifen induces cellular oxidative stress, and because changes in cell redox state can activate signaling pathways leading to the activation of activating protein-1 (AP-1), we investigated whether tamoxifen-resistant growth in vivo is associated with oxidative stress and/or activation of AP-1 in a xenograft model system where resistance is caused by tamoxifen-stimulated growth. METHODS: Control estrogen-treated, tamoxifen-sensitive, and tamoxifen-resistant MCF-7 xenograft tumors were assessed for oxidative stress by measuring levels of antioxidant enzyme (e.g., superoxide dismutase [SOD], glutathione S-transferase [GST], and hexose monophosphate shunt [HMS]) activity, glutathione, and lipid peroxidation. AP-1 protein levels, phosphorylated c-jun levels, and phosphorylated Jun NH(2)-terminal kinase (JNK) levels were examined by western blot analyses, and AP-1 DNA-binding and transcriptional activities were assessed by electrophoretic mobility shift assays and a reporter gene system. All statistical tests are two-sided. RESULTS: Compared with control estrogen-treated tumors, tamoxifen resistant tumors had statistically significantly increased SOD (more than threefold; P=.004) and GST (twofold; P=.004) activity and statistically significantly reduced glutathione levels (greater than twofold; P<.001) and HMS activity (10-fold; P<.001). Lipid peroxides were not significantly different between control and tamoxifen-resistant tumors. We observed no differences in AP-1 protein components or DNA-binding activity. However, AP-1-dependent transcription (P=.04) and phosphorylated c-Jun and JNK levels (P<.001) were statistically significantly increased in the tamoxifen-resistant tumors. CONCLUSION: Our results suggest that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with oxidative stress and the subsequent antioxidant response and with increased phosphorylated JNK and c-Jun levels and AP-1 activity, which together could contribute to tumor growth. (+info)