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PubMed1984

The effect of pyrroline-5-carboxylic acid on nucleotide metabolism in erythrocytes from normal and glucose-6-phosphate dehydrogenase-deficient subjects.

Pyrroline-5-carboxylate, the intermediate in the interconversion of proline, ornithine, and glutamate, increases 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) and purine nucleotide formation in intact human erythrocytes. We proposed that: 1) pyrroline-5-carboxylate is converted to proline by pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH, 2) NADP+ augments glucose-6-phosphate dehydrogenase activity, and 3) production of ribose-5-phosphate via the pentose shunt is increased. Since glucose-6-phosphate dehydrogenase plays a central role in this proposed mechanism, we examined the responsiveness of glucose-6-phosphate dehydrogenase-deficient erythrocytes to pyrroline-5-carboxylate. We compared erythrocytes from four Sardinian glucose-6-phosphate dehydrogenase-deficient subjects and four Sardinian normal controls. Without pyrroline-5-carboxylate treatment, the levels of pentose shunt activity, PP-ribose-P, and inosine monophosphate were comparable in the two populations. However, the response to pyrroline-5-carboxylate in erythrocytes from normal and glucose-6-phosphate dehydrogenase-deficient subjects was markedly different. In normal erythrocytes, pyrroline-5-carboxylate treatment increased pentose shunt activity 600%, PP-ribose-P formation 250%, and the incorporation of hypoxanthine into inosine monophosphate 260%. In contrast, pyrroline-5-carboxylate had no effect on glucose-6-phosphate dehydrogenase-deficient erythrocytes. These findings strongly support our proposed mechanism for the pyrroline-5-carboxylate effect on nucleotides. Furthermore, the markedly different capacities for nucleotide synthesis in the two populations with pyrroline-5-carboxylate treatment suggest a role for pyrroline-5-carboxylate-mediated modulation of nucleotide metabolism in normal cells.

Erythrocytes↗
PubMed1984

Reducing equivalents for mixed function oxidation in periportal and pericentral regions of the liver lobule in perfused livers from normal and phenobarbital-treated rats.

The supply of NADPH for cytochrome P-450-dependent mixed function oxidation from the pentose cycle and mitochondria in periportal and pericentral regions of the liver lobule was evaluated in perfused rat liver. Rates of 7-ethoxycoumarin O-deethylation in livers from fed, normal rats monitored with micro-light guides placed on periportal and pericentral regions were 1.2 mumol/g/hr in both regions of the liver lobule. In livers from fed, phenobarbital-treated rats, rates were 3.6 and 7.0 mumol/g/hr in periportal and pericentral regions, respectively. Following treatment of rats with 6-aminonicotinamide, an inhibitor of the pentose cycle, rates of 7-hydroxycoumarin production were approximately 0.9 mumol/g/hr in both regions of the lobule in livers from normal rats and 2.1 and 3.4 mumol/g/hr in periportal and pericentral regions, respectively, in livers from phenobarbital-treated rats. Based on the difference in rates of 7-hydroxycoumarin production in the presence and absence of 6-aminonicotinamide, we conclude that the pentose cycle supplies NADPH for 7-ethoxycoumarin metabolism at rates around 0.3 mumol/g/hr in both regions of the liver lobule in livers from normal rats and 1.5 and 3.6 mumol/g/hr in periportal and pericentral regions, respectively, in livers from phenobarbital-treated rats. Potassium cyanide, an inhibitor of mitochondrial oxidation, reduced rates of 7-ethoxycoumarin O-deethylation to approximately 0.6 mumol/g/hr in both regions of the liver lobule in livers from fed, normal rats and to around 0.2 mumol/g/hr after fasting or treatment with 6-aminonicotinamide. In livers from fasted, phenobarbital-treated rats, 7-hydroxycoumarin was produced at rates of 0.3 and 0.7 mumol/g/hr in periportal and pericentral regions, respectively, in the presence of KCN. Decreases in rates of 7-hydroxycoumarin production during KCN infusion indicate that the mitochondria supply about 0.7 mumol of NADPH/g/hr for 7-ethoxycoumarin metabolism in both regions in livers from normal rats and 1.3 and 2.7 mumol/g/hr in periportal and pericentral regions in livers from phenobarbital-treated rats. The sum of KCN and 6-aminonicotinamide-sensitive rates of 7-ethoxycoumarin metabolism closely approximated rates measured in the absence of the inhibitors. These data indicate that mitochondria supply 50 to 70% of the reducing equivalents for mixed function oxidation of 7-ethoxycoumarin in both regions of the liver lobule in livers from fed rats.

7-Alkoxycoumarin O-Dealkylase↗
PubMedDate not supplied

[Pentosephosphate shunt enzymatic activity in the liver in its disordered innervation].

Partial denervation of the liver evoked the dissection of subphrenic vagus nerves and nerves of the hepatogastric duodenal ligament tissue stimulates synthesis of pentoses in the liver due to the oxidative mechanism and inhibits the process of unoxidative formation of pentoses. Three-four weeks after the operation one may observe a 72% increase in the activity of glucose-6-phosphate dehydrogenase in the liver tissue and a 29 and 21% drop in the activity of transketolase and enzymes, respectively, participating in metabolism of ribose-5-phosphate. The content of soluble proteins in the liver with deneravation is unchanged. A correlation is observed between these changes and those in the content of nucleic acids, an opinion is confirmed on the autonomous character of the oxidative and unoxidative phases of the pentose cycle.

Animals↗
PubMed2005

Metabolic profiling by 13C-NMR spectroscopy: [1,2-13C2]glucose reveals a heterogeneous metabolism in human leukemia T cells.

Metabolic profiling is defined as the simultaneous assessment of substrate fluxes within and among the different pathways of metabolite synthesis and energy production under various physiological conditions. The use of stable-isotope tracers and the analysis of the distribution of labeled carbons in various intermediates, by both mass spectrometry and NMR spectroscopy, allow the role of several metabolic processes in cell growth and death to be defined. In the present paper we describe the metabolic profiling of Jurkat cells by isotopomer analysis using (13)C-NMR spectroscopy and [1,2-(13)C(2)]glucose as the stable-isotope tracer. The isotopomer analysis of the lactate, alanine, glutamate, proline, serine, glycine, malate and ribose-5-phosphate moiety of nucleotides has allowed original integrated information regarding the pentose phosphate pathway, TCA cycle, and amino acid metabolism in proliferating human leukemia T cells to be obtained. In particular, the contribution of the glucose-6-phosphate dehydrogenase and transketolase activities to phosphoribosyl-pyrophosphate synthesis was evaluated directly by the determination of isotopomers of the [1'-(13)C], [4',5'-(13)C(2)]ribosyl moiety of nucleotides. Furthermore, the relative contribution of the glycolysis and pentose cycle to lactate production was estimated via analysis of lactate isotopomers. Interestingly, pyruvate carboxylase and pyruvate dehydrogenase flux ratios measured by glutamate isotopomers and the production of isotopomers of several metabolites showed that the metabolic processes described could not take place simultaneously in the same macrocompartments (cells). Results revealed a heterogeneous metabolism in an asynchronous cell population that may be interpreted on the basis of different metabolic phenotypes of subpopulations in relation to different cell cycle phases.

Carbon Isotopes↗
PubMed2025

Biosynthesis of Ribose-5-Phosphate-Metabolic Regulator of Escherichia coli Viability.

Biosynthesis of ribose-5-phosphate (R5P) underlies all biosynthetic processes associated with biomass growth. Actively dividing cells continuously require building blocks for genome replication, synthesis of ribosomes and other derivatives containing R5P as a carbohydrate backbone. The main source of R5P in the cell is the pentose phosphate pathway (PPP), which is an anabolic sensor designed to coordinate the level of pentose phosphates and reduced NADPH required for anabolic processes. This review is devoted to a comparative analysis of R5P biosynthesis pathways among different domains of microorganisms, the features of PPP regulation in bacterial cells depending on physiological conditions, as well as genetic modifications of PPP and their effect on cell viability. We emphasize that ribose metabolism is a factor in the consolidation of cellular homeostasis under conditions of intensive biomass growth and the discrepancy between the processes of ribose synthesis and consumption is marked by spontaneous cell death.

Escherichia coli↗
PubMed1987

Impaired antioxidant defense in hemoglobin E-containing erythrocytes: a mechanism protective against malaria?

Red blood cell (RBC) antioxidant defense was investigated in eight individuals with hemoglobin E (Six EE and two E-B(+) thalassemia) and compared to that in six individuals with thalassemia and ten normal subjects. Individuals with hemoglobin E had increased incubated Heinz body formation (68% +/- 18%; p less than 0.001) compared to normal and thalassemic RBC (10% +/- 2% and 11% +/- 5%, respectively). Stimulated pentose phosphate shunt activity was increased in the thalassemic and decreased in the hemoglobin E RBC as compared to normal. The 2,3-diphosphoglycerate (DPG) content of the EE RBC was increased to 5.59 +/- 0.69 mumol/ml RBC as compared to normal (4.51 +/- 0.77; p less than 0.001). In the EE RBC, there was a direct correlation between Heinz body formation and DPG content (r = 0.73). Ascorbic and dehydroascorbic acid (0.1 and 1.0 mM) were able to decrease the degree of Heinz body formation in the hemoglobin E RBC. Ascorbic acid (0.1 mM) prolonged the response of the pentose shunt. Thus impaired antioxidant defense may account for the persistence of the hemoglobin E gene in areas where malaria is endemic. Oxidant medications should be used with caution in individuals of Southeast Asian origin.

2,3-Diphosphoglycerate↗
PubMed1993

Effects of intestinal insulin-like peptide on glucose catabolism in mealworm larval fat body in vitro: dependence on extracellular Ca2+ for its stimulatory action.

In vitro hormonally induced variations of glucose catabolism in mealworm fat body tissue were examined by a microradiorespirometric method. An insulin-like peptide (ILP) extracted from the midgut of last larval instar mealworm larvae significantly modified glucose catabolism and was dependent on energy metabolism and on the Ca2+ concentration in the culture medium. Using two different labelled substrate molecules, the stimulatory effects of ILP (compared with those of mammalian insulin) on the relative use of the pentose cycle as opposed to the glycolytic-citric acid cycle by the mealworm fat body were measured in vitro. Metabolic variations were evaluated using either [1-14C]glucose or [6-14C]glucose as substrates. Time course and dose-response curves of ILP and the hormonally induced variations in total CO2 and 14CO2 kinetics were determined. Modification in the specific radioactivity kinetics of 14CO2 derived from [1-14C] glucose and [6-14C]glucose molecules under hormonal effects were observed. As demonstrated in in vivo studies, ILP stimulated the relative utilization of the pentose cycle. However, this effect was observed much more rapidly, but for a shorter time, with fat body in vitro. Mammalian insulin produced similar, but not identical effects. Variations in transmembranous Ca2+ cellular exchanges, induced by either EGTA, nifedipine, or calcium ionophore ionomycin included in the culture medium, indicated that the stimulatory effects of ILP depends on this cation.

Animals↗
PubMed2002

Aerobic glucose metabolism of Saccharomyces kluyveri: growth, metabolite production, and quantification of metabolic fluxes.

The growth and product formation of Saccharomyces kluyveri was characterized in aerobic batch cultivation on glucose. At these conditions it was found that ethyl acetate was a major overflow metabolite in S. kluyveri. During the exponential-growth phase on glucose ethyl acetate was produced at a constant specific rate of 0.12 g ethyl acetate per g dry weight per hour. The aerobic glucose metabolism in S. kluyveri was found to be less fermentative than in S. cerevisiae, as illustrated by the comparably low yield of ethanol on glucose (0.08 +/- 0.02 g/g), and high yield of biomass on glucose (0.29 +/- 0.01 g/g). The glucose metabolism of S. kluyveri was further characterized by the new and powerful techniques of metabolic network analysis. Flux distributions in the central carbon metabolism were estimated for respiro-fermentative growth in aerobic batch cultivation on glucose and respiratory growth in aerobic glucose-limited continuous cultivation. It was found that in S. kluyveri the flux into the pentose phosphate pathway was 18.8 mmole per 100 mmole glucose consumed during respiratory growth in aerobic glucose-limited continuous cultivation. Such a low flux into the pentose phosphate pathway cannot provide the cell with enough NADPH for biomass formation which is why the remaining NADPH will have to be provided by another pathway. During batch cultivation of S. kluyveri the tricarboxylic acid cycle was working as a cycle with a considerable flux, that is in sharp contrast to what has previously been observed in S. cerevisiae at the same growth conditions, where the tricarboxylic acid cycle operates as two branches. This indicates that the respiratory system was not significantly repressed in S. kluyveri during batch cultivation on glucose.

Acetates↗
PubMed2001

Metabolic-flux analysis of continuously cultured hybridoma cells using (13)CO(2) mass spectrometry in combination with (13)C-lactate nuclear magnetic resonance spectroscopy and metabolite balancing.

Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO(2), and ammonia. In this study, the intracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste products derived from glucose. Continuously cultured hybridoma cells are grown in medium containing either 1-(13)C-, 2-(13)C-, or 6-(13)C-glucose. The uptake and production rates of amino acids, glucose, ammonia, O(2), and CO(2) as well as the cellular composition are measured. In addition, the (13)C distribution of the lactate produced and alanine produced by the hybridomas is determined by (1)H-NMR spectroscopy, and the (13)CO(2)/(12)CO(2) ratio is measured by on-line mass spectrometry. These data are used to calculate the intracellular fluxes of the glycolysis, the pentose phosphate pathway, the TCA cycle, and fluxes involved in amino acid metabolism. It is shown that: (i) approximately 20% of the glucose consumed is channeled through the pentose shunt; (ii) the glycolysis pathway contributes the most to lactate production, and most of the CO(2) is produced by the TCA cycle; (iii) the pyruvate-carboxylase flux is negligibly small; and (iv) the malic-enzyme flux is estimated to be 10% of the glucose uptake rate. Based on these flux data suggestions are made to engineer a more efficient glucose metabolism in mammalian cells.

Algorithms↗
PubMed2001

Effect of ethanol on the metabolism of primary astrocytes studied by (13)C- and (31)P-NMR spectroscopy.

Nuclear magnetic resonance was used as the primary technique to investigate the effect of ethanol (40, 80, and 160 mM) on the levels of high-energy phosphates, glycolytic flux, anaplerotic and oxidative fluxes to the tricarboxylic acid (TCA) cycle, the contribution of the pentose phosphate pathway (PPP), and the uptake and release of amino acids on primary cultures of rat astrocytes. On line (31)P-NMR spectroscopy showed that long-term exposure to ethanol caused a drop in the levels of ATP and phosphocreatine. The ratio between the fluxes through the pyruvate dehydrogenase and pyruvate carboxylase reactions also decreased, whereas the glycolytic flux and the ratio between formation of lactate and glucose consumption increased when cells were exposed to acute doses of ethanol. Flux through the pentose phosphate pathway was not affected. The uptake of cysteine and the release of glutamine were stimulated by ethanol, whereas the release of methionine was inhibited. Moreover, the fractional enrichment in serine was enhanced. The changes in the amino acid metabolism are interpreted as a response to oxidative stress induced by ethanol.

Adenine Nucleotides↗
PubMed2003

Proteome analysis of recombinant xylose-fermenting Saccharomyces cerevisiae.

Introduction of an active xylose utilization pathway into Saccharomyces cerevisiae, which does not naturally ferment pentose sugars, is likely to have a major impact on the overall cellular metabolism as the carbon introduced to the cells will now flow through the pentose phosphate pathway. The metabolic responses in the recombinant xylose-fermenting S. cerevisiae were studied at the proteome level by comparative two-dimensional gel electrophoresis of cellular proteins within a pH range of 3-10. Glucose-limited chemostat cultivations and corresponding chemostat cultivations performed in media containing xylose as the major carbon source were compared. The cultivations were studied in aerobic and anaerobic metabolic steady states and in addition at time points 5, 30 and 60 min after the switch-off of oxygen supply. We identified 22 proteins having a significant abundance difference on xylose compared to glucose, and 12 proteins that responded to change from aerobic to anaerobic conditions on both carbon sources. On xylose in all conditions studied, major changes were seen in the abundance of alcohol dehydrogenase 2 (Adh2p), acetaldehyde dehydrogenases 4 and 6 (Ald4p and Ald6p), and DL-glycerol 3-phosphatase (Gpp1p). Our results give indications of altered metabolic fluxes especially in the acetate and glycerol pathways in cells growing on xylose compared to glucose.

Aerobiosis↗
PubMed2001

Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability.

The yeast Saccharomyces cerevisiae efficiently ferments hexose sugars to ethanol, but it is unable to utilize xylose, a pentose sugar abundant in lignocellulosic materials. Recombinant strains containing genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from the xylose-utilizing yeast Pichia stipitis have been reported; however, such strains ferment xylose to ethanol poorly. One reason for this may be the low capacity of xylulokinase, the third enzyme in the xylose pathway. To investigate the potential limitation of the xylulokinase step, we have overexpressed the endogenous gene for this enzyme (XKS1) in S. cerevisiae that also expresses the P. stipitis genes for XR and XDH. The metabolism of this recombinant yeast was further investigated in pure xylose bioreactor cultivation at various oxygen levels. The results clearly indicated that overexpression of XKS1 significantly enhances the specific rate of xylose utilization. In addition, the XK-overexpressing strain can more efficiently convert xylose to ethanol under all aeration conditions studied. One of the important illustrations is the significant anaerobic and aerobic xylose conversion to ethanol by the recombinant Saccharomyces; moreover, this was achieved on pure xylose as a carbon. Under microaerobic conditions, 5.4 g L(-1) ethanol was produced from 47 g L(-1) xylose during 100 h. In fed-batch cultivations using a mixture of xylose and glucose as carbon sources, the specific ethanol production rate was highest at the highest aeration rate tested and declined by almost one order of magnitude at lower aeration levels. Intracellular metabolite analyses and in vitro enzyme activities suggest the following: the control of flux in a strain that overexpresses XKS1 has shifted to the nonoxidative steps of the pentose phosphate pathway (i.e., downstream of xylose 5-phosphate), and enzymatic steps in the lower part of glycolysis and ethanol formation pathways (pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase) do not have a high flux control in this recombinant strain. Furthermore, the intracellular ATP levels were found to be significantly lower for the XK strain compared with either the control strain under similar conditions or glucose-grown Saccharomyces. The ATP : ADP ratios were also lower for the XK strain, especially under microaerobic conditions (0.9 vs 6.4).

Aerobiosis↗
PubMed2002

Analysis of carbon metabolism in Escherichia coli strains with an inactive phosphotransferase system by (13)C labeling and NMR spectroscopy.

We have developed Escherichia coli strains that internalize glucose utilizing the GalP permease instead of the phosphoenolpyruvate:carbohydrate phosphotransferase system. It has been demonstrated that a strain with these modifications (PTS(-)Glc(+)) can direct more carbon flux into the aromatic pathway than the wild-type parental strain (N. Flores et al., 1996, Nat. Biotechnol. 14, 620-623; G. Gosset et al., 1996, J. Ind. Microbiol. 17, 47-52; J. L. Baéz et al., 2001, Biotechnol. Bioeng. 73, 530-535). In this study, we have determined and compared the carbon fluxes of a wild-type strain (JM101), a PTS(-)Glc(-) strain, and two isogenic PTS(-)Glc(+) derivatives named PB12 and PB13 by combining genetic, biochemical, and NMR approaches. It was determined that in these strains a functional glk gene in the chromosome is required for rapid glucose consumption; furthermore, glucokinase-specific activities were higher than in the wild-type strain. (13)C labeling and NMR analysis allowed the determination of differences in vivo which include higher glycolytic fluxes of 93.1 and 89.2% compared with the 76.6% obtained for the wild-type E. coli. In PB12 and PB13 we found a flux through the malic enzymes of 4 and 10%, respectively, compared to zero in the wild-type strain. While flux through the Pck enzyme was absent in PB12 and PB13, in the wild type it was 7.7%. Finally, it was found that in the JM101 and PB12 strains both the oxidative and the nonoxidative branches of the pentose phosphate pathway contributed to ribose 5-phosphate synthesis, whereas in PB13 this pentose was synthesized almost exclusively through the oxidative branch. The determined carbon fluxes correlate with biochemical and genetic characterizations.

Biomedical Engineering↗
PubMed1996

Microsequencing and cDNA cloning of the Calvin cycle/OPPP enzyme ribose-5-phosphate isomerase (EC 5.3.1.6) from spinach chloroplasts.

Ribose-5-phosphate isomerase (RPI) catalyses the interconversion of ribose-5-phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids.

Aldose-Ketose Isomerases↗
PubMedDate not supplied

Effects of triiodothyronine in spontaneously hypertensive rats. Studies on cardiac metabolism, function, and heart weight.

We have studied the effects of triiodothyronine (T3) on heart function, on the myocardial oxidative pentose phosphate pathway, and on heart weight in spontaneously hypertensive (SHR) rats. Another aim was to examine whether these T3-effects may be reversible. T3 was administered daily (0.2 mg/kg s.c.) for 14 days. Compared to the untreated SHR controls, T3 induced an increase in heart rate (beats/min) from 357 +/- 10 (n = 17) to 553 +/- 10 (n = 17), in the pressure-rate-product (mm Hg/min) from 78 400 +/- 4500 (n = 15) to 113 700 +/- 4800 (n = 15), and in the heart weight/body weight ratio (mg/g) from 4.2 +/- 0.2 (n = 20) to 5.8 +/- 0.2 (n = 19). The activity of myocardial glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the oxidative pentose phosphate pathway (units/g protein), was elevated from 4.2 +/- 0.2 (n = 9) to 7.0 +/- 0.6 (n = 9) after 14 days of T3-treatment, while the activity of 6-phosphogluconate dehydrogenase, one of the following enzymes in the pathway, was not altered appreciably. These changes returned to the respective control values when T3-treatment was discontinued for 14 days. Our results demonstrate that T3 had a positive chronotropic effect and induced an additional heart enlargement in an animal model with already established cardiac hyperfunction and hypertrophy. The effects on heart function and weight, which were fully reversible, were not as pronounced as in normal Sprague-Dawley rats.

Animals↗
PubMed2001

Effect of transketolase modifications on carbon flow to the purine-nucleotide pathway in Corynebacterium ammoniagenes.

Transketolase, one of the enzymes in the nonoxidative branch of the pentose phosphate pathway, operates to shuttle ribose 5-phosphate and glycolytic intermediates together with transaldolase, and might be involved in the availability of ribose 5-phosphate, a precursor of nucleotide biosynthesis. The tkt and tal genes encoding transketolase and transaldolase, respectively, were cloned from the typical nucleotide- and nucleoside-producing organism Corynebacterium ammoniagenes by a PCR approach using oligonucleotide primers derived from conserved regions of each amino acid sequence from other organisms. Enzymatic and molecular analyses revealed that the two genes were clustered on the genome together with the glucose 6-phosphate dehydrogenase gene (zwf). The effect of transketolase modifications on the production of inosine and 5'-xanthylic acid was investigated in industrial strains of C. ammoniagenes. Multiple copies of plasmid-borne tkt caused about tenfold increases in transketolase activity and resulted in 10-20% decreased yields of products relative to the parents. In contrast, site-specific disruption of tkt enabled both producers to accumulate 10-30% more products concurrently with a complete loss of transketolase activity and the expected phenotype of shikimate auxotrophy. These results indicate that transketolase normally shunts ribose 5-phosphate back into glycolysis in these biosynthetic processes and interception of this shunt allows cells to redirect carbon flux through the oxidative pentose pathway from the intermediate towards the purine-nucleotide pathway.

Amino Acid Sequence↗
PubMed1986

Biochemistry of reduction of nitro heterocycles.

Misonidazole is a metabolically active drug. Its addition to cells causes an immediate alteration in cellular electron transfer pathways. Under aerobic conditions the metabolic alterations can result in futile cycling with electron transfer to oxygen and production of peroxide. Thiol levels are extremely important in protecting the cell against the peroxide formation and potentially hazardous conditions for hydroxyl radical production. Nevertheless such electron shunting out of cellular metabolism will result in alterations in pentose cycle, glycolysis and cellular capacity to reduce metabolites to essential intermediates needed in DNA metabolism (i.e. deoxyribonucleotides). Glutathione must be depleted to very low levels before toxic effects of misonidazole and other nitro compounds are manifested in cell death via peroxidative damage. Under hypoxic conditions misonidazole also diverts the pentose cycle via its own reduction; however, unlike the aerobic conditions, there are a number of reductive intermediates produced that react with non-protein thiols such as GSH as well as protein thiols. The reaction with protein thiols results in the inhibition of glycolysis and other as yet undetermined enzyme systems. The consequences of the hypoxic pretreatment of cells with nitro compounds are increased vulnerability to radiation and chemotherapeutic drugs such as L-PAM, cis-platinum and bleomycin. The role that altered enzyme activity has in the cellular response to misonidazole and chemotherapeutic agents remains to be determined. It is also clear that the GSH depleted state not only makes cells more vulnerable to oxidative stress but also to hypoxic intermediates produced by the reduction of misonidazole beyond the one electron stage. The relevancy of the present work to the proposed use of thiol depletion in vivo to enhance the radiation or chemotherapeutic response of tumor tissue lies with the following considerations. Apparently, spontaneous peroxidative damage to normal tissue such as liver can occur with GSH depletion to 10-20% of control and with other normal tissue when GSH reaches 50% of control. This situation can obviously become more critical if peroxide producing drugs are administered. The only advantage to such combined drug treatments would lie in the possibility that tumors vary in their catalase and peroxidase activity and consequently may be more vulnerable to oxidative stress (cf. review by Meister. Our tumor model, the A549 human lung carcinoma cell in vitro, appears to be an exception because it has catalase, peroxidase and a high content of GSH.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Evidence for redox cycling of diquat in rat small intestine.

It has previously been established that acute diquat (1,1'-ethylene, 2,2'-bipyridilium) toxicity in the rat is associated with stimulation of net fluid secretion into the gastrointestinal tract. We have examined the possibility that the mechanism of diquat toxicity in the small intestine involves redox cycling of the bipyridyl leading to a disturbance of biochemical function and oxidative stress. Experiments performed in vitro showed that diquat (10 microM to 1 mM) produced an increase in activity of the pentose phosphate pathway in rat small intestinal tissue slices, suggesting that there was oxidation of NADPH even at concentrations of diquat which do not cause intestinal fluid secretion in anaesthetized rats. When the effect of diquat on pentose phosphate activity was measured in rats in situ at a dose which causes maximal fluid secretion [50 mM diquat dibromide (DQBr2)], production of 14CO2 from [1-14C]-glucose increased by 278 +/- 28% (N = 4) within 1 hr of exposure to diquat. Under these same conditions, the tissue content of NADPH in the proximal small intestine was significantly depleted, though there was no corresponding increase in NADP+ concentration. Diquat had no effect on tissue concentrations of either the reduced or oxidized forms of NAD. It is likely that NADPH oxidation at low diquat concentrations can be adequately compensated for by mechanisms within the tissue which protect against oxidative stress. However, the data also suggest that diquat-induced fluid secretion in the rat small intestine is associated with redox cycling of bipyridyl leading to depletion of NADPH.

Animals↗