Transketolase mutation in riboflavin-synthesizing strains of Bacillus subtilis

Unlike its predecessors B. subtilis rosR and 41, riboflavin producing B. subtilis 24 strain does not utilize pentose and gluconate and poorly assimilates glucose. Simultaneous addition of glutamic and shikimic acid restored its capacity to grow and produce riboflavin in medium with pentose and gluconate. This strain lacks the activity of transketolase, the key enzyme of the pentose phosphate cycle, and possesses normal ribulose-5-phosphate-epimerase and glucose phosphate isomerase activities. Like enterobacteria, B. subtilis has two different transport systems for glucose and mannose. The data are discussed from the viewpoint of increasing riboflavin production by transketolase mutants. Probable consequences of cell wall and cytoplasmatic membrane damage in B. subtilis with this mutation are discussed.     

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Control of pentose phosphate-cycle activity by cellular requirement for reduced nicotinamide adenine dinucleotide phosphate

By using inhibitors and stimulators of different metabolic pathways the interdependence of the pentose phosphate cycle and lipogenesis in isolated fat-cells was studied. Rotenone, which is known to inhibit electron transport in the respiratory chain, blocked glucose breakdown at the site of pyruvate dehydrogenase. Consequently, because of the lack of acetyl-CoA, fatty acid synthesis was almost abolished. A concomitant decrease in pentose phosphate-cycle activity was observed. Phenazine methosulphate stimulated pentose phosphate-cycle activity about five- to ten-fold without a considerable effect on fatty acid synthesis. The influence of rotenone on both the pentose phosphate cycle and lipogenesis could be overcome by addition of phenazine methosulphate, indicating that rotenone has no direct effect on these pathways. The decreased rate of the pentose phosphate cycle in the presence of rotenone therefore has to be considered as a consequence of decreased fatty acid synthesis. The rate of glucose catabolism via the pentose phosphate cycle in adipocytes appears to be determined by the requirement of NADPH for lipogenesis. Treatment of cells with 6-aminonicotinamide caused an accumulation of 6-phosphogluconate, indicating an inhibition of 6-phosphogluconate dehydrogenase. The rate of glucose metabolism via the pentose phosphate cycle as well as the rate of fatty acid synthesis, however, was not affected by 6-aminonicotinamide treatment and could still be stimulated by addition of insulin. Since even in cells from starved animals, in which the pentose phosphate-cycle activity is extremely low, no accumulation of 6-phosphogluconate was observed, it is concluded that the control of this pathway is achieved by the rate of regeneration of NADP at the site of glucose 6-phosphate dehydrogenase.     

The pentose phosphate cycle is regulated by NADPH/NADP ratio in rat liver

The changes in the activity of the pentose phosphate cycle produced by the activation or inhibition of different NADPH-consuming pathways have been studied. The inhibition of fatty acid synthesis by kynurenate produced to the same extent, inhibition of the pentose phosphate cycle activity and an increase (about twofold) in the NADPH/NADP ratio. The addition of ter-butyl-hydroperoxide or paraquat, which is metabolized via NADPH-consuming pathways, produced the activation of the pentose phosphate cycle and a decrease in the NADPH/NADP ratio (about threefold). The plot of the NADPH/NADP ratio versus the pentose phosphate cycle activity gave a straight line with a regression index of 0.999. The regulation of the pentose phosphate cycle mainly by the intracellular NADPH/NADP ratio is discussed.     

Gluconate catabolism in cowpea rhizobia: evidence for a ketogluconate pathway

Enzymatic and radiorespirometric analysis of several strains of cowpea rhizobia revealed the presence of key enzymes of the Entner-Doudoroff (ED) pathway with the operation of the hexose cycle for the dissimilation of gluconate. These bacteria lack the oxidative pentose phosphate (PP) pathway when grown on gluconate. Gluconate-grown cells possessed an operational tricarboxylic acid (TAC) cycle. Enzymes of an ancillary pathway, the ketogluconate (KG) pathway for gluconate catabolism were detected. The presence of this pathway was confirmed by techniques of thin-layer chromatography and radiorespirometry.Abbreviations ED Entner Doudoroff - PP pentose phosphate - EMP Embden-Meyerhof-Parnas - KG ketogluconate - TCA tricarboxylic acid - DKG diketogluconate - PFK phosphofructokinase     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known ;overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30-40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine-zinc-insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine-zinc-insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding.     

The role of glutathione in rat adipocyte pentose phosphate cycle activity

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 mm) had higher GSH contents (3.2 μg/10⁶ cells) than in the absence of glucose (2.3 μg/10⁶ cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H₂O₂ to a concentration of 60 μm to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 mm glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N-ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 mm GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 mm NADP⁺ was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP⁺] ratios may play a significant role in short-term control of the pentose phosphate cycle.     

Effect of carbon sources on the biosynthesis of ergot alkaloids and the activity of carbon metabolism enzymes in Penicillium sizovae

The study was aimed at finding out how different carbon sources influenced the growth of Penicillium sizovae, the biosynthesis of epoxyagroclavine-1 and agroclavine-1 as well as the activity of key enzymes involved in the citric acid cycle, the pentose phosphate pathway and the glyoxylate cycle. The fungal growth was shown to depend on the carbohydrate substrate: it had a two-phase profile when P. sizovae was cultivated on mannitol and glucose, but not on sorbitol. The quantitative content and composition of ergoalkaloids depended on the combination of carbohydrate and organic acid substrates. The overall productivity of the mycelium (epoxyagroclavin-1+agroclavin-1) was highest when mannitol and fumarate were used. A medium with sorbitol and fumaric acid was very selective in terms of epoxyagroclavine-1 synthesis. The high level of alkaloid biosynthesis correlated with the active functioning of the pentose phosphate cycle and with the low activity of the CAC.     

Chemostat culture of Bacillus caldotenax at 65°C: Activity and regulation of the main metabolic pathways

Bacillus caldotenax was cultivated in chemostat experiments at 65°C with a chemically defined minimal medium. Glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain were active as demonstrated by measuring the corresponding enzymes. No enzyme activity of the Entner-Doudoroff pathway could be detected. The specific activities of the citrate cycle enzymes were up to 10 times higher as compared to the enzymes of glycolysis. At dilution rates between 0.3 and 2.2 h^-1 none of the main metabolic pathways was regulated. In contrast the isocitrate lyase was regulated (drop of activity with increasing growth rates). As a result of a batch culture with glucose and acetate as carbon sources a regulation model was proposed: glucose, or a metabolite of glucose, represses the isocitrate lyase; in the absence of glucose acetate acts as an inducer.Abbreviations DCIP dichlorphenol indophenol - ED Entner-Doudoroff pathway - EMP Emden-Meyerhof-Parnas pathway - ICL isocitrate lyase - PP pentose phosphate pathway - TCC tricarbonic acid cycle     

Transfer of 1-pyrroline-5-carboxylate as oxidizing potential from hepatocytes to erythrocytes.

The interconversions of proline and 1-pyrroline-5-carboxylate form an intercellular cycle that is the basis of a metabolic interaction between hepatocytes and erythrocytes. The cycle transfers oxidizing potential from hepatocytes to erythrocytes, which stimulates pentose phosphate pathway in erythrocytes. This interaction depends on the differential metabolism of proline and 1-pyrroline-5-carboxylate in erythrocytes and hepatocytes and consists of the following: in hepatocytes proline oxidase converts proline into 1-pyrroline-5-carboxylate, which is released into the medium and taken up by erythrocytes; erythrocyte 1-pyrroline-5-carboxylate reductase converts 1-pyrroline-5-carboxylate into proline and concomitantly generates NADP+; the generated oxidizing potential drives glucose metabolism through the pentose phosphate pathway in erythrocytes; finally, erythrocytes release proline into the medium, enabling it to re-enter hepatocytes and repeat the cycle. The increased activity of the pentose phosphate pathway in erythrocytes may enhance the production of 5-phosphoribosyl pyrophosphate, a necessary moiety for the processing of purines.     

Study of certain carbohydrate metabolism enzymes in an active oxytetracycline producer strain and in an inactive mutant in relation to antibiotic biosynthesis]

The activity of the glycolysis enzymes, i.e. aldolase and pyruvate decarboxylase and the enzymes of the pentose cycle, i.e. transketolase were investigated in the process of cultivation of an active strain and inactive mutant of Act. rimosus under conditions favourable for oxytetracycline biosynthesis on starch medium and under unfavourable conditions on glucose medium. It was shown that the aldolase and transketolase activity in the inactive mutant was higher on the starch medium as compared to the active strain, while the activity of pyruvate dekarboxylase was lower. The above difference between the both strains was preserved on the glucose medium and the activity of aldolase and transketolase in both strains increased, while the activity of pyruvate dekarboxylase remained at the same level.     

Dehydrogenase activity of pentose and glucuronate pathways of glucose utilization in the rat brain during single and repeated cooling

Repeated supercoolings down to rectal temperatures (19-20 degrees C) results in the different changes in the dehydrogenase activity of pentose and glucuronate pathways in the rat brain: the activity of the pentose cycle oxidative enzymes (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) lowers considerably and that of VDP-glucose dehydrogenase rises. The dehydrogenase activity in the pentose cycle is found to be inhibited in rats cooled for the first time, the UDP-glucose dehydrogenase activity being preserved at the control level. In the adapted rats the cooling causes mobilization of the pentose cycle, the UDP-glucose dehydrogenase activity remains unchanged.     

Loss of [13C]glycerol carbon via the pentose cycle. Implications for gluconeogenesis measurement by mass isotoper distribution analysis

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.     

Effect of growth rate on the glucose metabolism of yeast grown in continuous culture. Radiorespirometric studies

Specifically labelled¹⁴C-d-glucose was used to estimate the percentage participation of glycolysis and pentose phosphate cycle in the glucose catabolism ofCandida utilis andSaccharomyces cerevisiae. The two yeasts were cultivated at various growth rates (0.1 to 0.5 h^?1) in a chemostat on synthetic medium limited with glucose under aerobic conditions. The results show a considerable increase in the percentage participation of pentose phosphate cycle in the glucose catabolized bySaccharomyces cerevisiae with the increase in specific growth rate. However, inCandida utilis, the specific growth rate does not influence significantly the part of glucose catabolized via pentose phosphate cycle, but its absolute values are relatively higher than inSaccharomyces cerevisiae. A rough quantitative estimate indicates that a maximum of 60 to 72% of the assimilated glucose is catabolized through the pentose phosphate cycle while inSaccharomyces cerevisiae the percentage participation of the pentose phosphate cycle varies from 24 to 60% (maximum) and 9 to 34% (minimum).     

The enzyme of carbon metabolism in the thermotolerant sulfobacillus strain K1

To determine enzymatic activities in the thermotolerant strain K1 (formerly "Sulfobacillus thermosulfidooxidans subsp. thermotolerans"), it was grown in a mineral medium with (1) thiosulfate and Fe2+ or pyrite (autotrophic conditions), (2) Fe2+, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. An increased content of carbon dioxide (up to 5 vol%) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide was fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO2 in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase decreased with the increasing content of CO2 in the medium.     

The pentose phosphate pathway: an antioxidant defense and a crossroad in tumor cell fate

The pentose phosphate pathway, one of the main antioxidant cellular defense systems, has been related for a long time almost exclusively to its role as a provider of reducing power and ribose phosphate to the cell. In addition to this "traditional" correlation, in the past years multiple roles have emerged for this metabolic cascade, involving the cell cycle, apoptosis, differentiation, motility, angiogenesis, and the response to anti-tumor therapy. These findings make the pentose phosphate pathway a very interesting target in tumor cells. This review summarizes the latest discoveries relating the activity of the pentose phosphate pathway to various aspects of tumor metabolism, such as cell proliferation and death, tissue invasion, angiogenesis, and resistance to therapy, and discusses the possibility that drugs modulating the pathway could be used as potential tools in tumor therapy.     

Metabolic network analysis on Phaffia rhodozyma yeast using 13C-labeled glucose and gas chromatography-mass spectrometry

Carotenoid production by microorganisms, as opposed to chemical synthesis, could fulfill an ever-increasing demand for 'all natural' products. The yeast Phaffia rhodozyma has received considerable attention because it produces the red pigment astaxanthin, commonly used as an animal feed supplement. In order to have a better understanding of its metabolism, labeling experiments with [1-(13)C]glucose were conducted with the wildtype strain (CBS5905T) and a hyper-producing carotenoid strain (J4-3) in order to determine their metabolic network structure and estimate intracellular fluxes. Amino acid labeling patterns, as determined by GC-MS, were in accordance with a metabolic network consisting of the Embden-Meyerhof-Parnas pathway, the pentose phosphate pathway, and the TCA cycle. Glucose was mainly consumed along the pentose phosphate pathway ( approximately 65% for wildtype strain), which reflected high NADPH requirements for lipid biosynthesis. Although common to other oleaginous yeast, there was no, or very little, malic enzyme activity for carbon-limited growth. In addition, there was no evidence of phosphoketolase activity. The central carbon metabolism of the mutant strain was similar to that of the wildtype strain, though the relative pentose phosphate flux was lower and the TCA cycle flux in accordance with the biomass yield being lower.     

Use of the flux model of amino acid metabolism of Escherichia coli

A program implementing a flux model of Escherichia coli metabolism was used to analyze the effects of the addition of amino acids (tryptophan, tyrosine, phenylalanine, leucine, isoleucine, valine, histidine, lysine, threonine, cysteine, methionine, arginine, proline) to minimal medium or media lacking nitrogen, carbon, or both. The overall response of the metabolic system to the addition of various amino acids to the minimal medium is similar. Glycolysis and the synthesis of pyruvate with its subsequent degradation to acetate via acetyl-CoA become more efficient, whereas the fluxes through the pentose phosphate pathway and the TCA cycle decrease. If amino acids are used as the sole source of carbon, nitrogen, or both, the changes in the flux distribution are determined mainly by the carbon limitation. The phosphoenolpyruvate to glucose-6-phosphate flux increases; the flux through the pentose phosphate path is directed towards ribulose-5-phosphate. Other changes are determined by the compounds that are the primary products of catabolism of the added amino acid.     

Evaluation of Thallium Acetate-Citrate Medium for Isolation of Enterococci

Thallous acetate-citrate (TAC) agar, a selective medium for isolating enterococci from frozen foods, was evaluated. Two types of bacteria, micrococci and group N streptococci, were responsible for erroneously high enterococcus counts on TAC agar. Selectivity of the medium was improved by the addition of 0.01% sodium azide.     

The regulation of peroxisomal enzyme systems of Tetrahymena pyriformis by fatty acid composition, glucose and oxygen in the medium

The effect of the chain length of fatty acids on peroxisomal enzyme activities of Tetrahymena pyriformis was investigated. The growth of cells and the activities of peroxisomal enzymes were inhibited markedly by the addition of medium-chain fatty acids (C6-C12) to the culture medium, whereas the addition of longer-chain fatty acids (C14-C18) resulted in a slight increase of growth and in the marked stimulation of enzyme activities concerned with fatty acid beta-oxidation and the glyoxylate cycle in peroxisomes. Peroxisomal beta-oxidation (fatty acyl-CoA oxidase) was more potent towards longer-chain fatty acids than the mitochondrial activity (fatty acyl-CoA dehydrogenase). The induction of the peroxisomal beta-oxidation system by palmitate was repressed both by the addition of glucose and the aeration of the culture medium, whereas that of the peroxisomal glyoxylate cycle was repressed only by the addition of glucose to the medium. These results indicate that peroxisomal enzyme systems related to the beta-oxidation of fatty acids and the glyoxylate cycle are regulated by the compositions of fatty acids, glucose, and oxygen in the medium.     

Effect of orally administered secondary autoxidation products of linoleic acid on carbohydrate metabolism in rat liver

The effects of orally administered secondary autoxidation products of linoleic acid in rat liver were investigated. Their administration led to two toxic effects on hepatic carbohydrate metabolism, as compared to the administration of saline or linoleic acid used as controls. One effect was depletion of glucose 6-phosphate and fructose 6-phosphate caused by the reduction of glycolysis and glycogenolysis, accompanied by decreases in glycogen synthesis and pentose phosphate cyclic activity. The reduction in these metabolic systems seems unlikely to occur because phosphofructokinase was regulated by ATP or citrate enzymatically, because their accumulation in the liver was not detected in the secondary products. Another toxic effect was the depletion of oxaloacetate and isocitrate caused by the reduction in enzyme activity of the mitochondrial citrate cycle. On the basis of these results, the hepatotoxic effects of secondary products are discussed as follows: the incorporated secondary products impaired the activities of hexokinase and phosphoglucomutase in the liver. The reduction in these enzyme activities resulted in the depletion of glucose 6-phosphate and fructose 6-phosphate, which led ultimately to decreases in the activities of phosphofructokinase, the pentose phosphate cycle, and glycogen synthesis. Moreover, the secondary products disturbed the mitochondrial membrane, resulting in a decrease in the activity of the citrate cycle, which was accompanied by depletion of its metabolites.