Decrease of NADH in HeLa cells in the presence of transferrin or ferricyanide

The short-term incubation of HeLa cells in the presence of diferric transferrin or ferricyanide, which are reduced externally by the transplasma membrane reductase, produces a stoichiometric decrease in NADH and increase in NAD+, which is stimulated by insulin. The NADP/NADPH ratio does not change during 15 min incubation with the oxidants. The total pyridine nucleotide pool of HeLa cells is not affected. Incubation with apotransferrin and ferrocyanide, which cannot act as oxidants for transmembrane electron transport, does not change the pyridine nucleotide concentrations in the cells. Our results show that NADH can act as the internal electron donor for the reduction of external oxidants by the transmembrane reductase. It appears that oxidation of NADH by the transmembrane electron transport using ferricyanide or iron transferrin as external electron acceptors is sufficient to stimulate growth in HeLa cells.     

Effect of Lactacidosis on Pyridine Nucleotide Stability During Ischemia in Mouse Brain

Brain levels of NADH and NAD+ were measured in three models of cerebral ischemia to determine whether degradation of the pyridine nucleotides is enhanced in models that generate high concentrations of lactic acid. Complete ischemia (decapitation), in which lactate increased to 14 mmol/kg, caused a gradual decrease in the NAD pool to 50% of control by 2 h. During focal ischemia (occlusion of the middle cerebral artery), the decrease in the NAD pool was less pronounced (82% of control at 2 h) despite the accentuated accumulation of lactate to 33 mmol/kg. In a third model (unilateral hypoxia-ischemia), pretreatment of animals with glucose augmented the ischemic elevation of lactate from 30 mmol/kg to 40 mmol/kg and greatly impaired restoration of energy metabolites during recirculation. However, glucose pretreatment had no effect on the size of the NAD pool during ischemia or early recovery. These results, therefore, demonstrate that the pyridine nucleotide pool is not rapidly degraded during ischemic insults that accumulate high concentrations of lactic acid. The stability of the NAD pool may have been enhanced by the limited increase in brain levels of NADH that occurred in these models of incomplete ischemia.     

Regulation of nicotinamide adenine dinucleotide phosphate levels in yeast

The steady-state levels and redox states of pyridine nucleotide pools have been studied in yeast as a function of external growth conditions. Yeast grown aerobically on 0.8% glucose show two distinct phases of logarithmic growth, a first phase utilizing glucose with ethanol accumulation, and a second phase utilizing ethanol. During growth on glucose, the size of the NADP pool (NADP⁺ + NADPH) is maintained at approximately 12% the size of the NAD pool (NAD⁺ + NADH). Upon exhaustion of glucose, the mechanism(s) that maintain the levels of NADP relative to NAD are altered, resulting in a rapid 2- to 2.5-fold decrease in the size of the NADP pool relative to the size of the NAD pool. The lower levels of NADP are maintained during growth on ethanol. The NAD pool is approximately 50% NADH during both the glucose and ethanol phases of growth, while the NADP pool is approximately 67 and 48% NADPH during the glucose and ethanol phases of growth, respectively. Rapid media transfer experiments show that the decrease in NADP is reversible, that it does not require the net synthesis of pyridine nucleotide or protein, and that changes in the size of the NADP pool relative to the total pyridine nucleotide pool are correlated with changes in the redox state of the NADP pool.     

Cofactor engineering in Saccharomyces cerevisiae: Expression of a H2O-forming NADH oxidase and impact on redox metabolism

The pyridine nucleotides NAD(H) and NADP(H) play major roles in the formation of by-products. To analyse how Saccharomyces cerevisiae (S. cerevisiae) metabolism during growth on glucose might be altered when intracellular NADH pool is decreased, we expressed noxE encoding a water-forming NADH oxidase from Lactococcus lactis (L. lactis) in the S. cerevisiae strain V5. During batch fermentation under controlled microaeration conditions, expression of the NADH oxidase under the control of a yeast promoter lead to large decreases in the intracellular NADH concentration (five-fold) and NADH/NAD+ ratio (six-fold). This increased NADH consumption caused a large redistribution of metabolic fluxes. The ethanol, glycerol, succinate and hydroxyglutarate yields were significantly reduced as a result of the lower NADH availability, whereas the formation of more oxidized metabolites, acetaldehyde, acetate and acetoin was favoured. The biomass yield was low and consumption of glucose, at concentration above 10%, was impaired. The metabolic redistribution in cells expressing the NADH oxidase was a consequence of the maintenance of a redox balance and of the management of acetaldehyde formation, which accumulated at toxic levels early in the process.     

Glyoxylic acid prevents NAD+ and NADH depletion in K562 cells cultured at limiting dilution

K562 erythroleukemic cells cultured at low population density in the absence of serum die within 12-24 hours, unless 0.1 mM glyoxylic acid is added to the culture medium. Earlier events, preceding cell death and occurring within 2 hours culture, are: a) a marked drop of both the NAD+/NADH ratio and the NAD+ concentration, which is prevented by 10mM benzamide, b) an increased biosynthesis of NAD+, leading to extensive depletion of cellular ATP. In the presence of 0.1 mM glyoxylic acid the NAD+/NADH ratio as well as their absolute concentrations remain unchanged, while NAD+ biosynthesis is absent. A NAD+/NADH glycohydrolase activity is present in the cell extract, inhibited by 10 mM benzamide and with a higher affinity for NADH than for NAD+. Preservation of a high NAD+/NADH ratio by glyoxylic acid apparently prevents enzyme activity and the related loss of pyridine nucleotides.     

Levels of oxidized and reduced pyridine nucleotides in dormant spores and during growth, sporulation, and spore germination of Bacillus megaterium.

Dormant spores of Bacillus megaterium contained no detectable reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) despite significant levels of the oxidized forms of these nucleotides (NAD and NADP). During the first minutes of spore germination there was rapid accumulation of NADH and NADPH. However, this accumulation followed the fall in optical density that is characteristic of the initiation of spore germination. Accumulation of NADH and NADPH early in germination was not blocked by fluoride or cyanide, and it occurred even when germination was carried out in the absence of an exogenous source of reducing power. In addition to pyridine nucleotide reduction, de novo synthesis also began early in germination as the pyridine nucleotide levels increased to those found in growing cells. Midlog-phase cells grown in several different media had 20 to 35 times as much total pyridine nucleotide as did dormant spores. However, as growth and sporulation proceeded, the NADH plus NAD level fell four- to fivefold whereas the NADPH plus NADP level fell by a lesser amount. From min 10 of spore germination until midway through sporulation the value for the ratio of NADH/NAD is about 0.1 (0.03 to 0.18) while the ratio of NADPH/ANDP is about 1.4 (0.3 to 2.4). Comparison of these ratios in log-phase versus stationary phase (sporulation) growth in all three growth media tested did not reveal any common pattern of changes.     

Pyridine nucleotide oxidation by a plasma membrane fraction from red beet (Beta vulgaris L.) storage tissue

The potential role of pyridine nucleotide oxidation in the energization and/or regulation of membrane transport was examined using sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue. In this system, pyridine nucleotide oxidation, which was enhanced in the presence of ferricyanide, occurred. In the presence or absence of ferricyanide, the oxidation of NADH was several-fold greater than the oxidation of NADPH, indicating that it was the preferred substrate for oxidation in this system. Ferricyanide reduction coupled to NADH oxidation did not require the transmembrane movement of reducing equivalents since ferricyanide incorporated inside the vesicles could not be reduced by NADH added externally to the vesicles, unless the vesicles were made leaky by the addition of 0.05% (v/v) Triton X-100. Using fluorescent probes for the measurement of transmembrane pH gradients and membrane potentials, it was determined that NADH oxidation did not result in the production of a proton electrochemical gradient or have any effect upon the proton electrochemical gradient produced by the plasma membrane H+-ATPase. The oxidation of NADH in the presence of ferricyanide did result in the acidification of the reaction medium. This acidification was unaffected by the addition of Gramicidin D and stimulated by the addition of 0.05% (v/v) Triton X-100, suggesting a scalar (nonvectorial) production of protons in the oxidation/reduction reaction. The results of this study suggest that the oxidation of pyridine nucleotides by plasma membrane vesicles is not related to energization of transport at the plasma membrane or modulation of the activity of the plasma membrane H+-ATPase.     

Relationship between ion requirements for respiration and membrane transport in a marine bacterium.

Intact cells of the marine bacterium Alteromonas haloplanktis 214 oxidized NADH, added to the suspending medium, by a process which was stimulated by Na+ or Li+ but not K+. Toluene-treated cells oxidized NADH at three times the rate of untreated cells by a mechanism activated by Na+ but not by Li+ or K+. In the latter reaction, K+ spared the requirement for Na+. Intact cells of A. haloplanktis oxidized ethanol by a mechanism stimulated by either Na+ or Li+. The uptake of alpha-aminoisobutyric acid by intact cells of A. haloplanktis in the presence of either NADH or ethanol as an oxidizable substrate required Na+, and neither Li+ nor K+ could replace it. The results indicate that exogenous and endogenous NADH and ethanol are oxidized by A. haloplanktis by processes distinguishable from one another by their requirements for alkali metal ions and from the ion requirements for membrane transport. Intact cells of Vibrio natriegens and Photobacterium phosphoreum oxidized NADH, added externally, by an Na+-activated process, and intact cells of Vibrio fischeri oxidized NADH, added externally, by a K+-activated process. Toluene treatment caused the cells of all three organisms to oxidize NADH at much faster rates than untreated cells by mechanisms which were activated by Na+ and spared by K+.     

Isolation and partial characterization of a mutant of Escherichia coli lacking pyridine nucleotide transhydrogenase.

A mutant of Escherichia coli lacking pyridine nucleotide transhydrogenase (EC 1.6.1.1) was isolated by assaying activity in clones of cells mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine. The mutant is missing both energy-independent and energy-dependent transhydrogenase, but has normal NADH dehydrogenase and ATPase activities. Compared to the parental strain, the mutant has normal growth rates with glucose, glycerol, or succinate aerobically and with glucose or glycerol plus fumarate anaerobically. The aerobic growth yield with limiting glucose concentrations is also normal. These growth properties indicate that the enzyme is not an essential source of NADPH or ATP in vivo.     

Inhibitory effect of mannose on erythrocyte defense against oxidants

The erythrocyte can phosphorylate a variety of hexoses. Since it can consume mannose and glucose equivalently in the hereditary deficiencies of hexokinase and phosphoglucose isomerase and since erythrocyte defense against oxidants is impaired in a variety of hereditary hemolytic anemias, we tested the hypothesis that mannose may be a significant alternative to glucose as a fuel for this defense system. Unexpectedly, mannose inhibited defense against oxidants as manifested by increased Heinz body formation when both normal and high-reticulocyte erythrocytes were incubated with acetylphenylhydrazine (APH). Using APH as the oxidant, mannose-incubated erythrocytes had decreased reduced glutathione stability and impaired hexose oxidation by the pentose shunt compared to glucose-incubated erythrocytes. After incubation with mannose and APH, normal erythrocytes showed a decrease in ATP content. Approximately 25% of the consumed mannose accumulated in the erythrocytes as mannose 6-phosphate. Erythrocytes incubated with mannose and APH displayed a significant loss of redox potential as manifested by decreased NADH/(NADH + NAD+) and NADPH/(NADPH + NADP+) ratios. Since phosphomannose isomerase is the rate-limiting step for mannose metabolism, our results suggest that mannose impairs erythrocyte defense against oxidants by causing ATP depletion and by impairing the regeneration of reduced pyridine nucleotides by the Embden-Meyerhof and pentose phosphate pathways.     

Alterations in membrane function in an Escherichia coli mutant tolerant to colicins Ia and Ib.

An Escherichia coli mutant (tolI) previously shown to be tolerant to colicins Ia and Ib is defective in several functions of the bacterial cytoplasmic membrane. When compared with its parental strain, X36, whole cells of tolI show reduced rates of respiration with succinate, malate, or lactate as the substrate but near-normal rates with glucose or glycerol. Cell membrane preparations prepared from tolI cells exhibit reduced succinate and D-lactate oxidase activity but elevated levels of reduced-form nicotinamide adenine dinucleotide (NADH) oxidase. tolI cells have reduced levels of succinate and D-lactate dehydrogenase but normal levels of NADH dehydrogenase. Glycerol-grown tolI cells and membrane vesicles prepared from such cells are defective in the active transport of several amino acids and thiomethyl-beta-D-galactoside; however, they accumulate higher levels of alpha-methylglucoside when compared with X36 whole cells or vesicles. Although tolI cells adsorb less colicin Ia at high colicin concentrations than do X36 cells, it is shown that the adsorption of an Ia molecule to tolI cells has a lower probability of eliciting cell death than does Ia adsorption to strain X36 cells. It is concluded that a single mutation can lead to an alteration in several aspects of cytoplasmic membrane function and colicin I sensitivity.     

Evidence for age-related changes in pyridine nucleotide content of isolated rat islets

The purpose of this study was to document the effect of age on alpha-glycerophosphate activity and pyridine nucleotide concentration in pancreatic islets isolated from rats. In order to do this, islets were isolated from pancreases of 2 and 12 month-old rats, and measurements made of alpha-glycerophosphate activity and of NAD+ and NADH, determinations were made following incubation at both basal (5.6 mM) and elevated glucose concentrations (28 mM). The results indicated that islet alpha-glycerophosphate dehydrogenase activity was decreased (P less than 0.001) by approximately 50% in the older rats. This was associated with an increase in mean (+/- SEM) basal NADH content (pmol/microgram DNA) in 12 month-old (4.48 +/- 0.31) as compared to 2 month-old rats (2.73 +/- 0.49). Although mean (+/- SEM) basal NAD+ levels (pmol/microgram DNA) were the same in 2 and 12 month-old rats (29.4 +/- 2.5 and 30.8 +/- 2.8, respectively), NAD+ content following incubation at elevated levels of glucose declined (absolutely and relatively) to a significantly greater degree in the younger rats. The incremental rise in islet NADH concentration following incubation at the elevated glucose concentration was similar in the two groups, but the relative increase was only approximately half as great in islets from 12 month-old rats. These data indicate that the age-related decline in the activity of alpha-glycerophosphate dehydrogenase, the enzyme regulating the glycerophosphate shuttle system in 12 month-old rats, is associated with alterations in islet pyridine nucleotide composition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of preculturing conditions on growth of Lactobacillus rhamnosus on medium containing glucose and citrate

Lactobacillus rhamnosus can metabolize citrate through a citrate inducible transport system. The growth curves of L. rhamnosus on medium containing glucose and citrate was found to be highly dependent on preculturing conditions. It exhibited diauxic growth when precultured on glucose, but demonstrated simultaneous consumption when cultured on citrate. The maximum specific growth rate for cells growing on glucose + citrate was 0.38 h-1, which was higher than the growth rate on individual substrates (0.28 h-1). Simultaneous consumption also yielded higher net flavour compounds, diacetyl and acetoin. Flux analysis indicated that L. rhamnosus requires oxygen for balancing excess NADH through NADH oxidase. The flux analysis provided insights into the metabolic network of L. rhamnosus.     

Regulation of dinitrogen fixation in intact Azotobacter vinelandii

1. In intact Azotobacter vinelandii the influence of oxygen on the levels of oxidized nicotinamide adenine dinucleotides and adenine nucleotides in relation to nitrogenase activity was investigated.

2. The hypothesis that a high (NADH + NADPH)/(NAD⁺ + NADP⁺) is the driving force for the transport of reducing equivalents to nitrogenase in intact A. vinelandii was found to be invalid. On the contrary, with a decreasing ratio of reduced to oxidized pyridine nucleotides, the nitrogenase activity of the whole cells increases.

3. By measuring oxidative phosphorylation and using 9-amino acridine as a fluorescent probe, it could be demonstrated that respiration-coupled transport of reducing equivalents to the nitrogenase requires a high energy level of the plasma membrane or possibly coupled to it, a high pH gradient over the cytoplasmic membrane. Furthermore nitrogen fixation is controlled by the presence of oxygen and the ATP/ADP ratio.     

Generation of a proton potential by succinate dehydrogenase of Bacillus subtilis functioning as a fumarate reductase.

The membrane fraction of Bacillus subtilis catalyzes the reduction of fumarate to succinate by NADH. The activity is inhibited by low concentrations of 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO), an inhibitor of succinate: quinone reductase. In sdh or aro mutant strains, which lack succinate dehydrogenase or menaquinone, respectively, the activity of fumarate reduction by NADH was missing. In resting cells fumarate reduction required glycerol or glucose as the electron donor, which presumably supply NADH for fumarate reduction. Thus in the bacteria, fumarate reduction by NADH is catalyzed by an electron transport chain consisting of NADH dehydrogenase (NADH:menaquinone reductase), menaquinone, and succinate dehydrogenase operating in the reverse direction (menaquinol:fumarate reductase). Poor anaerobic growth of B. subtilis was observed when fumarate was present. The fumarate reduction catalyzed by the bacteria in the presence of glycerol or glucose was not inhibited by the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or by membrane disruption, in contrast to succinate oxidation by O2. Fumarate reduction caused the uptake by the bacteria of the tetraphenyphosphonium cation (TPP+) which was released after fumarate had been consumed. TPP+ uptake was prevented by the presence of CCCP or HOQNO, but not by N,N'-dicyclohexylcarbodiimide, an inhibitor of ATP synthase. From the TPP+ uptake the electrochemical potential generated by fumarate reduction was calculated (Deltapsi = -132 mV) which was comparable to that generated by glucose oxidation with O2 (Deltapsi = -120 mV). The Deltapsi generated by fumarate reduction is suggested to stem from menaquinol:fumarate reductase functioning in a redox half-loop.     

Inhibition of NADH oxidation by pyridine derivatives

The neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an impurity in an illicit drug, is expressed after its oxidation to 1-methyl-4-phenylpyridinium by monoamine oxidase. The pyridinium is concentrated by carrier-mediated transport into the mitochondria where it inhibits NADH dehydrogenase and, hence, ATP synthesis. Some structurally related compounds have been tested for their effect on the oxidation of NAD+-linked substrates in intact mitochondria, and for the inhibition of the accumulation of the pyridinium into mitochondria and of NADH dehydrogenase activity in a membrane preparation. Some pyridine derivatives are more inhibitory to NADH dehydrogenase than is 1-methyl-4-phenylpyridinium but these are not concentrated into mitochondria by the uptake system. 4-Phenylpyridine, one of the most effective inhibitors, both occurs naturally and is an environmental pollutant.     

Mitochondrial stress causes increased succination of proteins in adipocytes in response to glucotoxicity

2SC [S-(2-succino)-cysteine] is a chemical modification formed by a Michael addition reaction of fumarate with cysteine residues in proteins. Formation of 2SC, termed 'succination' of proteins, increases in adipocytes grown in high-glucose medium and in adipose tissues of Type?2 diabetic mice. However, the metabolic mechanisms leading to increased fumarate and succination of protein in the adipocyte are unknown. Treatment of 3T3 cells with high glucose (30?mM compared with 5?mM) caused a significant increase in cellular ATP/ADP, NADH/NAD+ and Δψm (mitochondrial membrane potential). There was also a significant increase in the cellular fumarate concentration and succination of proteins, which may be attributed to the increase in NADH/NAD+ and subsequent inhibition of tricarboxylic acid cycle NAD+-dependent dehydrogenases. Chemical uncouplers, which dissipated Δψm and reduced the NADH/NAD+ ratio, also decreased the fumarate concentration and protein succination. High glucose plus metformin, an inhibitor of complex I in the electron transport chain, caused an increase in fumarate and succination of protein. Thus excess fuel supply (glucotoxicity) appears to create a pseudohypoxic environment (high NADH/NAD+ without hypoxia), which drives the increase in succination of protein. We propose that increased succination of proteins is an early marker of glucotoxicity and mitochondrial stress in adipose tissue in diabetes.     

Evidence for an alternative and non-phosphorylating pathway for NADH reoxidation in a yeast strain resistant to glucose repression

A yeast strain (SP1) resistant to glucose repression modified simultaneously in the fermentative and in the oxidative pathways (loss of alcohol dehydrogenase I and over production of cytochrome a + a3, being insensitive to the glucose effect) developed a secondary mitochondrial hydrogen pathway. Oxidative phosphorylation was measured with exogenous NADH as substrate on mitochondria derived from repressed or derepressed cells. In this strain, antimycin A promotes a partial inhibition of NADH oxidation but a complete inhibition of phosphorylation. Amytal partially inhibits oxidation of NADH but not phosphorylation. KCN inhibits NADH oxidation in a biphasic way (first level 0.1 mM, second level 5 mM) but phosphorylation was fully inhibited by 0.1 mM KCN. This alternative but non-phosphorylating pathway is insensitive to salicyl hydroxamate. The external NADH dehydrogenase, like cytochrome c oxidase is partially insensitive to catabolite repression. These results provide evidence for the presence in strain SP1 of an alternative mitochondrial pathway, going from the external NADH dehydrogenase to an oxidase, different from the normal NADH dehydrogenase ubiquinone pathway.     

On the mechanism of the glucose-induced ATP catabolism in ascites tumour cells and its reversal by pyruvate.

Addition of glucose to Ehrlich-Landschütz ascites tumour cells preincubated for 30-60 min in phosphate-buffered Krebs-Ringer salt solution ("starved cells") resulted within 1-2 min in an approx. 90% decline of their ATP content and a massive accumulation of fructose 1,6-bisphosphate. These alterations, which took place under both aerobic and anaerobic conditions, were followed by a gradual spontaneous recovery with restoration of normal ATP and fructose 1,6-bisphosphate values. The transient derangement of the energy metabolism after glucose addition to starved ascites tumour cells by preventable by simultaneous addition of pyruvate or 2-oxobutyrate, or by preincubating the cells in the presence of glucose. The protective effect of pyruvate was duplicated by addition of phenazine methosulphate or NAD+ to the incubation medium. The data seem to warrant the conclusion that the glucose-induced ATP depletion is determined by a blockade of glycolysis at the stage of glyceraldehyde phosphate dehydrogenase caused by the failure of the cells to oxidize the NADH produced in the same reaction. The continued unrestrained action of 6-phosphofructokinase results in accumulation of fructose 1,6-bisphosphate, which constitutes a trap for the high-energy phosphate bonds of ATP. The primary metabolic disturbance appears to consist of a transient inhibition of pyruvate kinase with the resultant inability of the cells to maintain an unimpaired supply of pyruvate, as required for the lactate dehydrogenase-mediated oxidation of NADH. The regulatory mechanism underlying this phenomenon is discussed.     

Adenine and pyridine nucleotides in the erythrocyte of different mammalian species

Adenine (ATP, ADP, AMP) and pyridine nucleotides (NADP+, NADPH, NAD+, NADH) concentrations have been determined by HPLC in the erythrocytes from five different mammalian species (pig, rat, mouse, rabbit and cow) and compared to those in human red blood cells. Two different extraction procedures have been used and the results obtained are compared and discussed. A good correlation between the different abilities of the erythrocytes of the six species to utilize glucose and the NAD+/NADH ratio was found, with high NAD+/NADH ratio in the red blood cell of the species with high glucose utilization rates. The levels of all the glycolytic enzymes and some of the pentose phosphate shunt enzymes were also determined.