Soluble and chloroplast malate dehydrogenase isoenzymes of Triticum aestivum

Three isoenzymes of malate dehydrogenase have been isolated from 9-day-old wheat shoots. The microbody (peroxisome) and chloroplast MDH are similar in their electrophoretic behaviour. The mitochondrial MDH, soluble MDH and chloroplast MDH differ in K_m values for malate and NAD. The activity of MDH isoenzymes with NAD⁺-analogues as substrate was in the order 3-AP-NAD⁺ > 3-AP-deam NAD⁺ > NAD⁺ > TN-NAD⁺ and deam NAD⁺. The thermal stabilities of the isoenzymes were significantly different: C-MDH > m-MDH > S-MDH.     

Lactate dehydrogenase isoenzymes of sperm cells and testes

1. The kinetic and metabolic properties of lactate dehydrogenase isoenzyme LDH_x from human sperm cells and rat testes were studied. 2. LDH_x shows a sensitivity to inhibition by stilboestrol diphosphate, urea and guanidinium chloride different from that of the LDH-H₄ and LDH-M₄ isoenzymes. 3. About 10 and 20% of the total lactate dehydrogenase activity of testes and sperm cells respectively were associated with particulate fractions. In sperm cells 11% was localized in the middle piece and 18·8% in the head fraction. LDH_x was found in all particulate fractions of sperm cells. The middle piece contained 41·0% of total LDH_x activity and showed high succinate dehydrogenase activity. 5. The pH-dependence of lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated. Lactate dehydrogenase in sperm cells has maximal activity with NADH as coenzyme at pH7·5 and with NADPH as coenzyme at pH6·0. At pH6·0 a 10% greater oxidation of NADPH than of NADH was found. At acid pH lactate hydrogenase may function as an enzyme bringing about transhydrogenation from NADPH to NAD⁺. 6. In agreement with the stoicheiometry of the lactate de- hydrogenase reaction, the lactate/pyruvate concentration ratio decreased with increasing pH. 7. The lactate/pyruvate and NAD⁺/NADH concentration ratios were estimated with glucose, fructose and sorbitol as substrates and as a function of time after addition of these substrates. During a 20min. period after the addition of the substrates, changes in lactate/pyruvate and NAD⁺/NADH concentration ratios were noticed. Increasing concentration of the substrates mentioned gave rise to asymptotic increases in lactate and pyruvate. 8. Sorbitol did not act as a substrate for LDH_x. 9. The findings described are consistent with the idea that LDH_x is different from other known lactate dehydrogenase isoenzymes, but that it has a metabolic function similar to that of the isoenzymes of other tissues.     

Exogenous NAD Effects on Plant Mitochondria: A Reinvestigation of the Transhydrogenase Hypothesis

Addition of NAD⁺ to purified potato (Solanum tuberosum L.) mitochondria respiring α-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O₂ uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD⁺ (NAP₄-NAD⁺), an inhibitor of NAD⁺ uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD⁺-stimulated malate-cytochrome c reductase activity, and reduction of added NAD⁺ by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD⁺ reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD⁺ by storing on ice for 72 hours, was completely dependent on added NAD⁺, and the effect of NAD⁺ on these mitochondria was prevented by incubating them with NAP₄-NAD⁺. External NAD⁺ reduction by these mitochondria was not affected by NAP₄-NAD⁺. We conclude that all effects of exogenous NAD⁺ on plant mitochondrial respiration can be attributed to net uptake of the NAD⁺ into the matrix space.     

PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity

Sirtuin1 (SIRT1) deacetylase and poly(ADP-ribose)-polymerase-1 (PARP-1) respond to environmental cues, and both require NAD⁺ cofactor for their enzymatic activities. However, the functional link between environmental/oxidative stress-mediated activation of PARP-1 and SIRT1 through NAD⁺ cofactor availability is not known. We investigated whether NAD⁺ depletion by PARP-1 activation plays a role in environmental stimuli/oxidant-induced reduction in SIRT1 activity. Both H₂O₂ and cigarette smoke (CS) decreased intracellular NAD⁺ levels in vitro in lung epithelial cells and in vivo in lungs of mice exposed to CS. Pharmacological PARP-1 inhibition prevented oxidant-induced NAD⁺ loss and attenuated loss of SIRT1 activity. Oxidants decreased SIRT1 activity in lung epithelial cells; however increasing cellular NAD⁺ cofactor levels by PARP-1 inhibition or NAD⁺ precursors was unable to restore SIRT1 activity. SIRT1 was found to be carbonylated by CS, which was not reversed by PARP-1 inhibition or selective SIRT1 activator. Overall, these data suggest that environmental/oxidant stress-induced SIRT1 down-regulation and PARP-1 activation are independent events despite both enzymes sharing the same cofactor.     

Effect of selected compounds on the activity of glutamate dehydrogenase from triticale roots

The influence of selected factors on the activity of highly purified GDH in triticale roots was investigated in vitro. In the presence of 2-ME, NADH-GDH activity increased by 400 %, while NADPH-GDH activity rose by 500 %. No effect of reducing factors on NAD(P)⁺-GDH reaction was detected. The sulphydryl groups inhibitors, such as p-chloromercuribenzoate (p-CMB) and iodoacetamide, proved the strongest inhibitors of the aminative reaction. Metal-binding compounds: ethylenediaminetetraacetic acid disodium salt (EDTA) and Zinkov also considerably inhibited NAD(P)H-GDH activity. Diisopropylfluorophosphate (DFP) and pepstatin A, the inhibitors specific for -OH serine and COO⁻ aspartic acid groups respectively, caused a non-significant NAD(P)H-GDH activity decrease. Cd²⁺, Co²⁺, Hg²⁺, Mg²⁺, Pb²⁺ and Zn²⁺ ions strongly inhibited the amination reaction, whereas their inhibiting effect upon NAD⁺-GDH activity was negligible. Among the applied ions, only Ca²⁺ activated NADH-GDH.     

The effect of glutamine and asparagine on net NH4 + uptake in young wheat plants

Wheat plants grown during 10 days in the absence of N were pretreated with 1.0 eq m^-3 of methionine, asparagine or glutamine and/or 1.0 eq m^-3 MSX⁴ or 0.17 eq m^-3 DON. Net NH₄ ⁺ uptake was measured both in the presence or in the absence of the amino acid or enzyme inhibitor used in the pretreatment. The effect of met, asn and gln on net K⁺ uptake was also studied using K⁺-depleted plants. Changes in the contents of root free NH₄ ⁺, asn, gln and the activities of GS, PEP-carboxylase, NAD⁺-GDH and NADH-GDH were determined. Net NH₄ ⁺ uptake in gln and asn pretreated plants was markedly, and sometimes completely suppressed provided uptake was measured in the presence of the amides. On the other hand, the met pretreated plants absorbed only 35% less NH₄ ⁺ than the control. When NH₄ ⁺ uptake was measured in the absence of the amino acids, only those plants pretreated with asn showed a marked suppression of net uptake during the first 120 min. None of the 3 amino acids tested significantly inhibited K⁺ uptake. Free NH₄ ⁺ concentration in roots of N-starved plants increased after 4 h incubation with gln, asn or MSX in the absence of external NH₄ ⁺. Nevertheless, no correlation was observed between root NH₄ ⁺ concentration and the extent of net NH₄ ⁺ uptake suppression. The inhibitory effect exerted by asn decreased when it was supplied together with MSX or DON. Pretreatments with gln or asn in the absence of external NH₄ ⁺ significantly increased the level of asn in the roots, while that of gln remained unchanged. It is concluded that asn and gln specifically suppress net NH₄ ⁺ uptake in wheat, although it is not clear wether they act only from the root exterior, or through an endogenous pool exhibiting fast turn-over.Abbreviations AUR ammonium uptake rate - DON 6-diazo-5-oxo-L-norleucine - GDH glutamic dehydrogenase - GOGAT oxoglutarate- glutamine aminotransferase - GS glutamine synthetase - MSX L-methionine sulfoximine - PEP phosphoenolpyruvate - PVPP polyvinylpolypyrrolidone     

Glycerol phosphate dehydrogenase in mammalian peroxisomes

Peroxisomes isolated on sucrose density gradients from homogenates of rat, chicken, or dog livers and rat kidney contained NAD⁺:α-glycerol phosphate dehydrogenase. Since the amount of sucrose in the peroxisomal fraction inhibited the enzyme activity about 70%, it was necessary to remove the sucrose by dialysis. About 8.4% of the total dehydrogenase of rat livers was in the surviving intact peroxisomes after homogenation. If corrected for particle breakage, this represented approximately 21% of the total activity. About 9.5% of the total enzyme was isolated in rat kidney peroxisomes, and because of severe particle rupture may represent over half of the total activity. No glycerol phosphate dehydrogenase was found in spinach leaf peroxisomes. A specific activity of 326 nmoles min^?1 mg^?1 protein in the rat liver peroxisomal fraction was at least twice that in the cytoplasm. NAD⁺:α-glycerol phosphate dehydrogenase was also present in a membrane fraction which was not identified, but none was in the mitochondria. The liver peroxisomal and cytoplasmic NAD⁺:α-glycerol phosphate dehydrogenase moved similarly on polyacrylamide gels and each resolved into two adjacent bands.Malate dehydrogenase was not found in peroxisomes from liver and kidney of rats and pigs, but 1–2% of the total particulate malate dehydrogenase was present in the peroxisomal area of the gradient from dog livers. However, this malate dehydrogenase in dog peroxisomal fractions did not exactly coincide with the peroxisomal marker, catalase. Malate dehydrogenase in dog liver mitochondria and in the peroxisomal fraction had similar pH optima and K_m values and migrated similarly to the anode at pH 6.5 on starch gels as a major and a minor band. The cytoplasmic malate dehydrogenase had a different pH optimum and K_m value and resolved into five different isoenzymes by electrophoresis. It is concluded that NAD⁺:α-glycerol phosphate dehydrogenase is in peroxisomes of liver and kidney, whereas malate dehydrogenase, present in peroxisomes of plants, is apparently absent in animal peroxisomes.     

Prolonged root hypoxia effects on enzymes involved in nitrogen assimilation pathway in tomato plants

In order to investigate the effects of root hypoxia (1–2% oxygen) on the nitrogen (N) metabolism of tomato plants (Solanum lycopersicum L. cv. Micro-Tom), a range of N compounds and N-assimilating enzymes were performed on roots and leaves of plants submitted to root hypoxia at the second leaf stage for three weeks. Obtained results showed that root hypoxia led to a significant decrease in dry weight (DW) production and nitrate content in roots and leaves. Conversely, shoot to root DW ratio and nitrite content were significantly increased. Contrary to that in leaves, glutamine synthetase activity was significantly enhanced in roots. The activities of nitrate and nitrite reductase were enhanced in roots as well as leaves. The higher increase in the NH₄⁺ content and in the protease activities in roots and leaves of hypoxically treated plants coincide with a greater decrease in soluble protein contents. Taken together, these results suggest that root hypoxia leaded to higher protein degradation. The hypoxia-induced increase in the aminating glutamate dehydrogenase activity may be considered as an alternative N assimilation pathway involved in detoxifying the NH₄⁺, accumulated under hypoxic conditions. With respect to hypoxic stress, the distinct sensitivity of the enzymes involved in N assimilation is discussed.Key words: tomato, hypoxia, nitrogen, glutamine synthetase, protease, glutamate dehydrogenase     

Sex-related differences in human plasma NAD+/NADH levels depend on age

Nicotinamide adenine dinucleotide (NAD) is a coenzyme in metabolic reactions and cosubstrate in signaling pathways of cells. While the intracellular function of NAD is well described, much less is known about its importance as an extracellular molecule. Moreover, there is only little information about the concentration of extracellular NAD and the ratio between its oxidized (NAD⁺) and reduced (NADH) form in humans. Therefore, our study aimed at the analysis of total NAD and NAD⁺/NADH ratio in human plasma depending on sex and age. First, an enzymatic assay was established for detecting NAD⁺ and NADH in human plasma samples. Then, plasma NAD was analyzed in 205 probands without severe diseases (91 men, 114 women) being 18–83 years old. The total plasma NAD concentration was determined with median 1.34 µM (0.44–2.88 µM) without difference between men and women. Although the amounts of NAD⁺ and NADH were nearly balanced, women had higher plasma NAD⁺/NADH ratios than men (median 1.33 vs. 1.09, P<0.001). The sex-related difference in the plasma NAD⁺/NADH ratio reduces with increasing age, an effect that was more obvious for two parameters of the biological age (skin autofluorescence, brachial-femoral pulse wave velocity (PWV)) than for the chronological age. However, plasma values for total NAD and NAD⁺/NADH ratio did not generally alter with increasing age. In conclusion, human plasma contains low micromolar concentrations of total NAD with higher NAD⁺/NADH redox ratios in adult but not older women compared with same-aged men.     

Effect of (NH 4 ) SO 4 and glycerol on the preservation of the "NAD + -independent" activity of D-glucose-6-phosphate, L-myo-inositol-1-phosphate cycloaldolase from Neurospora crassa

Partially purified preparations of glucocycloaldolase from Neurospora crassa show an absolute requirement for NAD⁺. The experimental findings in this paper indicate that the conditions of isolation used determine whether or not glucocycloaldolase activity can be detected in the absence of added NAD⁺. The protective action of glycerol and (NH₄)₂SO₄ on this so-called “NAD⁺-independent” activity was studied.     

Genetically encoded fluorescent indicator for imaging NAD/NADH ratio changes in different cellular compartments

Background

The ratio of NAD⁺/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD⁺/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD⁺/NADH are fundamentally new approach for studying the NAD⁺/NADH dynamics.

Methods

We developed a genetically encoded probe for the nicotinamide adenine dinucleotide, NAD(H), redox state changes by inserting circularly permuted YFP into redox sensor T-REX from Thermus aquaticus. We characterized the sensor in vitro using spectrofluorometry and in cultured mammalian cells using confocal fluorescent microscopy.

Results

The sensor, named RexYFP, reports changes in the NAD⁺/NADH ratio in different compartments of living cells. Using RexYFP, we were able to track changes in NAD⁺/NADH in cytoplasm and mitochondrial matrix of cells under a variety of conditions. The affinity of the probe enables comparison of NAD⁺/NADH in compartments with low (cytoplasm) and high (mitochondria) NADH concentration. We developed a method of eliminating pH-driven artifacts by normalizing the signal to the signal of the pH sensor with the same chromophore.

Conclusion

RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments.

General significance

RexYFP has several advantages over existing NAD⁺/NADH sensors such as smallest size and optimal affinity for different compartments. Our results show that normalizing the signal of the sensor to the pH changes is a good strategy for overcoming pH-induced artifacts in imaging.     

Nickel and Al-excess inhibit nitrate reductase but upregulate activities of aminating glutamate dehydrogenase and aminotransferases in growing rice seedlings

The present study was undertaken to examine the influence of toxic levels of Ni and Al, on the activities of key nitrogen assimilatory enzymes in roots and shoots of growing rice seedlings. When seedlings of two inbred rice (Oryza sativa L.) cvs. Malviya-36 and Pant-12, sensitive to both Ni and Al, were raised in sand cultures containing 200 and 400 μM NiSO₄ or 80 and 160 μM Al₂(SO₄)₃, a marked inhibition in the activities of NO₃ ⁻ assimilatory enzymes NR and GS was observed in roots as well as shoots during a 5–20 day growth period. Both Ni and Al treatments, in growth medium, stimulated the activity of aminating glutamate dehydrogenase (NADH-GDH) whereas the activity of deaminating GDH (NAD⁺-GDH) decreased under metal toxicities. The activities of the aminotransferases studied; alanine aminotransferase (AlaAT) and aspartate amino transferase (AspAT) increased due to Ni and Al treatments. Results suggest that both Ni and Al treatments impair N assimilation in rice seedlings by inhibiting the activities of NR and GS and that GDH appears to play a role in assimilation of NH₄ ⁺ in metal stress conditions. Further, higher activity of aminotransferases in metal stressed seedlings might be helpful in meeting higher demand of amino acids under stressed conditions.     

NAD-linked alcohol dehydrogenase 1 regulates methylglyoxal concentration in Candida albicans

We purified a fraction that showed NAD⁺-linked methylglyoxal dehydrogenase activity, directly catalyzing methylglyoxal oxidation to pyruvate, which was significantly increased in glutathione-depleted Candida albicans. It also showed NADH-linked methylglyoxal-reducing activity. The fraction was identified as a NAD⁺-linked alcohol dehydrogenase (ADH1) through mass spectrometric analyses. In ADH1-disruptants of both the wild type and glutathione-depleted cells, the intracellular methylglyoxal concentration increased significantly; defects in growth, differentiation, and virulence were observed; and G2-phase arrest was induced.     

Nutritional Energy Stimulates NAD+ Production to Promote Tankyrase-Mediated PARsylation in Insulinoma Cells

The poly-ADP-ribosylation (PARsylation) activity of tankyrase (TNKS) regulates diverse physiological processes including energy metabolism and wnt/β-catenin signaling. This TNKS activity uses NAD⁺ as a co-substrate to post-translationally modify various acceptor proteins including TNKS itself. PARsylation by TNKS often tags the acceptors for ubiquitination and proteasomal degradation. Whether this TNKS activity is regulated by physiological changes in NAD⁺ levels or, more broadly, in cellular energy charge has not been investigated. Because the NAD⁺ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in vitro is robustly potentiated by ATP, we hypothesized that nutritional energy might stimulate cellular NAMPT to produce NAD⁺ and thereby augment TNKS catalysis. Using insulin-secreting cells as a model, we showed that glucose indeed stimulates the autoPARsylation of TNKS and consequently its turnover by the ubiquitin-proteasomal system. This glucose effect on TNKS is mediated primarily by NAD⁺ since it is mirrored by the NAD⁺ precursor nicotinamide mononucleotide (NMN), and is blunted by the NAMPT inhibitor FK866. The TNKS-destabilizing effect of glucose is shared by other metabolic fuels including pyruvate and amino acids. NAD⁺ flux analysis showed that glucose and nutrients, by increasing ATP, stimulate NAMPT-mediated NAD⁺ production to expand NAD⁺ stores. Collectively our data uncover a metabolic pathway whereby nutritional energy augments NAD⁺ production to drive the PARsylating activity of TNKS, leading to autoPARsylation-dependent degradation of the TNKS protein. The modulation of TNKS catalytic activity and protein abundance by cellular energy charge could potentially impose a nutritional control on the many processes that TNKS regulates through PARsylation. More broadly, the stimulation of NAD⁺ production by ATP suggests that nutritional energy may enhance the functions of other NAD⁺-driven enzymes including sirtuins.     

O-methylhydroxylamine as a reagent for NAD+ modification in urocanase.

The inhibition of urocanase from Pseudomonas putida by O-methylhydroxylamine has been characterized as being due to the formation of an adduct between CH₃ONH₂ and NAD⁺, the latter of which has been recently shown to be a tightly bound coenzyme for this urocanase. Inhibition is maximal at pH 8.5 and is blocked by the presence of the substrate analog imidazole propionate. Loss of catalytic activity corresponds directly with the binding of 1 mol of ¹⁴CH₃ONH₂ per mole of enzyme, and partial reversibility of the modification, achieved by dialysis at pH 7.5, is accompanied by concomitant restoration of enzymatic activity. No incorporation of ¹⁴CH₃ONH₂ into urocanase is seen when enzyme-bound NAD⁺ is first converted to NADH or when NAD⁺ is removed by prior treatment of urocanase with 8 m urea. Stability and spectral properties of the CH₃ONH · NAD adduct are consistent with previous data reported for the product of the hydroxylamine reaction with NAD⁺. It is concluded that other urocanases which exhibit inhibition by hydroxylamine may likewise contain NAD⁺ as an essential coenzyme and that the use of ¹⁴CH₃ONH₂ as a reversible modification reagent for NAD⁺ should prove helpful for studies on the role of NAD⁺ in the urocanase catalytic process.     

Effects of overexpression of NAPRTase,NAMNAT, and NAD synthetase in the NAD(H) biosynthetic pathways on the NAD(H) pool,NADH/NAD+ ratio,and succinic acid production with different carbon sources by metabolically engineered Escherichia coli

Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose anaerobically due to the inability to regenerate NAD⁺. To regulate NAD(H) pool size and NADH/NAD⁺ ratio, overexpression of the enzymes in the NAD(H) biosynthetic pathways in BA002 was investigated. The results clearly demonstrate that the increased NAD(H) pool size and the decreased NADH/NAD⁺ ratio improved the glucose consumption and cell growth, which improved succinic acid production. When the pncB and the nadD genes were co-overexpressed in CA102, the ratio of NADH/NAD⁺ was decreased from 0.60 to 0.12, and the concentration of NAD(H) was the highest among that of all the strains. Moreover, the dry cell weight (DCW), glucose consumption, and the concentration of succinic acid in CA102 were also the highest. Based on the sufficient NAD⁺ supply after gene modification in the NAD(H) biosynthetic pathways, reductive carbon sources with different amounts of NADH can further change the distribution of metabolites. When sorbitol was used as a carbon source in CA102, the byproducts were lower than those of glucose fermentation, and the yield of succinic acid was increased.     

Sulfhydryl groups in urocanase: Relationship to nicotinamide adenine dinucleotide binding

This study concerned the role of the sulfhydryl groups in urocanase of Pseudomonas putida. When p-chloromercuribenzoate was added to the enzyme, two sulfhydryl groups reacted at once with little inhibition; the enzyme slowly became inhibited while further sulfhydryls reacted. After the p-chloromercuribenzoate inhibition occurred, if a thiol was subsequently added, most of the original activity was recovered. As the incubation time with p-chloromercuribenzoate was increased, the thiol became less effective in reversing the inhibition. However, if NAD⁺ (10 μm) was added with the thiol, 60–90% of the initial activity was restored even after long p-chloromercuribenzoate incubations. Restoration of activity by NAD⁺ was concentration dependent and specific for NAD⁺. Radioactive NAD⁺ could be bound to urocanase. These results confirm the coenzyme role for NAD⁺ in urocanase. In urea, p-chloromercuribenzoate titration of urocanase measured 11.9 -SH groups per molecule. Sulfite-modified enzyme treated with p-chloromercuribenzoate and dialyzed was substantially photoactivated in the presence of a thiol; that is, NAD⁺ was not required to restore activity. From these results, it is proposed that this enzyme contains two reactive —SH groups and that an essential —SH group is involved in NAD⁺ binding. Forces present in the sulfite-modified enzyme prevent the release of the NAD⁺ in the presence of mercurials.