The oxidation of reduced nicotinamide nucleotides by hydrogen peroxide in the presence of lactoperoxidase and thiocyanate, iodide or bromide

Lactoperoxidase (EC 1.11.1.7) catalysed the oxidation of NADH by hydrogen peroxide in the presence of either thiocyanate, iodide or bromide. In the presence of thiocyanate, net oxidation of thiocyanate occurred simultaneously with the oxidation of NADH, but in the presence of iodide or bromide, only the oxidation of NADH occurred to a significant extent. In the presence of thiocyanate or bromide, NADH was oxidized to NAD(+) but in the presence of iodide, an oxidation product with spectral and chemical properties distinct from NAD(+) was formed. Thiocyanate, iodide and bromide appeared to function in the oxidation of NADH by themselves being oxidized to products which in turn oxidized NADH, rather than by activating the enzyme. Iodine, which oxidized NADH non-enzymically, appeared to be an intermediate in the oxidation of NADH in the presence of iodide. NADPH was oxidized similarly under the same conditions. An assessment was made of the rates of these oxidation reactions, together with the rates of other lactoperoxidase-catalysed reactions, at physiological concentrations of thiocyanate, iodide and bromide. The results indicated that in milk and saliva the oxidation of thiocyanate to a bacterial inhibitor was likely to predominate over the oxidation of NADH.     

Inhibitory Effect of Ellagitannin Metabolites on IgE-Mediated Allergic Responses in RBL-2H3 Cells

A flavoprotein functional as an NADH oxidase has NADH peroxidase activity in the presence of free-FAD. The NADH peroxidase activity was observed in cell free extracts from aerobically grown cells in the presence of free FAD. The enzyme that has this NADH peroxidase activity was purified to homogeneity, and had exactly the same properties as the NADH oxidase.     

Cooperative interaction of reduced pyridine nucleotides with estrogen synthetase of human placental microsomes

The estrogen synthetase present in human placental microsomes appears to be dependent on the cooperative interaction of the reduced cofactors NADPH and NADH for optimal activity. Using steady-state concentrations of either cofactor, it was found that while the estrogen synthetase activity followed hyperbolic saturation kinetics with NADPH (K_mapp = 14 μM), the enzyme followed sigmoidal saturation kinetics when the cofactor was NADH, with the half-maximum velocity attained at a cofactor concentration of 1.1 mm. The maximum velocity obtained with NADPH as the cofactor was greater than with corresponding concentrations of NADH. Estrogen synthetase activity in the presence of NADH was not due to NADPH contamination. NADH, in the presence of small concentrations of NADPH (0.5 to 5 μm), stimulated significantly the rate of estrogen formation from androstenedione by placental microsomes and, in addition, the enzyme saturation kinetics changed from sigmoidal to hyperbolic, thus mimicking the effect of NADPH. Estrogen synthetase activity, measured in the presence of 1 mm NADH, was stimulated in a dose-dependent manner by NADPH (K_mapp = 0.4 μM NADPH) and, when the enzyme was measured in the presence of 5 μm NADPH, the activity was stimulated in a dose-dependent manner by NADH (K_mapp = 45 μM NADH). Estrogen synthetase activity measured in the presence of NADH, without and with NADPH (1 μm) remained linear both with time of incubation for approximately 15 min and with microsomal protein concentration up to 3 mg/ml. The apparent K_m of estrogen synthetase for androstenedione, when measured in the presence of NADH, was 1 μm. The synergistic interaction between NADH and NADPH in stimulating placental estrogen synthetase activity observed in vitro may, conceivably, take place in vivo in the intact placenta.     

The kinetics of NADH oxidation by complex I of isolated plant mitochondria

The oxidation of matrix NADH in the presence and absence of rotenone was investigated in submitochondrial particles prepared from purified beetroot ( Beta vulgaris L.) mitochondria. The submitochondrial particles oxidised NADH using oxygen and artificial electron acceptors such as ferricyanide (FeCN) and short-chain analogues of ubiquinone(UQ)-10, although the NADH-FeCN reductase activity was not inhibited by rotenone. NADH-oxygen reductase activity in the presence and absence of rotenone displayed different affinities for NADH (145 ± 37 and 24 ± 9 μ M , respectively). However, in the presence of 0.15 m M UQ-1 where any contribution from non-specific sites of UQ-reduction was minimal, the rotenone-insensitive oxygen uptake was stimulated dramatically and the K_m(NADH) decreased from 167 ± 55 μ M to 11 ± 1 μ M ; a value close to that determined for the total oxygen uptake which itself was virtually unaffected by the addition of UO-1 [K_m(NADH) of 13 ± 3 μ M ).
The similar affinity of NADH-oxygen reductase for NADH when UQ-1 was present in both the presence and absence of rotenone, suggested that there may be only one NADH binding site involved in the two activities. A quantitative two-stage model for Complex I is postulated with one NADH binding site and two sites of UQ-reduction (one of which is insensitive to rotenone) with a common intermediate 'P' whose level of reduction can influence the NADH binding site. The poor affinity that rotenone-insensitive NADH-oxygen reductase activity displayed for NADH results from a limitation on the interaction of its UQ-reduction site with UQ-10 in the membrane; possibly from a low concentration of UQ-10 around this site or from steric hindrance restricting the access of UQ-10 to this reduction site.     

Salts- induced oxidase activity of lipoamide dehydrogenase from pig heart.

A weak NADH oxidase activity of lipoamide dehydrogenase at neutral pH is increased as much as 15-fold by the addition of KI or (NH4)2SO4. The addition of NAD+ shifts the optimum pH for the KI-induced oxidase activity from 6.3 to 5.5 without changing the maximum activity. The optimum pH is similarly shifted to 5.6 when sulfhyldryl groups of the enzyme are oxidized in the presence of small amount of cupric ion. The NADH: lipoamide and NADH: p-benzoquinone reductase activities are strongly inhibited by KI but both are increased by the presence of (NH4)2SO4. The known intermediate having a charge-transfer band at 530 nm can be seen upon an addition of NADH to the enzyme in the presence of (NH4)2SO4 but not in the presence of KI. The enzyme flavin is reductase by a stoichiometric amount of NADH when KI is present.     

The influence of NADH on the ethylmorphine-N-demethylation in liver microsomes from control and phenobarbital-treated rats or different ages.

The ethylmorphine-N-demethylation by liver microsomes from control and phenobarbital-treated rats of different ages was investigated by means of adding NADPH in combination with NADH to the incubation medium. The rate of ethylmorphine-N-demethylation in the presence of NADPH without NADH is greater in adult than in young rats and greater in induced that in control rats. The higher the activity of ethylmorphine metabolism with NADPH alone the more it is abolsutely enhanced by NADH. The relative increase in ethylmorphine metabolism caused by NADH is equal in all groups of animals. It is concluded that there are no differences in the introduction of the second electron from NADH to the oxygenated cytochrome P-450 but there are differences in the concentration of cytochrome-substrate complex and, consequently, in the oxygenated cytochrome-substrate complex. The enhancing effect of NADH is higher at lower NADPH concentrations. In the presence of NADH, the NADPH concentrations necessary to obtain a msximum metabolic rate are lower than without NADH.     

Absence of NADH channeling in coupled reaction of mitochondrial malate dehydrogenase and complex I in alamethicin-permeabilized rat liver mitochondria

A simple in situ model of alamethicin-permeabilized isolated rat liver mitochondria was used to investigate the channeling of NADH between mitochondrial malate dehydrogenase (MDH) and NADH:ubiquinone oxidoreductase (complex I). Alamethicin-induced pores in the mitochondrial inner membrane allow effective transport of low molecular mass components such as NAD+/NADH but not soluble proteins. Permeabilized mitochondria demonstrate high rates of respiration in the presence of malate/glutamate and NAD+ due to coupled reaction between MDH and complex I. In the presence of pyruvate and lactate dehydrogenase, an extramitochondrial competitive NADH utilizing system, respiration of permeabilized mitochondria with malate/glutamate and NAD+ was completely abolished. These data are in agreement with the free diffusion of NADH and do not support the suggestion of direct channeling of NADH from MDH to complex I.     

Effects of electron transport inhibitors and uncouplers on denitrification in Paracoccus denitrificans

Abstract Gaschromatographic analysis shows that whole cells of Paracoccus denitrificans produce dinitrogen in the absence and nitrous oxide in the presence of thiocyanate during nitrate reduction. NADH nitrate reductase activity in vesicles is much more sensitive to thiocyanate than either NADH oxidase activity in vesicles or reduction of nitrate by endogenous substrates in whole cells. NADH nitrate reductase activity is not inhibited and NADH oxidase activity is partially inhibited by antimycin A in vesicles. Production of nitrous oxide from nitrate in cells is completely inhibited by the simultaneous presence of thiocyanate and Triton X-100. Carbonylcyanide m -chlorophenylhydrazone does not cause a lag phase in reduction of nitrate by NADH in vesicles, in contrast to the situation in cells.     

Investigation of methaemoglobin reduction by extracellular NADH in mammalian erythrocytes

The effect of extracellular NADH on the rate of reduction of nitrite-induced methaemoglobin in erythrocytes from man, cattle, dog, horse, grey kangaroo, pig and sheep was investigated. Extracellular NADH was found to enhance the rate of methaemoglobin reduction in man, dog, pig and kangaroo erythrocytes, but had essentially no effect on the rate of methaemoglobin reduction in erythrocytes from cattle, horse and sheep. In erythrocytes of those animals affected by extracellular NADH the rate of reduction of metHb in the presence of NADH was the same or greater than that observed in the presence of nutrients such as glucose and inosine. The combination of nutrient and NADH produced a more profound increase in the rate of methaemoglobin reduction. The rate of methaemoglobin reduction in all cases was significantly less than that observed with methylene blue, the standard treatment of methaemoglobinaemia. Extracellular NADH was found to indirectly increase the intracellular NADH concentration through displacement of the pseudo-equilibrium of the intracellular LDH reaction and relied upon the presence of sufficient LDH activity released into the extracellular medium through haemolysis. The lack of response of cattle, horse and sheep RBCs to extracellular NADH was found to derive mainly from their low extracellular LDH activity, but also correlated with their lower NADH-methaemoglobin reductase activity compared to the other species.     

Studies of Auxin Protectors VIII. Evidence that Auxin Protectors Act as Cellular Poisers

Auxin protectors completely inhibit the peroxidase-catalyzed oxidation of indoleacetic acid (IAA). Presumably only when the protector substance itself has been oxidized, does IAA oxidation begin. Reduced nicotinamide-adenine dinucleotide (NADH) mimics the native auxin protectors: In the presence of NADH, the peroxidase-catalyzed oxidation of IAA does not begin until almost all the NADH has been oxidized. Auxin protectors slow the oxidation of NADH in the presence of the peroxidase complex (enzyme plus manganese). However, in the absence of the peroxidase complex, protectors actually accelerate the spontaneous oxidation of NADH. Protectors can also accelerate the oxidation of the dye 2,6-dichlorophenol-indophenol, especially in the presence of manganese. Protector oxidized by boiling with traces of hydrogen peroxide will act as an electron acceptor in the peroxidase-catalyzed oxidation of NADH. The reversible redox role of auxin protectors implies that they can act as cellular poisers.     

Mutagenesis in Salmonella after NADH-dependent microsomal activation of dimethylnitrosamine

The mutagenic activity of dimethylnitrosamine activated by rat-liver microsomes in the presence of NADH was compared with that obtained with NADPH. 3 histidine auxotrophic strains of Salmonella underwent reversions after activation with NADH as the sole coenzyme. All 3 tester strains showed a dose-response relationship with dimethylnitrosamine (10-125 mumoles per plate) after NADH-supported activation. With NADH as the sole coenzyme, the most sensitive strain, hisG46, showed a 105-fold increase in mutagenesis frequency as compared with the 230-fold increase obtained with NADPH. Activation of dimethylnitrosamine in the presence of NADH and NADPH, in combination, produced mutagenesis at frequencies above those seen with NADH alone, but less than or equal to those seen with NADPH as the only coenzyme during the activation step. Experiments in vitro showed that microsomal incorporation of carbon from [14C]dimethylnitrosamine was highest in the presence of NADPH, lowest with NADH and reached intermediate levels when both coenzymes were present. The source of the microsomes in all experiments was liver from rats pre-treated with Aroclor 1254.     

The reduced nicotinamide adenine nucleotide-activated phosphoenolpyruvate carboxylase from Pseudomonas MA. Further studies on regulatory properties.

Phosphoenolpyruvate carboxylase from Pseudomonas MA grown on methylamine as a sole carbon source is an allosteric enzyme activated by NADH. Activation is accompanied by a change in the sedimentation value of the enzyme from 12 S to 9 S. In this paper ADP is shown to be an inhibitor of the enzyme. ADP has its most potent effect on the NADH-activated enzyme. Kinetics of ADP inhibition in the presence of NADH and of NADH activation in the presence of ADP are allosteric. The presence of ADP prevents the decrease in sedimentation value in the presence of NADH. Cross-linking studies indicate that the 12 S form of the enzyme is a tetramer of identically sized subunits and that the 9 S form corresponds to a dimer. The cross-linked enzyme is active and is activated by NADH and inhibited by ADP. It is proposed that NADH and ADP are a regulatory pair for this enzyme and reflect the energy status of the organism, allowing the carboxylase to control the flow of carbon into anabolic or catabolic pathways.     

Amperometric assay of coenzyme-dependent oxidoreductase enzymes in a flow-through cell.

We have demonstrated that a simple electrochemical cell can serve as a detector of NADH concentration in a flow system thereby providing an assay technique for NADH dependent enzymes. When this is applied to NADH produced by enzymatic reaction, then a reproducible measure of enzyme activity is obtained. This method of enzyme activity assay is applicable to a number of oxidoreductase enzymes which employ NAD+ or NADP+ as coenzymes to achieve substrate modification. The presence of electroactive species in samples of human serum has proved a serious problem in the electrochemical analysis of serum activity. These species produce a large background anode current at the anode voltage appropriate for NADH oxidation. The presence of this high current limits the usefulness of amplification of the current output to detect small changes in NADH concentration.     

Conformational changes and the regulation of glutamate-dehydrogenase activity

1. The effect of NADH and the non-competitive inhibitor GTP on the optical-rotatory-dispersion properties of glutamate dehydrogenase has been studied. 2. Analysis of the data in terms of the a(0) and b(0) parameters of the Moffitt-Yang equation indicates that a conformational change is induced either by NADH or by GTP in the presence of small amounts of NADH. 3. Sedimentation measurements under comparable conditions showed that the enzyme reversibly dissociates into sub-units but that this dissociation is only secondary to the conformational changes. 4. Fluorescence measurements showed that the binding constant of NADH and the number of binding sites on the enzyme increased in the presence of GTP. 5. This is confirmed by studies of fluorescence polarization, which in addition showed that the movement of NADH on the enzyme surface is more restricted in the presence of GTP. 6. The relation of these results to possible regulatory mechanisms is discussed.     

Thiols mediate superoxide-dependent NADH modification of glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is covalently modified by NAD in the presence of nitric oxide (NO) and dithiothreitol. Replacement of NAD with NADH in the presence of SIN-1 (3-morpholinosydnonimine) and dithiothreitol increased modification 25-fold. We now demonstrate that in contrast to NO-mediated attachment of NAD, covalent attachment of NADH to GAPDH proceeds in the presence of low molecular weight thiols, independent of NO. Removal of oxygen and transition metal ions inhibited modification, consistent with a role for reactive oxygen species; inhibition by superoxide dismutase, stimulation by xanthine oxidase/hypoxanthine, and the lack of an effect of catalase supported the hypothesis that superoxide, generated from thiol oxidation, was involved. Electrospray mass spectrometry showed covalent linkage of the NADH molecule to GAPDH. Characterization of the product of phosphodiesterase cleavage demonstrated that linkage to GAPDH occurred through the nicotinamide of NADH. Lys-C digestion of GAPDH, followed by peptide isolation by high performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight analysis, and Edman sequencing, demonstrated that NADH attachment occurred at Cys-149, the active-site thiol. This thiol linkage was stable to HgCl2. Thus, linkage of GAPDH to NADH, in contrast to NAD, occurs in the presence of thiol, is independent of NO, and is mediated by superoxide.     

Reversible inactivation of maize leaf nitrate reductase

Preincubation of maize leaves crude extracts with NADH resulted in a progressive accumulation of nitrite which mimicked a rapid and lineal activation of nitrate reductase. Nevertheless, in partially purified preparations it was found that preincubation at pH 8.8 with NADH promoted a gradual inactivation of nitrate reductase. At pH 7.5, the enzyme was not inactivated by the presence of NADH alone, but, with the simultaneous presence of a low concentration of cyanide, a fast inactivation took place. The NADH-cyanide-inactivated nitrate reductase remained inactive after removing the excess of NADH and cyanide by filtration through Sephadex G-25. However, it could be readily reactivated by incubation with ferricyanide or by a short exposure to light in the presence of FAD. Prolonged irradiation caused a progressive inactivation of the photoreactivated enzyme.     

Enzymatic and energetic properties of the aerobic respiratory chain-linked NADH oxidase system in the marine bacterium Pseudomonas nautica

Membranes of Pseudomonas nautica, grown aerobically on a complex medium, oxidized both NADH and deamino-NADH as substrates. The activity of membrane-bound NADH oxidase was activated by monovalent cations including Na+, Li+, and K+. The activation by Na+ was higher than that by Li+ and K+. The maximum activity of NADH oxidase was obtained at about pH 9.0 in the presence of 0.08 M NaCl. The NADH oxidase activity was completely inhibited by 60 microM 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), while the NADH:quinone oxidoreductase activity was about 37% inhibited by 60 microM HQNO. The activities of NADH oxidase and NADH:quinone oxidoreductase were about 40% inhibited by 60 microM rotenone. The fluorescence quenching technique revealed that electron transfer from NADH to ubiquinone-1 (Q-1) or oxygen generated a membrane potential (deltapsi) which was larger and more stable in the presence of Na+ than in the absence of Na+. However, the All was highly sensitive to a protonophore, carbonyl-cyanide m-chlorophenylhydrazone (CCCP) even at alkaline pH.     

HQNO-sensitive NADH:quinone oxidoreductase of Bacillus cereus KCTC 3674

The enzymatic properties of NADH:quinone oxidoreductase were examined in Triton X-100 extracts of Bacillus cereus membranes by using the artificial electron acceptors ubiquinone-1 and menadione. Membranes were prepared from B. cereus KCTC 3674 grown aerobically on a complex medium and oxidized with NADH exclusively, whereas deamino-NADH was determined to be poorly oxidized. The NADH oxidase activity was lost completely by solubilization of the membranes with Triton X-100. However, by using the artificial electron acceptors ubiquinone-1 and menadione, NADH oxidation could be observed. The activities of NADH:ubiquinone-1 and NADH:menadione oxidoreductase were enhanced approximately 8-fold and 4-fold, respectively, from the Triton X-100 extracted membranes. The maximum activity of FAD-dependent NADH:ubiquinone-1 oxidoreductase was obtained at about pH 6.0 in the presence of 0.1M NaCl, while the maximum activity of FAD-dependent NADH:menadione oxidoreductase was obtained at about pH 8.0 in the presence of 0.1 M NaCl. The activities of the NADH:ubiquinone-1 and NADH:menadione oxidoreductase were very resistant to such respiratory chain inhibitors as rotenone, capsaicin, and AgNO(3), whereas these activities were sensitive to 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Based on these results, we suggest that the aerobic respiratory chain-linked NADH oxidase system of B. cereus KCTC 3674 possesses an HQNO-sensitive NADH:quinone oxidoreductase that lacks an energy coupling site containing FAD as a cofactor.     

Inhibition of hepatic adenylate cyclase by NADH

Adenylate cyclase activity in isolated rat liver plasma membranes was inhibited by NADH in a concentration-dependent manner. Half-maximal inhibition of adenylate cyclase was observed at 120 microM concentration of NADH. The effect of NADH was specific since adenylate cyclase activity was not altered by NAD+, NADP+, NADPH, and nicotinic acid. The ability of NADH to inhibit adenylate cyclase was not altered when the enzyme was stimulated by activating the cyclase was not altered when the enzyme was stimulated by activating the Gs regulatory element with either glucagon or cholera toxin. Similarly, inhibition of Gi function by pertussis toxin treatment of membranes did not attenuate the ability of NADH to inhibit adenylate cyclase activity. Inhibition of adenylate cyclase activity to the same extent in the presence and absence of the Gpp (NH) p suggested that NADH directly affects the catalytic subunit. This notion was confirmed by the finding that NADH also inhibited solubilized adenylate cyclase in the absence of Gpp (NH)p. Kinetic analysis of the NADH-mediated inhibition suggested that NADH competes with ATP to inhibit adenylate cyclase; in the presence of NADH (1 mM) the Km for ATP was increased from 0.24 +/- 0.02 mM to 0.44 +/- 0.08 mM with no change in Vmax. This observation and the inability of high NADH concentrations to completely inhibit the enzyme suggest that NADH interacts at a site(s) on the enzyme to increase the Km for ATP by 2-fold and this inhibitory effect is overcome at high ATP concentrations.     

Stimulation of NADH oxidation by xanthine oxidase and polyvanadate in presence of some dehydrogenases and flavin compounds

The rates of NADH oxidation in presence of xanthine oxidase increase to a small and variable extent on addition of high concentrations of lactate dehydrogenase and other dehydrogenases. This heat stable activity is similar to polyvanadate-stimulation with respect to pH profile and SOD sensitivity. Isocitric dehydrogenase (NADP-specific) showed heat labile, SOD-sensitive polyvanadate-stimulated NADH oxidation activity. Polyvanadate-stimulated SOD-sensitive NADH oxidation was also found to occur with riboflavin, FMN and FAD in presence of a non-specific protein, BSA, suggesting that some flavoproteins may possess this activity.