A study on the mechanism of the vanadate-dependent NADH oxidation

The mechanism of the vanadate (V(_v))-dependent oxidation of NADH was different in phosphate buffers and in phosphate-free media. In phosphate-free media (aqueous medium or HEPES buffer) the vanadyl (V(_v)) generated by the direct V(_v)-dependent oxidation of NADH formed a complex with V(_v). In phosphate buffers V(_v) autoxidized instead of forming a complex with V(_v). The generated superoxide radical (O₂⁻) initiated, in turn, a high-rate free radical chain oxidation of NADH. Phosphate did not stimulate the V(_v)-dependent NADH oxidation catalyzed by O₂⁻-generating systems. Monovanadate proved to be a stronger catalyzer of NADH oxidation as compared to polyvanadate.     

Importance of hydroxyl radical in the vanadium-stimulated oxidation of NADH

Vanadium compounds are known to stimulate the oxidation of NAD(P)H, but the mechanism remains unclear. This reaction was studied spectrophotometrically and by electron spin resonance spectroscopy (ESR) using vanadium in the reduced state (+4, vanadyl) and the oxidized state (+5, vanadate). In 25 mM sodium phosphate buffer at pH 7.4, vanadyl was slightly more effective in stimulating NADH oxidation than was vanadate. Addition of a superoxide generating system, xanthine/xanthine oxidase, resulted in a marked increase in NADH oxidation by vanadyl, and to a lesser extent, by vanadate. Decreasing the pH with superoxide present increased NADH oxidation for both vanadate and vanadyl. Addition of hydrogen peroxide to the reaction mixture did not change the NADH oxidation by vanadate, regardless of concentration or pH. With vanadyl however, addition of hydrogen peroxide greatly enhanced NADH oxidation which further increased with lower pH. Use of the spin trap DMPO in reaction mixtures containing vanadyl and hydrogen peroxide or a superoxide generating system resulted in the detection by ESR of hydroxyl. In each case, the hydroxyl radical signal intensity increased with vanadium concentration. Catalase was able to inhibit the formation of the DMPO--OH adduct formed by vanadate plus superoxide. These results show that the ability of vanadium to act in a Fenton-type reaction is an important process in the vanadium-stimulated oxidation of NADH.     

Characterization of vanadate-dependent NADH oxidation stimulated by Saccharomyces cerevisiae plasma membranes.

Plasma membrane-stimulated vanadate-dependent NADH oxidation has been characterized in Saccharomyces cerevisiae. This activity is specific for vanadate, because molybdate, a similar metal oxide, did not substitute for vanadate in the reaction. Vanadate-dependent plasma membrane-stimulated NADH oxidation activity was dependent on the concentrations of vanadate, NADH, and NADPH and required functional plasma membranes; no stimulation occurred in the presence of boiled membranes or bovine serum albumin. The dependence of membrane-stimulated vanadate-dependent NADH oxidation was not linearly dependent on added membrane protein. The activity was abolished by the superoxide anion scavenger superoxide dismutase and was stimulated by paraquat and NADPH. These data are consistent with the previously proposed chain reaction for vanadate-dependent NADH oxidation. The role of the plasma membrane appears to be to stimulate superoxide radical formation, which is coupled to NADH oxidation by vanadate. 51V-nuclear magnetic resonance studies are consistent with the hypothesis that a phosphovanadate anhydride is the stimulatory oxyvanadium species in the phosphate buffers used at pHs 5.0 and 7.0. In phosphate buffers, compared with acetate buffers, the single vanadate resonance was shifted upfield at both pH 5.0 and pH 7.0, which is characteristic of the phosphovanadate anhydride. Since the cell contains an excess of phosphate to vanadate, the phosphovanadate anhydride may be involved in membrane-mediated vanadate-dependent NADH oxidation in vivo.     

The oxidation of NADH by vanadate plus sugars

Sugars and sugar phosphates enable vanadate to catalyze the oxidation of NADH. Superoxide dismutase inhibits this oxidation. Incubation of sugars with vanadate, prior to addition of NADH, accelerates this oxidation of subsequently added NADH and eliminates the lag phase otherwise noted. Incubation of sugars with vanadate also results in the reduction of vanadate to vanadyl, with appearance of a blue-green color probably associated with a vanadyl-vanadate complex. It appears that sugars reduce vanadate to vanadyl which, in turn, reduces O2 to O2- and that vanadate plus O2- then catalyzes the oxidation of NAD(P)H by a free radical chain reaction. Such oxidation of NAD(P)H may account for several of the biological effects of vanadate.     

The oxidatin of NADH by vanadate plus sugars

Sugars and sugar phosphates enable vanadate to catalyze the oxidation of NADH. Superoxide dismutase inhibits this oxidation. Incubation of sugars with vanadate, prior to addition of NADH, accelerates this oxidation of subsequently added NADH and eliminates the lag phase otherwise noted. Incubation of sugars with vanadate also results in the reduction of vanadate to vanadyl, with appearance of a blue-green color probably associated with a vanadyl-vanadate complex. It appears that sugars reduce vanadate to vanadyl which, in turn, reduces O₂ to O₂⁻ and that vanadate plus O₂⁻ then catalyzes the oxidation of NAD(P)H by a free radical chain reaction. Such oxidation of NAD(P)H may account for several of the biological effects of vanadate.     

Vanadate-stimulated NADH oxidation in microsomes

Addition of vanadate, stimulated oxidation of NADH by rat liver microsomes. The products were NAD⁺ and H₂O₂. High rates of this reaction were obtained in the presence of phosphate buffer and at low pH values. The yellow-orange colored polymeric form of vanadate appears to be the active species and both ortho- and meta-vanadate gave poor activities even at mM concentrations.The activity as measured by oxygen uptake was inhibited by cyanide, EDTA, mannitol, histidine, ascorbate, noradrenaline, adriamycin, cytochrome c, Mn²⁺, superoxide dismutase, horseradish peroxidase and catalase. Mitochondrial outer membranes possess a similar activity of vanadate-stimulated NADH oxidation. But addition of mitochondria and some of its derivative particles abolished the microsomal activity. In the absence of oxygen, disappearance of NADH measured by decrease in absorbance at 340 nm continued at nearly the same rate since vanadate served as an electron acceptor in the microsomal system. Addition of excess catalase or SOD abolished the oxygen uptake while retaining significant rates of NADH disappearance indicating that the two activities are delinked. A mechanism is proposed wherein oxygen receives the first electron from NAD radical generated by oxidation of NADH by phosphovanadate and the consequent reduced species of vanadate (V^iv) gives the second electron to superoxide to reduce it H₂O₂. This is applicable to all membranes whereas microsomes have the additional capability of reducing vanadate.     

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

Summary Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK _equil of 6 × 10^–17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.Abbreviations AFR ascorbate free radical - DHA dehydroascorbic acid - EDTA ethylenediaminetetraacetic acid - DTPA diethylenetri-aminepentaacetic acid - TEMPO 2,2,6,6-tetramethylpiperidinoxy     

A vanadate-stimulated NADH oxidase in erythrocyte membrane generates hydrogen peroxide

Summary Oxidation of NADH by rat erythrocyte plasma membrane was stimulated by about 50-fold on addition of decavanadate, but not other forms of vanadate like orthovanadate, metavanadate aad vanadyl sulphate. The vanadate-stimulated activity was observed only in phosphate buffer while other buffers like Tris, acetate, borate and Hepes were ineffective. Oxygen was consumed during the oxidation of NADH and the products were found to be NAD⁺ and hydrogen peroxide. The reaction had a stoichiometry of one mole of oxygen consumption and one mole of H₂O₂ production for every mole of NADH that was oxidized.Superoxide dismutase and manganous inhibited the activity indicating the involvement of superoxide anions. Electron spin resonance in the presence of a spin trap, 5, 5-dimethyl pyrroline N-oxide, indicated the presence of superoxide radicals. Electron spin resonance studies also showed the appearance of V^IV species by reduction of V^V of decavanadate indicating thereby participation of vanadate in the redox reaction. Under the conditions of the assay, vanadate did not stimulate lipid peroxidation in erythrocyte membranes. Extracts from lipid-free preparations of the erythrocyte membrane showed full activity. This ruled out the possibility of oxygen uptake through lipid peroxidation. The vanadate-stimulated NADH oxidation activity could be partially solubilized by treating erythrocyte membranes either with Triton X-100 or sodium cholate. Partially purified enzyme obtained by extraction with cholate and fractionation by ammonium sulphate and DEAE-Sephadex was found to be unstable.     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

Vanadate-stimulated NADH oxidation in plasma membrane

The rate of NADH oxidation with oxygen as the acceptor is very low in mouse liver plasma membrane and erythrocyte membrane. When vanadate is added, this rate is stimulated 10- to 20-fold. The absorption spectrum of vanadate does not change with the disappearance of NADH. The reaction is inhibited by superoxide dismutase, and there is no activity under an argon atmosphere. This indicates that oxygen is the electron acceptor and the reaction is mediated by superoxide. The vanadate stimulation is not limited to plasma membrane. Golgi apparatus and endoplasmic reticulum show similar increase in NADH oxidase activity when vanadate is added. The endomembranes have significant vanadate-stimulated activity with both NADH and NADPH. The vanadate-stimulated NADH oxidase in plasma membrane is inhibited by compounds, which inhibit NADH dehydrogenase activity: catechols, anthracycline drugs and manganese. This activity is stimulated by high phosphate and sulfate anion concentrations.     

Vanadate-stimulated NADH oxidation by xanthine oxidase: an intrinsic property

Vanadate-dependent oxidation of NADH by xanthine oxidase does not require the presence of xanthine and therefore is not due to cooxidation. Addition of NADH or xanthine had no effect on the oxidation of the other substrate. Oxidation of NADH was high at acid pH and oxidation of xanthine was high at alkaline pH. The specific activity was relatively very high with NADH. Concentration-dependent oxidation of NADH Concentration-dependent oxidation of NADH was obtained in the presence of the polymeric form of vanadate, but not orthovanadate or metavanadate. Both NADH and NADPH were oxidized, as in the nonenzymatic system. Oxidation of NADH, but not xanthine, was inhibited by KCN, ascorbate, MnCl2, cytochrome c, mannitol, Tris, epinephrine, norepinephrine, and triiodothyronine. Oxidation of NADH was accompanied by uptake of oxygen and generation of H2O2 with a stoichiometry of 1:1:1 for NADH:O2:H2O2. A 240-nm-absorbing species was formed during the reaction which was different from H2O2 or superoxide. A mechanism of NADH oxidation is suggested wherein Vv and O2 receive one electron each successively from NADH followed by VIV giving the second electron to superoxide and reducing it to H2O2.     

Ascorbate free radical and its role in growth control

Summary Ascorbate and its free radical potentiates proliferation of HL-60 cells in serum-limiting media. Dehydroascorbate does not affect growth. This stimulation of growth is due to a general shortening of the cell cycle. The incubation of HL-60 cells with ascorbate free radical produces a significant change of the redox potential of cells. The presence of cells in culture media avoids the total oxidation of ascorbate, and also HL-60 cells induce the short-term stabilization of ascorbate. Ascorbate free radical potentiates also the onset of DNA synthesis in CCL39 cells induced by fetal calf serum, although itself does not affect quiescense to proliferation transition. This transition induced by fetal calf serum also potentiates the capacity of CCL39 cells to stabilize ascorbate. We discuss here the role of ascorbate free radical on growth control by its reduction by the plasma membrane redox system and its meaning for cell physioslogy.     

Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells

Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H₂O₂ to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H₂O₂ was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.     

The oxidation of NADH by tetravalent vanadium

Vanadyl (V_(IV)) salts autoxidize in neutral aqueous solution yielding O₂⁻ plus vanadate (V_(V)) and these, in turn, cause the oxidation of NADH, by a free radical chain reaction. This oxidation of NADH was inhibited by superoxide dismutase, but not by a scavenger of HO.. When H₂O₂ was present V_(IV)) caused rapid oxidation of NADH by a process which was unaffected by superoxide dismutase but was inhibited by a scavenger of HO.. This appeared to be dependent upon reduction of H₂O₂ to OH⁻ plus HO., by V_(IV)), followed by oxidation of NADH by HO.. Since there are reductants, within cells, capable of reducing V_(V)) to V_(IV), these reactions are likely to contribute to the toxicity of vanadate.     

Erythrocytes reduce extracellular ascorbate free radicals using intracellular ascorbate as an electron donor

Ascorbate is readily oxidized in aqueous solution by ascorbate oxidase. Ascorbate radicals are formed, which disproportionate to ascorbate and dehydroascorbic acid. Addition of erythrocytes with increasing intracellular ascorbate concentrations decreased the oxidation of ascorbate in a concentration-dependent manner. Concurrently, it was found, utilizing electron spin resonance spectroscopy, that extracellular ascorbate radical levels were decreased. Control experiments showed that these results could not be explained by leakage of ascorbate from the cells, inactivation of ascorbate oxidase, or oxygen depletion. Thus, this means that intracellular ascorbate is directly responsible for the decreased oxidation of extracellular ascorbate. Exposure of ascorbate-loaded erythrocytes to higher levels of extracellular ascorbate radicals resulted in the detection of intracellular ascorbate radicals. Moreover, efflux of dehydroascorbic acid was observed under these conditions. These data confirm the view that intracellular ascorbate donates electrons to extracellular ascorbate free radical via a plasma membrane redox system. Such a redox system enables the cells to effectively counteract oxidative processes and thereby prevent depletion of extracellular ascorbate.     

Inactivation of glucose oxidase by diperoxovanadate-derived oxidants.

Inactivation of glucose oxidase occurred in the presence of bromide, vanadate, H(2)O(2), and phosphate (the bromide system), and this was prevented by NADH or phenol red, a bromine acceptor. Glucose oxidase present during the reaction between diperoxovanadate and a reduced form of vanadate, vanadyl (the vanadyl system), but not added after mixing the reactants, was inactivated, and this was accompanied by a loss of binding of the dye, Coomassie blue, to the protein. The transient intermediate of the type OVOOV(O(2)), known to form in these reactions and used in the oxidation of bromide ion and NADH, appears to be responsible for inactivating glucose oxidase. In both systems, the inactivation of the enzyme was prevented by histidine and DTT, known to quench singlet-oxygen. By direct measurement of 1270-nm emission of singlet-oxygen, its generation was demonstrated in the bromide system, and in the reaction of hypohalous acids with diperoxovanadate, but not in the vanadyl system. By themselves both hypohalous acids, HOCl and HOBr inactivated glucose oxidase, and their prior reaction with H(2)O(2) during which singlet-oxygen was released, protected the enzyme. The results provide support for possible oxidative inactivation of glucose oxidase by diperoxovanadate-derived oxidants.     

Initiation of a superoxide-dependent chain oxidation of lactate dehydrogenase-bound NADH by oxidants of low and high reactivity

In cells, NADH and NADPH are mainly bound to dehydrogenases such as lactate dehydrogenase (LDH). In cell-free systems, the binary LDH-NADH complex has been demonstrated to produce reactive oxygen species via a chain oxidation of NADH initiated and propagated by superoxide. We studied here whether this chain radical reaction can be initiated by oxidants other than LDH largely increased the oxidation of NADH (but not of NADPH) by O(2), H(2)O(2) and during the intermediacy of HNO(2). LDH also increased the oxidation of NADH by peroxynitrite. The increases in NADH oxidation were completely prevented by superoxide dismutase (SOD). In contrast, the nitrogen dioxide-dependent oxidation of NADH and NADPH was decreased by LDH in a SOD-independent manner. These experimental data strongly indicate that oxidation of LDH-bound NADH can be induced from reaction of either weak oxidants with LDH-bound NADH or of strong oxidants with free NADH thus yielding which is highly effective to propagate the chain. Our results underline the importance of SOD in terminating superoxide-dependent chain reactions in cells under oxidative stress.     

Effects of vanadate on the oxidation of NADH by xanthine oxidase

Vanadate (V(V)) stimulates the oxidation of NADH by xanthine oxidase and superoxide dismutase eliminates the effect of V(V). Paraquat stimulates both the oxidation of NADH by xanthine oxidase and the V(V) enhancement of that oxidation. Xanthine, which is a better substrate for xanthine oxidase than is NADH, causes a V(V)-dependent co-oxidation of NADH which is transient and eliminated by SOD. Urate inhibits the V(V)-stimulated oxidation of NADH by xanthine oxidase or by Rose Bengal plus light. Measurement of rates of both O2- production and V(V)-stimulated NADH oxidation showed that many molecules of NADH were oxidized per O2-. These chain lengths were an inverse function of overall reaction rate. Minimum chain lengths, calculated on the basis of 100% univalent reduction of O2 to O2-, were smaller than measured average chain lengths by a factor of five. All of these results are in accord with the view that V(V) does not directly affect the activity of the enzyme, but rather catalyzes the free radical chain oxidation of NADH by O2-. It was further shown that phosphate was not involved and that the active form of V(V) was orthovanadate, rather than decavanadate.     

Reaction of vanadate ion with chlorpromazine,formation of vanadyl ion and chlorpromazine free radical

The red colored product, which was identified as a chlorpromazine (CPZ) free radical, was observed in the reaction of CPZ with the vanadate ion (+5 oxidation state). The product and the mechanism for the reaction were characterized from optical and EPR spectrometries. Optimal conditions for generation of the free radical were determined as reaction time within one minute of pH 6 and free radical stabilizing time of 30 minutes by acidifying with HCl. Under these conditions, the stoichiometry for the reaction was found to be 1:1, indicating the involvement of one electron transfer from CPZ to the vanadate ion to form the free radical and vanadyl ion (+4 oxidation state). A possible reaction scheme was proposed:

The implications of this reaction were discussed with regard to the pharmacological action of the vanadate ion and CPZ.