Inhibition of (Na,K)-ATPase by tetravalent vanadium

Vanadyl, the tetravalent state of vanadium and a divalent cation, VO2+, was a relatively powerful inhibitor of highly purified membrane-bound sodium and potassium ion transport adenosine triphosphatase. The sensitivity of the ATPase activity to vanadyl characteristically correlated positively with the specific activity of the enzyme preparation. Inhibition ranged from nearly complete inhibition at less than 5 microM vanadyl for some of the purest fractions (specific activity approximately 45 mumol/min/mg of protein) to no observable inhibition at 300 microM vanadyl in one crude preparation of the enzyme with a specific activity of 10 mumol/min/mg of protein. The level of free vanadyl was reduced by incubation with these membranes, but this reduction was not sufficient to account for the low sensitivity to vanadyl observed in crude preparations. A reduction in specific activity by partial inactivation of a sensitive preparation by treatment with FeCl3 and ascorbate reduced its sensitivity to vanadyl. Anionic ligands of the enzyme, vanadate or ATP, increased the rate of recovery from inhibition after chelation of free vanadyl. At pH 6.1, the inhibition was characteristically fully reversible (t1/2 approximately 10 min), whereas at pH 8.1 it was stable for hours. The degree and stability of enzyme inhibition by vanadyl increased for several hours during incubation of the vanadyl-enzyme mixture, and at pH 6.1 the properties of the inhibitor itself also changed with time. Preincubation of the ion at that pH for 5 h before addition of the enzyme produced a more stable inhibition. The time- and pH-dependent changes in the degree and stability of enzyme inhibition probably relate to the complex chemistry of the vanadyl ion in solution.     

Enhanced sensitivity of insulin-resistant adipocytes to vanadate is associated with oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4).

Vanadate (sodium orthovanadate), an inhibitor of phosphotyrosine phosphatases (PTPs), mimics many of the metabolic actions of insulin in vitro and in vivo. The potential of vanadate to stimulate glucose transport independent of the early steps in insulin signaling prompted us to test its effectiveness in an in vitro model of insulin resistance. In primary rat adipocytes cultured for 18 h in the presence of high glucose (15 mm) and insulin (10(-7) m), sensitivity to insulin-stimulated glucose transport was decreased. In contrast, there was a paradoxical enhanced sensitivity to vanadate of the insulin-resistant cells (EC(50) for control, 325 +/- 7.5 microm; EC(50) for insulin-resistant, 171 +/- 32 microm; p < 0.002). Enhanced sensitivity was also present for vanadate stimulation of insulin receptor kinase activity and autophosphorylation and Akt/protein kinase B Ser-473 phosphorylation consistent with more effective PTP inhibition in the resistant cells. Investigation of this phenomenon revealed that 1) depletion of GSH with buthionine sulfoximine reproduced the enhanced sensitivity to vanadate while preincubation of resistant cells with N-acetylcysteine (NAC) prevented it, 2) intracellular GSH was decreased in resistant cells and normalized by NAC, 3) exposure to high glucose and insulin induced an increase in reactive oxygen species, which was prevented by NAC, 4) EPR (electron paramagnetic resonance) spectroscopy showed a decreased amount of vanadyl (+4) in resistant and buthionine sulfoximine-treated cells, which correlated with decreased GSH and increased vanadate sensitivity, while total vanadium uptake was not altered, and 5) inhibition of recombinant PTP1B in vitro was more sensitive to vanadate (+5) than vanadyl (+4). In conclusion, the paradoxical increased sensitivity to vanadate in hyperglycemia-induced insulin resistant adipocytes is due to oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4). Thus, sensitivity of PTP inhibition and glucose transport to vanadate is regulated by cellular redox state.     

A novel effect of vanadium ions: inhibition of succinyl-CoA synthetase

Effect of vanadate and vanadyl ions on the ATP-dependent succinyl-CoA synthetase (A-SCS) solubilized by Lubrol-PX from the rat brain mitochondria was tested. Vanadate added to the assay medium at 10(-5) mol.l-1 and 10(-4) mol.l-1 concentrations inhibited the enzyme activity by about 50% and 94%, respectively. When the enzyme was solubilized from the mitochondria preincubated with 10(-4) mol.l-1 and 10(-3) mol.l-1 vanadate, the residual inhibitions were 55% and 100% respectively. The vanadyl cation also induced inhibition of the A-SCS activity but the effect was less expressed. At 10(-4) mol.l-1 concentration only 20% inhibition was achieved. The A-SCS solubilized from the mitochondrial subfractions (perikaryal, light and heavy synaptosomal) differed neither in the activity of A-SCS nor in the susceptibility toward action of vanadium ions. A strong dependence of the vanadate inhibition on the concentration of succinate was observed. The above effect (50% inhibition) could be demonstrated only at saturating concentration of succinate (50 mmol.l-1). The mechanism of vanadium ions action as well as differences between vanadate and vanadyl ions effects are discussed.     

Mechanism of inhibition of glycolysis by vanadate

Vanadate is known to inhibit several phosphatases including Na+, K+-ATPase, alkaline phosphatase, and glyceraldehyde-3-P dehydrogenase. Inhibition presumably results because vanadium adopts a stable structure which resembles the transition state of phosphate during the reactions involving these enzymes. We performed experiments to further examine the effects of vanadate (VO3-4) on erythrocyte (red blood cells (RBC] glycolytic intermediates. RBC obtained from human subjects were centrifuged and washed with lactated Ringer's 5% dextrose. 31P nuclear magnetic resonance analysis of the RBC revealed the characteristic peaks for the 3-phosphate and 2-phosphate of 2,3-diphosphoglycerate (DPG), inorganic phosphate (Pi), and ATP. Incubation of RBC with 10(-6) M VO3-4 led to a disappearance of ATP and 2,3-DPG while the peak for Pi increased. By the end of 4 h over 90% of the VO3-4 had been reduced to VO2+ (vanadyl) in the RBC. The effects of 10(-4) M iodoacetamide and 10(-5) M ethacrynic acid, known inhibitors of glyceraldehyde-3-P dehydrogenase that act by interactions with sulfhydryl groups (-SH) of the enzyme, were similar to those of VO3-4. Incubation with vanadyl did not affect the peaks for Pi, 2-DPG, or 3-DPG. Furthermore, using electron spin resonance we demonstrated that in the presence of glyceraldehyde-3-P dehydrogenase, VO3-4 is reduced to VO2+. The findings demonstrate that VO3-4 inhibits glycolysis at micromolar concentrations and that the ion is reduced to VO2+ in the cell. The similarity of the effect of VO3-4 to those of iodoacetamide and ethacrynic acid suggests that interactions with -SH groups is its mechanism of inhibition. Since under physiological conditions intracellular VO3-4 concentrations are in the micromolar range and may exist in oxidized and/or reduced forms, VO3-4 could regulate the activity of glyceraldehyde-3-P dehydrogenase through changes in the redox state of the enzyme rather than by substituting for the PO3-4 ion.     

Recent advances into vanadyl, vanadate and decavanadate interactions with actin

Although the number of papers about "vanadium" has doubled in the last decade, the studies about "vanadium and actin" are scarce. In the present review, the effects of vanadyl, vanadate and decavanadate on actin structure and function are compared. Decavanadate (51)V NMR signals, at -516 ppm, broadened and decreased in intensity upon actin titration, whereas no effects were observed for vanadate monomers, at -560 ppm. Decavanadate is the only species inducing actin cysteine oxidation and vanadyl formation, both processes being prevented by the natural ligand of the protein, ATP. Vanadyl titration with monomeric actin (G-actin), analysed by EPR spectroscopy, reveals a 1:1 binding stoichiometry and a K(d) of 7.5 μM(-1). Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC(50) of 68 and 300 μM, respectively, as analysed by light scattering assays, whereas no effects were detected for vanadate up to 2 mM. However, only vanadyl (up to 200 μM) induces 100% of G-actin intrinsic fluorescence quenching, whereas decavanadate shows an opposite effect, which suggests the presence of vanadyl high affinity actin binding sites. Decavanadate increases (2.6-fold) the actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Both vanadium species increased the ε-ATP exchange rate (k = 6.5 × 10(-3) s(-1) and 4.47 × 10(-3) s(-1) for decavanadate and vanadyl, respectively). Finally, (1)H NMR spectra of G-actin treated with 0.1 mM decavanadate clearly indicate that major alterations occur in protein structure, which are much less visible in the presence of ATP, confirming the preventive effect of the nucleotide on the decavanadate interaction with the protein. Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl. By affecting actin structure and function, vanadium can regulate many cellular processes of great physiological significance.     

Bleomycin stimulates both membrane (Na+-K+) ATPase and electrogenic (Na+-K+) pump and partially removes the inhibition by vanadium ions

Bleomycin 2 X 10(-6) and 6 X 10(-6) mol.1(-1) increased the activity of specific (Na+-K+) ATPase of the rat brain microsomes. It also stimulated the electrogenic (Na+-K+) pump in intact skeletal muscle cells. The blocking effect of vanadyl (+4V) on membrane (Na+-K+) ATPase was eliminated completely by the drug, but the action of vanadate (+5V) was counteracted only partially. Electron paramagnetic resonance spectra revealed the formation of a +4V - bleomycin complex which is still able to activate the (Na+-K+) ATPase.     

Reduction of vanadate to vanadyl by a strain of Saccharomyces cerevisiae

Three strains of Saccharomyces cerevisiae, SC-1, DBVPG 6173 and DBVPG 6037, were studied for vanadate resistance in complex Sabouraud medium since they did not thrive in different minimal media (yeast nitrogen base with and without amino acids). The strain SC-1 was resistant up to 16 mm of vanadate, whereas the strains DBVPG 6173 and DBVPG 6037 were inhibited by 8 mm and 4 mm vanadate, respectively. The vanadate resistance in strain SC-1 was constitutive and due to the reduction of this oxyanion to vanadyl, which was detected by EPR spectroscopy and visible spectroscopy. The transformation of vanadate to vanadyl took place during the exponential growth phase; 10 mm of vanadate was reduced to vanadyl outside the cells since the oxyanion was not detected in the cell biomass and only a negligible concentration of vanadyl (25 nmoles mg cells dry weight) was found in the biomass. The other two vanadate-sensitive yeast strains only accumulated vanadate and did not reduce the oxyanion to vanadyl.     

Evidence for a regulatory action of vanadate on protein phosphorylation in brain microvessels

We investigated the action of vanadate on protein phosphorylation in microvessels isolated from rat brain. We found that a stimulation of protein phosphorylation from 32P-ATP occurs, in the presence of different concentrations of vanadate, 10(-3) M being the most effective dose. This action was time-dependent, and it was more evident after 60 s of treatment. The contribution of ATPase inhibition caused by vanadate appears to be negligible. In addition a stimulation of cAMP-dependent protein kinase activity was observed. The pattern of protein phosphorylation showed that exposure to 10(-3) M vanadate resulted in a nonspecific stimulation of protein phosphorylation concomitantly with a selective inhibition of the 55 KDa protein phosphorylation. The nature of this protein is also discussed.     

Metabolism of added orthovanadate to vanadyl and high-molecular-weight vanadates by Saccharomyces cerevisiae

The effect of vanadium oxides on living systems may involve the in vivo conversion of vanadate and vanadyl ions. The addition of 5 mM orthovanadate (VO4(3-), V(V)), a known inhibitor of the (Na,K)-ATPase, to yeast cells stopped growth. In contrast, the addition of 5 mM vanadyl (VO2+, V(IV) stimulated growth. Orthovanadate addition to whole cells is known to stimulate various cellular processes. In yeast, both ions inhibited the plasma membrane Mg2+ ATPase and were transported into the cell as demonstrated with [48V]VO4(3-) and VO2+. ESR spectroscopy has been used to measure the cell-associated paramagnetic vandyl ion, while 51V NMR has detected cell-associated diamagnetic vanadium (e.g. V(V)). Cells were exposed to both toxic (5 mM) and nontoxic (1 mM) concentrations of vanadate in the culture medium. ESR showed that under both conditions, vanadate became cell associated and was converted to vanadyl which then accumulated in the cell culture medium. 51V NMR studies showed the accumulation of new cell-associated vanadium resonances identified as dimeric vanadate and decavanadate in cells exposed to toxic amounts of medium vanadate (5 mM). These vanadate compounds did not accumulate in cells exposed to 1 mM vanadate. These studies confirm that the inhibitory form of vanadium usually observed in in vitro experiments is vanadate, in one or more of its hydrated forms. These data also support the hypothesis that the stimulatory form of vanadium usually observed in whole cell experiments is the vanadyl ion or one or more of its liganded derivatives.     

Do vanadium ions exert any specific effect on brain protein phosphrylation

It has been shown previously (1) that vanadate stimulates phosphorylation of the overall protens from the synaptic membranes of rat cerebral cortex. The aim of the present experiments was to investigate whether the action of vanadate and also of vanadyl ions could exert any specific effect on endogenous phosphorylation of proteins from subcellular fractions of the rat brain cortex. Both vanadate and vanadyl ions stimulate phosphorylation of the overall proteins from synaptic membranes and to lesser extent from mitochondria. An attempt was made to estimate the contribution of inhibition of ATPase activity to nonspecific stimulation of phosphate labeling in the synaptic membrane fraction. A band of M_r approx. 37 kDa from synaptic membranes was particularly sensitive to vanadate. In mitochondria both vanadate and vanadyl caused a marked, concentration dependent inhibition of phosphorylation of a band corresponding to M_r approx. 34 kDa. The effect was confined exclusively to the mitochondrial fractions (total, perikaryal and two synaptic types). It was absent in all subcellular fractions tested, including the nuclear one. Phosphorylation of the mitochondrial 34 kDa band is not influenced by cyclic AMP, Ca-calmodulin, shift of pH from 6.6 to 8.1. Alkaline hydrolysis removed almost all phosphatelabeled bands of mitochondria, including that of 34 kDa.     

Inhibition of insulin receptors by vanadate and ouabain

Insulin binding studies were performed, using cells from 5 non-obese, non-diabetic subjects, on four separate days: 2 were paired control studies to demonstrate precision, and 2 other sets were binding studies in which one incubation solution was a control and the other contained either vanadate, (10(-4) M) or ouabain (10(-4) M). For both substances tracer binding of 125I insulin was reduced significantly, 27% by vanadate and 30% by ouabain. Furthermore, at all points on the binding curve these substances inhibited binding by 18-98%, in a pattern consistent with reduced receptor number. The concentrations of vanadate or ouabain which we used did not change cell volume or inhibit trypan blue dye exclusion, as an index of cell viability. Because vanadate and ouabain inhibit Na+K+ATPase and have largely dissimilar effects on a variety of cell systems, our observations may reflect specific involvement of Na+K+ATPase in binding or closely related processes.     

The fate of cytoplasmic vanadium. Implications on (NA,K)-ATPase inhibition.

The fate of vanadate (+5 oxidation state of vanadium) taken up by the red cell was studied using EPR spectroscopy. The appearance of an EPR signal indicated that most of the cytoplasmic vanadate is reduced to the +4 oxidation state with axial symmetry characteristic of vanadyl ions. The signal at 23 degrees C was characteristic of an immobilized system indicating that the vanadyl ions in the cytoplasm are associated with a large molecule. [48V]Vanadium eluted with hemoglobin when the lysate from Na3[48V[O4-treated red cells was passed through a Sephadex G-100 column and rabbit anti-human hemoglobin serum caused a hemoglobin-specific precipitation of 48V when added to the red cell lysate. Both results indicate that hemoglobin is the protein which binds cytoplasmic vanadyl ions. However, neither sodium vanadate nor vanadyl sulfate bound to purified hemoglobin in vitro. Finally, transient kinetics of vanadyl sulfate interaction with the sodium-and potassium-stimulated adenosine triphosphatase showed that the +4 oxidation state of vanadium is less effective than the +5 oxidation state in inhibiting this enzyme. These results indicate that oxidation-reduction reactions in the cytoplasm are capable of relieving vanadate inhibition of cation transport.     

Interaction of RNase A with VO3- and VO2+ ions. Metal ion binding mode and protein secondary structure

Some of vanadyl complexes have shown potential to inhibit RNase activity by acting as transition state analogue, while at the same time not inhibiting DNase. To gain an insight into the interaction of protein with vanadate (VO3-) and vanadyl (VO2+) ions, the present study was designed to examine the binding of ribonuclase A (RNase A) with NaVO3 and VOSO4 in aqueous solution at physiological pH with metal ion concentrations of 0.001 mM to 1 mM, and protein concentration of 2% w/v. Absorption spectra and Fourier transform infrared (FTIR) spectroscopy with self-deconvolution and second derivative resolution enhancement were used to determine the cation binding mode, association constant and the protein secondary structure in the presence of vanadate and vanadyl ions in aqueous solution. Spectroscopic results show that an indirect metal ion interaction occurs with the polypeptide C = O, C-N (via H2O) with overall binding constants of K(VO3-) = 3.93x10(2) M(-1) and K(VO2+) = 4.20x10(3) M(-1). At high metal ion concentrations, major protein secondary structural changes occur from that of the alpha-helix 29% (free enzyme) to 23-24%; beta-sheet (pleated and anti) 50% (free enzyme) to 64-66% and turn 21% (free enzyme) to 10-12% in the metal-RNase complexes. The observed structural changes indicate a partial protein unfolding in the presence of high metal ion concentration.     

Vanadium-induced Cl(-)-secretion in rabbit descending colon is mediated by prostaglandins.

Vanadium in the 4+ (vanadyl-ion) and 5+ (vanadate-ion) oxidation state stimulates furosemide-sensitive electrogenic Cl- secretion in isolated epithelia of rabbit descending colon. This effect is associated with an increased release of prostaglandin E2 from the tissue. Inhibitors of phospholipase A2 or cyclooxygenase abolish both vanadium-induced release of prostaglandin E2 and Cl- secretion. Neuronal mechanisms are not likely to be involved, as tetrodotoxin does not affect the vanadate induced Cl- secretion. Although vanadate is known to inhibit Na+,K(+)-ATPase activity, no inhibition of active Na+ transport was observed in intact colonic epithelia suggesting a rapid intracellular reduction of vanadate ions to vanadyl ions which have no inhibitory effect on the Na+,K(+)-ATPase. The present findings therefore indicate that vanadate stimulated colonic Cl- secretion involves intracellular conversion of vanadate to vanadyl and release of prostaglandin E2.     

Kinetics of vanadate dissociation: estimation of the rate by inhibitor inactivation

Vanadate (+5) is a potent inhibitor of a variety of ATPases including dynein ATPase. We describe a method useful for estimating the functional dissociation rate of vanadate from the active site which does not rely on classical physical separation techniques. The method involves spectrophotometrically monitoring the enzymatic activity as the inhibitor dissociates from the enzyme and is inactivated by norepinephrine. Norepinephrine effectively reverses vanadate inhibition by reducing vanadate (+5) to oxovanadium (+4). This reduction by norepinephrine is sufficiently fast for these purposes--addition of vanadate after norepinephrine shows no inhibition of ATPase activity. The mathematical estimation procedure is generally useful for estimation of dissociation rates of other reversible inhibitors which can be quickly inactivated after dissociation from the enzyme. The rate of dissociation of vanadate from dynein with ATP and 2-N3ATP as substrates using this method was estimated to be in the ranges 0.0023-0.0042 and 0.0057-0.0075 s-1, respectively. These rates permit estimation of the rates of vanadate association with dynein by using the reported dissociation constant for vanadate. The results are consistent with the very fast and potent inhibition of dynein ATPase activity observed.     

Decavanadate as a biochemical tool in the elucidation of muscle contraction regulation

Recently reported decameric vanadate (V(10)) high affinity binding site in myosin S1, suggests that it can be used as a tool in the muscle contraction regulation. In the present article, it is shown that V(10) species induces myosin S1 cleavage, upon irradiation, at the 23 and 74 kDa sites, the latter being prevented by actin and the former blocked by the presence of ATP. Identical cleavage patterns were found for meta- and decavanadate solutions, indicating that V(10) and tetrameric vanadate (V(4)) have the same binding sites in myosin S1. Concentrations as low as 50 muM decavanadate (5 muM V(10) species) induces 30% of protein cleavage, whereas 500 muM metavanadate is needed to attain the same extent of cleavage. After irradiation, V(10) species is rapidly decomposed, upon protein addition, forming vanadyl (V(4+)) species during the process. It was also observed by NMR line broadening experiments that, V(10) competes with V(4) for the myosin S1 binding sites, having a higher affinity. In addition, V(4) interaction with myosin S1 is highly affected by the products release during ATP hydrolysis in the presence or absence of actin, whereas V(10) appears to be affected at a much lower extent. From these results it is proposed that the binding of vanadate oligomers to myosin S1 at the phosphate loop (23 kDa site) is probably the cause of the actin stimulated myosin ATPase inhibition by the prevention of ATP/ADP exchange, and that this interaction is favoured for higher vanadate anions, such as V(10).     

Does vanadyl affect adenylate cyclase?

While the stimulatory effect of vanadate, an anion of pentavalent vanadium, on adenylate cyclase (AC) has been repeatedly demonstrated in various tissues only a few studies have been hitherto devoted to the effect of vanadyl, a cation of tetravalent vanadium, but these have provided contradictory results. In the present experiments synaptic plasma membranes from normal rat cerebral cortex were used for estimation of the vanadyl effect (in the concentration range from 10(-5) mol.1(-1) to 10(-3) mol.1(-1) on the basal adenylate cyclase activity. Four types of incubation media were used. In the presence of Tris-maleate and creatine phosphate + creatine phosphokinase (CP + CK) maximal stimulation (33%) was reached at 10(-4) mol.1(-1). In the same buffer but in absence or (CP + CK) maximum was already obtained at 10(-5) mol.1(-1) (49%); at 10(-3) mol.1(-1) no effect was observed. In Tric.HCl buffer with (CP + CK) maximal stimulation appeared at 10(-5) mol.1(-1), whereas at 10(-3) mol.1(-1) inhibition (-25%) was observed. In a medium containing Tris.HCl without (CP + CK) the biphasic nature of vanadyl effect was less markedly expressed: maximal stimulation (+55%) occurred at 10(-4) mol.1(-1). Thus vanadyl stimulates AC, but at relatively low concentrations (10(-5)-10(-4); at higher concentration it tends to exert an inhibitory action. Vanadate had a qualitatively similar effect, but the stimulation was more pronounced and the tendency to inhibition was shifted to higher concentrations.     

Organic anions stabilize the reactivated motility of sperm flagella and the latency of dynein 1 ATPase activity

Substitution of any of a variety of organic anions, including acetate, propionate, lactate, gluconate, and succinate, for chloride in the reactivation medium improves the motility of demembranated sperm of Tripneustes gratilla. At the optimum concentration of 0.20 N, all of these anions improve the duration of motility, with lactate and gluconate being the best. The Michaelis constant for beat frequency (Kmf) is lower (0.11-0.14 mM at 22 degrees C) in most of the organic anions than it is in Cl- (0.20 mM), and the minimum ATP concentration required to support oscillatory beating is reduced from 10 microM in chloride to 2 microM in acetate, which together indicate a greater affinity of the axonemal ATPase for MgATP2- in the organic anions media. The maximal beat frequency, fmax, is as high as 42 Hz in 0.2 N succinate compared to 31 Hz in Cl-, whereas the mean bend angle averages 2.8 rad in acetate compared to 2.4 rad in Cl-; these values give a calculated average velocity of tubule sliding of approximately 15 micron/s in acetate and succinate, which is approximately 30% greater than the value of 11 micron/s observed in chloride. The reactivated sperm are sixfold more sensitive to vanadate inhibition in 0.2 M acetate than they are in 0.15 M Cl-. The specific ATPase activity of soluble dynein 1, which increases more than 15-fold between 0 and 1.0 N Cl-, undergoes only a twofold activation over the same range of organic anion concentration, and, like the reactivated motility, is up to 50-fold more sensitive to vanadate. This greater apparent mechanochemical efficiency and the increased sensitivity to vanadate inhibition in the organic anions suggest that they, unlike chloride, do not promote the spontaneous dissociation of ADP and PO4(3-) from the dynein-ADP-PO4 kinetic intermediate in the dynein crossbridge cycle. The use of organic anion media may lead to significant improvements in reactivation of other motile and transport systems.     

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.