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.     

The vanadate-stimulated oxidation of NAD(P)H by biomembranes is a superoxide-initiated free radical chain reaction

Rat liver microsomes catalyze a vanadate-stimulated oxidation of NAD(P)H, which is augmented by paraquat and suppressed by superoxide dismutase, but not by catalase. NADPH oxidation was a linear function of the concentration of microsomes in the absence of vanadate, but was a saturating function in the presence of vanadate. Microsomes did not catalyze a vanadate-stimulated oxidation of reduced nicotinamide mononucleotide (NMNH), but gained this ability when NADPH was also present. When the concentration of NMNH was much greater than that of NADPH a minimal average chain length could be calculated from 1/2 the ratio of NMNH oxidized per NADPH added. The term chain length, as used here, signifies the number of molecules of NMNH oxidized per initiating event. Chain length could be increased by increasing [vanadate] and [NMNH] and by decreasing pH. Chain lengths in excess of 30 could easily be achieved. The Km for NADPH, arrived at from saturation of its ability to trigger NMNH oxidation by microsomes in the presence of vanadate, was 1.5 microM. Microsomes or the outer mitochondrial membrane was able to catalyze the vanadate-stimulated oxidation of NADH or NADPH but only the oxidation of NADPH was accelerated by paraquat. The inner mitochondrial membrane was able to cause the vanadate-stimulated oxidation of NAD(P)H and in this case paraquat stimulated the oxidation of both pyridine coenzymes. Our results indicate that vanadate stimulation of NAD(P)H oxidation by biomembranes is a consequence of vanadate stimulation of NAD(P)H or NMNH oxidation by O-2, rather than being due to the existence of vanadate-stimulated NAD(P)H oxidases or dehydrogenases.     

Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases

The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH:cytochrome b5 (EC 1.6.2.2) and NADPH:cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD+ and NADP+, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O2- than O2-2. We suggest that a ferroheme-O2 and various heme-oxy radical complexes (mainly ferroheme-O-2 complex) play a crucial role in NAD(P)H oxidation.     

Superoxide is responsible for the vanadate stimulation of NAD(P)H oxidation by biological membranes

Vandate augments the oxidation of NAD(P)H, but not of NMNH, by rat liver microsomes. Paraquat increases the vanadate effect on NADPH, but not on NADH, oxidation. Substoichiometric levels of NADPH caused the co-oxidation of NADH or NMNH and SOD inhibited in all cases. The ratio of NADH or NMNH co-oxidized per NADPH added allowed estimation of average chain length, which increased as the pH was lowered from 8.0 to 7.1. The initial rate of this co-oxidation of NMNH was a saturating function of the concentration of microsomes, reflecting a decrease in chain length with an increase in number of concomitant reaction chains, and due to increasing radical-radical termination reactions. Mitochondrial outer membranes behaved like the microsomal membranes, but mitochondrial inner membranes catalyzed a rapid oxidation of NADH which could be augmented by vanadate, whose action was enhanced by paraquat and inhibited by antimycin or rotenone. These and related observations support the view that vanadate stimulates NAD(P)H oxidation by biological membranes, not by virtue of interacting with enzymes, but rather by interacting with O-2.     

Superoxide-independent reduction of vanadate by rat liver microsomes/NAD(P)H: vanadate reductase activity.

It has been reported that vanadate-stimulated oxidation of NAD(P)H by microsomal systems can proceed anaerobically, in contrast to the general notion that the oxidation proceeds exclusively by an O(2-)-dependent free radical chain mechanism. The current study indicates that microsomal systems are endowed with a vanadate-reductase property, involving a NAD(P)H-dependent electron transport cytochrome P450 system. Our ESR measurements demonstrated the formation of a vanadium(IV) species in a mixture containing vanadate, rat liver microsomes, and NAD(P)H. This vanadium(IV) species was identified as the vanadyl ion (VO2+) by comparison with the ESR spectrum of VOSO4. The initial rate of vanadium(IV) formation depends linearly on the concentration of microsomes. The Michaelis-Menten constants were found to be: km = 1.25 mM and Vmax = 0.066 mumol (min)-1 (mg microsomes)-1, respectively. Pretreatment of the microsomes with carbon monoxide or K3Fe(CN)6 reduced vanadium(IV) generation, suggesting that the NAD(P)H-dependent electron transport cytochrome P450 system plays a significant role in the microsomal reduction of vanadate. Measurements under argon or in the presence of superoxide dismutase caused only minor (less than 10%) reductions in vanadium(IV) generation. The VO2+ species was also detected in NAD(P)H oxidation by fructose plus vanadate, a reaction known to proceed via an O(2-)-mediated chain mechanism. However, the amount of vanadium(IV) generated by this reaction was an order of magnitude smaller than that by the microsomal system and was inhibitable by superoxide dismutase, affirming the conclusion that the microsomal/NAD(P)H system is endowed with the (O(2-)-independent) vanadium(V) reductase property.     

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Characteristics and actions of NAD(P)H oxidase on the sarcoplasmic reticulum of coronary artery smooth muscle

It has been reported that nonmitochondrial NAD(P)H oxidases make an important contribution to intracellular O2-* in vascular tissues and, thereby, the regulation of vascular function. Topological analyses have suggested that a well-known membrane-associated NAD(P)H oxidase may not release O2-* into the cytosol. It is imperative to clarify the source of intracellular O2-* associated with this enzyme and its physiological significance in vascular cells. The present study hypothesized that an NAD(P)H oxidase on the sarcoplasmic reticulum (SR) in coronary artery smooth muscle (CASM) regulates SR ryanodine receptor (RyR) activity by producing O2-* locally. Western blot analysis was used to detect NAD(P)H oxidase subunits in purified SR from CASM. Fluorescent spectrometric analysis demonstrated that incubation of SR with NADH time dependently produced O2-*, which could be substantially blocked by the specific NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin and by SOD or its mimetic tiron. This SR NAD(P)H oxidase activity was also confirmed by HPLC analysis of conversion of NADH to NAD+. In experiments of lipid bilayer channel reconstitution, addition of NADH to the cis solution significantly increased the activity of RyR/Ca2+ release channels from these SR preparations from CASM, with a maximal increase in channel open probability from 0.0044 +/- 0.0005 to 0.0213 +/- 0.0018; this effect of NADH was markedly blocked in the presence of SOD or tiron or the NAD(P)H oxidase inhibitors diphenylene iodonium, N-vanillylnonanamide, and apocynin. These results suggest that a local NAD(P)H oxidase system on SR from CASM regulates RyR/Ca2+ channel activity and Ca2+ release from SR by producing O2-*.     

NAD(P)H oxidation elicits anion superoxide formation in radish plasmalemma vesicles

Radish plasmalemma-enriched fractions show an NAD(P)H-ferricyanide or NAD(P)H-cytochrome c oxidoreductase activity which is not influenced by pH in the 4.5-7.5 range. In addition, at pH 4.5-5.0, NAD(P)H elicits an oxygen consumption (NAD(P)H oxidation) inhibited by catalase or superoxide dismutase (SOD), added either before or after NAD(P)H addition. Ferrous ions stimulate NAD(P)H oxidation, which is again inhibited by SOD and catalase. Hydrogen peroxide does not stimulate NADH oxidation, while it does stimulate Fe2+-induced NADH oxidation. NADH oxidation is unaffected by salicylhydroxamic acid and Mn2+, is stimulated by ferulic acid, and inhibited by KCN, EDTA and ascorbic acid. Moreover, NADH induces the conversion of epinephrine to adrenochrome, indicating that anion superoxide is formed during its oxidation. These results provide evidence that radish plasma membranes contain an NAD(P)H-ferricyanide or cytochrome c oxidoreductase and an NAD(P)H oxidase, active only at pH 4.5-5.0, able to induce the formation of anion superoxide, that is then converted to hydrogen peroxide. Ferrous ions, sparking a Fenton reaction, would stimulate NAD(P)H oxidation.     

Properties of a coenzyme, pyrroloquinoline quinone: generation of an active oxygen species during a reduction-oxidation cycle in the presence of NAD(P)H and O2

The oxidation of NAD(P)H by pyrroloquinoline quinone (PQQ) was non-enzymatically carried out at physiological pH in the presence of O2. The PQQ-NAD(P)H system requires about 1 mol of O2 for the oxidation of 1 mol of NAD(P)H. The oxidation of NAD(P)H occurred at a pseudo-first-order rate with respect to NAD(P)H and was of zero order with respect to PQQ concentration in in the presence of O2: k0[PQQ] [NAD(P)H] = k1 [NAD(P)H], where k0[PQQ] = k1, in which [PQQ] represents the initial concentration of PQQ. k0 values for NADH and NADPH were 3.4.10(2) M-1.min-1 and 2.0.10(2) M-1.min-1, respectively, at 25 degrees C and at 258 microM O2 (initial concentration). The system produced O-2, probably by the interaction of PQQ.H and/or NAD(P).with O2, during the oxidation of NAD(P)H. PQQH2 and PQQ.H were easily oxidized to PQQ in the presence of O2, yielding H2O2.     

Chlorination of NADH: similarities of the HOCl-supported and chloroperoxidase-catalyzed reactions

The chloroperoxidase-catalyzed reactions of NAD(P)H with H2O2 in the presence of Cl- or Br- have been characterized. With 1 mol H2O2 per mol of NADH, one atom of 36Cl was incorporated into the 264-nm-absorbing intermediate product. This species was oxidized enzymatically by a second mole of H2O2 to a species distinct from NAD+, which retained one Cl atom. Spectroscopically identical species were also produced by reaction of NADH with one and two molar ratios of HOCl, respectively. These data indicate that, with respect to halogenation activities, chloroperoxidase functions similarly to myeloperoxidase, i.e., produces HOCl as the first product of Cl- oxidation by H2O2. Moreover, rapid chlorination of NAD(P)H followed by oxidation may be an important and highly lethal microbicidal effect of HOCl produced by myeloperoxidase in activated neutrophils.     

The crystal structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis: insights into the conversion of O2 into two water molecules by the flavoenzyme

The FAD-dependent NAD(P)H oxidase from Lactobacillus sanfrancisensis (L.san-Nox2) catalyzes the oxidation of 2 equivalents of either NADH or NADPH and reduces 1 equivalent of O(2) to yield 2 equivalents of water. During steady-state turnover only 0.5% of the reducing equivalents are detected in solution as hydrogen peroxide, suggesting that it is not released from the enzyme after the oxidation of the first equivalent of NAD(P)H and reaction with O(2). Here we report the crystal structure of L.san-Nox2 to 1.8 A resolution. The enzyme crystallizes as a dimer with each monomer consisting of a FAD binding domain (residues 1-120), a NAD(P)H binding domain (residues 150-250), and a dimerization domain (residues 325-451). The electron density for the redox-active Cys42 residue located adjacent to the si-face FAD is consistent with oxidation to the sulfenic acid (Cys-SOH) state. The side chain of Cys42 is also observed in two conformations; in one the sulfenic acid is hydrogen bonded to His10 and in the other it hydrogen bonds with the FAD O2' atom. Surprisingly, the NAD(P)H binding domains each contain an ADP ligand as established by electron density maps and MALDI-TOF analysis of the ligands released from heat-denatured enzyme. The ADP ligand copurifies with the enzyme, and its presence does not inhibit enzyme activity. Consequently, we hypothesize that either NADPH or NADH substrates bind via a long channel that extends from the enzyme exterior and terminates at the FAD re-face. A homology model of the NADH oxidase from Lactococcus lactis (L.lac-Nox2) was also generated using the crystal structure of L.san-Nox2, which reveals several important similarities and differences between the two enzymes. HPLC analysis of ligands released from denatured L.lac-Nox2 indicates that it does not bind ADP, which correlates with the specificity of the enzyme for oxidation of NADH.     

Reaction of peroxynitrite with reduced nicotinamide nucleotides, the formation of hydrogen peroxide.

NAD(P)H acts as a two-electron reductant in physiological, enzyme-controlled processes. Under nonenzymatic conditions, a couple of one-electron oxidants easily oxidize NADH to the NAD(.) radical. This radical reduces molecular oxygen to the superoxide radical (O-(2)) at a near to the diffusion-controlled rate, thereby subsequently forming hydrogen peroxide (H(2)O(2)). Because peroxynitrite can act as a one-electron oxidant, the reaction of NAD(P)H with both authentic peroxynitrite and the nitric oxide ((. )NO) and O-(2) releasing compound 3-morpholinosydnonimine N-ethylcarbamide (SIN-1) was studied. Authentic peroxynitrite oxidized NADH with an efficiency of approximately 25 and 8% in the absence and presence of bicarbonate/carbon dioxide (HCO(3)(-)/CO(2)), respectively. NADH reacted 5-100 times faster with peroxynitrite than do the known peroxynitrite scavengers glutathione, cysteine, and tryptophan. Furthermore, NADH was found to be highly effective in suppressing peroxynitrite-mediated nitration reactions even in the presence of HCO(3)(-)/CO(2). Reaction of NADH with authentic peroxynitrite resulted in the formation of NAD(+) and O-(2) and, thus, of H(2)O(2) with yields of about 3 and 10% relative to the added amounts of peroxynitrite and NADH, respectively. Peroxynitrite generated in situ from SIN-1 gave virtually the same results; however, two remarkable exceptions were recognized. First, the efficiency of NADH oxidation increased to 60-90% regardless of the presence of HCO(3)(-)/CO(2), along with an increase of H(2)O(2) formation to about 23 and 35% relative to the amounts of added SIN-1 and NADH. Second, and more interesting, the peroxynitrite scavenger glutathione (GSH) was needed in a 75-fold surplus to inhibit the SIN-1-dependent oxidation of NADH half-maximal in the presence of HCO(3)(-)/CO(2). Similar results were obtained with NADPH. Hence, peroxynitrite or radicals derived from it (such as, e.g. the bicarbonate radical or nitrogen dioxide) indeed oxidize NADH, leading to the formation of NAD(+) and, via O-(2), of H(2)O(2). When peroxynitrite is generated in situ in the presence of HCO(3)(-)/CO(2), i.e. under conditions mimicking the in vivo situation, NAD(P)H effectively competes with other known scavengers of peroxynitrite.     

Cytochemical localization of hydrogen peroxide production in the rat uterus

A reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent H2O2-generating activity of the rat uterus was investigated both electron cytochemically and biochemically. We tried to cytochemically demonstrate H2O2 generation from the oxidation of reduced NADH or NADPH using the cerium method. NADPH oxidation resulted in electron-dense deposits on the apical plasma membrane covering the microvilli of the surface epithelium of the lightly fixed endometrium. In control specimens incubated in a medium from which substrate was omitted, no such deposits were observed. The reduction of ferricytochrome c due to NADH oxidation was spectrophotometrically detected in the lightly fixed uterus. Absorption at 550 nm increased with the addition of NADH, but not with that of NAD. The reaction was weakened by preheating and adversely affected by the addition of superoxide dismutase, but it was not inhibited by adding 50 mM sodium azide. These results suggest that a kind of NAD(P)H oxidase, generating H2O2 via superoxide formation, may possibly be present on the apical plasma membrane of the rat endometrial epithelium.     

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.     

The effect of inhibitors of glucose utilization on the chemiluminescence and fluorescence of murine peritoneal macrophages]

Using the inhibitors of glucose utilization and mitochondrial oxidation we found that the blue autofluorescence (AF) of murine peritoneal macrophages (MP) originates predominantly from the cytosolic NAD(P)H. AF intensity correlates with the changes in glycolytic enzymes activity. The luminol-dependent chemiluminescence (CL) intensity which characterizes the amount of O2- generating by MP upon the addition of formyl-methionyl-leucyl-phenylalanine (FMLP) is inhibited in the case of maximum activity of glycolytic enzymes. Incubation of MP with non-convertible analog of glucose, such as 2-deoxyglucose increases the CL intensity under the influence of FMLP. A conclusion is drawn that amount of O2- generating by MP depends on the intracellular concentration of sugars.     

Generation of superoxide by the mitochondrial Complex I

Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.     

Formation of reduced nicotinamide adenine dinucleotide peroxide

Incubation of NADH at neutral and slightly alkaline pH leads to the gradual absorption of 1 mol of H+. This uptake of acid requires oxygen and mainly yields anomerized NAD+ (NAD+), with only minimal formation od acid-modified NADH. The overall stoichiometry of the reaction is: NADH + H+ + 1/2O2 leads to H2O + NAD+, with NADH peroxide (HO2-NADH+) serving as the intermediate that anomerizes and breaks down to give NAD+ and H2O2. The final reaction reaction mixture contains less than 0.1% of the generated H2O2, which is nonenzymically reduced by NADH. The latter reaction is inhibited by catalase, leading to a decrease in the overall rate of acid absorption, and stimulated by peroxidase, leading to an increase in the overall rate of acid absorption. Although oxygen can attack NADH at either N-1 or C-5 of the dihydropyridine ring, the attack appears to occur primarily at N-1. This assignment is based on the inability of the C-5 peroxide to anomerize, whereas the N-1 peroxide, being a quaternary pyridinium compound, can anomerize via reversible dissociation of H2O2. The peroxidase-catalyzed oxidation of NADH by H2O2 does not lead to anomerization, indicating that anomerization occurs prior to the release of H2O2. Chromatography of reaction mixtures on Dowex 1 formate shows the presence of two major and several minor neutral and cationic degradation products. One of the major products is nicotinamide, which possibly arises from breakdown of nicotinamide-1-peroxide. The other products have not been identified, but may be derived from other isomeric nicotinamide peroxides.     

Chorion peroxidase-mediated NADH/O(2) oxidoreduction cooperated by chorion malate dehydrogenase-catalyzed NADH production: a feasible pathway leading to H(2)O(2) formation during chorion hardening in Aedes aegypti mosquitoes

A specific chorion peroxidase is present in Aedes aegypti and this enzyme is responsible for catalyzing chorion protein cross-linking through dityrosine formation during chorion hardening. Peroxidase-mediated dityrosine cross-linking requires H(2)O(2), and this study discusses the possible involvement of the chorion peroxidase in H(2)O(2) formation by mediating NADH/O(2) oxidoreduction during chorion hardening in A. aegypti eggs. Our data show that mosquito chorion peroxidase is able to catalyze pH-dependent NADH oxidation, which is enhanced in the presence of Mn(2+). Molecular oxygen is the electron acceptor during peroxidase-catalyzed NADH oxidation, and reduction of O(2) leads to the production of H(2)O(2), demonstrated by the formation of dityrosine in a NADH/peroxidase reaction mixture following addition of tyrosine. An oxidoreductase capable of catalyzing malate/NAD(+) oxidoreduction is also present in the egg chorion of A. aegypti. The cooperative roles of chorion malate/NAD(+)oxidoreductase and chorion peroxidase on generating H(2)O(2) with NAD(+) and malate as initial substrates were demonstrated by the production of dityrosine after addition of tyrosine to a reaction mixture containing NAD(+) and malate in the presence of both malate dehydrogenase fractions and purified chorion peroxidase. Data suggest that chorion peroxidase-mediated NADH/O(2) oxidoreduction may contribute to the formation of the H(2)O(2) required for chorion protein cross-linking mediated by the same peroxidase, and that the chorion associated malate dehydrogenase may be responsible for the supply of NADH for the H(2)O(2) production.     

Vanadate-stimulated oxidation of NAD(P)H

Vanadate stimulates the oxidation of NAD(P)H by biological membranes because such membranes contain NAD(P)H oxidases which are capable of reducing dioxygen to O₂⁻ and because vanadate catalyzes the oxidation of NAD(P)H by O₂⁻, by a free radical chain mechanism. Dihydropyridines, such as reduced nicotinamide mononucleotide (NMNH), which are not substrates for membrane-associated NAD(P)H oxidases, are not oxidized by membranes plus vanadate unless NAD(P)H is present to serve as a source of O₂⁻. When [NMNH] greatly exceeds [NAD(P)H], in such reaction mixtures, one can observe the oxidation of many molecules of NMNH per NAD(P)H consumed. This reflects the chain length of the free radical chain mechanism. We have discussed the mechanism and significance of this process and have tried to clarify the pertinent but confusing literature.