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.     

Interaction of menadione and duroquinone with Q-cycle during DT-diaphorase function]

The interaction of quinones (menadione and duroquinone) with DT-diaphorase and mitochondrial electron transport chain translocators at low (120 mosM) and high (400 mosM) values of the medium tonicity in the quinone concentration range of 6-90 microM was studied. It was shown that with a rise in menadione (K3) concentration the number of electron transport carriers interacting with it increase. At K3 concentration of 6 microM the latter is reduced by DT-diaphorase and fully oxidized via the Q-cycle. At K3 concentration of 15 microM the latter is also reduced by DT-diaphorase via the Q-cycle, but in this case the oxidation is incomplete (about 30% K3H2 is oxidized by the terminal part of the respiratory chain). At 90 microM K3 50% of quinone is reduced by DT-diaphorase and 50% by the respiratory chain NADH dehydrogenase complex enzymes; about 30% of K3H2 is oxidized via the Q-cycle, about 20%--by the terminal part of the respiratory chain and about 50%--by O2 without cytochrome oxidase. Unlike menadione, duroquinone (6-90 microM) is reduced only by DT-diaphorase and is oxidized in all cases by cytochrome oxidase. It was shown that the increase in the mitochondrial matrix volume in low tonicity media decreases the rate of the DT-diaphorase shunt operation.     

The plasma membrane NADH oxidase of soybean has vitamin K(1) hydroquinone oxidase activity

Isolated plasma membrane vesicles and the plasma membrane NADH oxidase partially purified from soybean plasma membrane vesicles exhibited a cyanide-insensitive vitamin K(1) hydroquinone oxidase activity with isolated plasma membrane vesicles. Reduced vitamin K(1) (phylloquinol) was oxidized at a rate of about 10 nmol/min/mg protein as determined by reduced vitamin K(1) reduction or oxygen consumption. The K(m) for reduced K(1) was 350 microM. With the partially purified enzyme, reduced vitamin K(1) was oxidized at a rate of about 600 nmol/min/mg protein and the K(m) was 400 microM. When assayed in the presence of 1 mM KCN, activities of both plasma membrane vesicles and of the purified protein were stimulated (0.1 microM) or inhibited (0.1 mM) by the synthetic auxin growth factor 2, 4-dichlorophenoxyacetic acid. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via reduced vitamin K(1) to acceptors (molecular oxygen or protein disulfides) at the cell surface.     

Inhibition of plasma membrane NADH oxidase activity and growth of HeLa cells by natural and synthetic retinoids

Several retinoids, both natural and synthetic, were evaluated for their ability to modulate NADH oxidase activity of plasma membranes of cultured HeLa cells and the growth of HeLa cells in culture. Both NADH oxidase activity and the growth of cells were inhibited by the naturally-occurring retinoids all trans-retinoic acid (tretinoin) and retinol as well as by the synthetic retinoids, trans-acitretin, 13-cis-acitretin, etretinate and arotonoid ethylester (Ro 13-6298). For all retinoids tested, inhibition of NADH oxidase activity and inhibition of growth were correlated closely. With tretinoin, etretinate and arotonoid ethylester, NADH oxidase activity and cell growth were inhibited in parallel in proportion to the logarithm of retinoid concentration over the range of concentrations 10-8 to 10-5 M. Approximately 70% inhibition of both NADH oxidase activity and growth was reached at 10 µM. With retinol, trans-acitretin and 13-cis-acitretin, inhibition of NADH oxidase activity and growth also were correlated but maximum inhibition of both was about 40% at 10 µM. The possibility is suggested that inhibition of the plasma membrane NADH oxidase activity by retinoids may be related to their mechanism of inhibition of growth of HeLa cells in culture. (Mol Cell Biochem 166: 101-109, 1997)     

Mechanisms of chromium toxicity in mitochondria

The oxygen consumption of isolated rat heart mitochondria was potently depressed in presence of 10-50 microM Na2CrO4 when NAD-linked substrates were oxidized. The succinate stimulated respiration and the oxidation of exogeneous NADH in sonicated mitochondria were not affected by chromate at this concentration range. A rapid and persistent drop (40% in 2 min) in the mitochondrial NADH level was observed after chromate addition (30 microM) under conditions which generally should promote regeneration of NADH. Experiments with bis-(2-ethyl-2-hydroxybutyrato)oxochromate(V) and vanadyl induced reduction of Cr(VI) in presence of excess NADH were performed. These experiments indicated that NADH may be directly oxidized by Cr(V) at physiological pH. The activity of 10 different enzymes were measured after lysis of intact mitochondria pretreated with chromate (1-100 microM). Na2CrO4 at a very low level (3-5 microM) was sufficient for 50% inhibition of alpha-ketoglutarate dehydrogenase. Higher concentrations (20-70 microM) was necessary for similar effect on beta-hydroxybutyrate and pyruvate dehydrogenase. The other enzymes tested were unaffected. Thus, the chromate toxicity in mitochondria may be due to NADH depletion as a result of direct oxidation by Cr(V) as well as reduced formation of NADH due to specific enzyme inhibition.     

A growth factor- and hormone-stimulated NADH oxidase from rat liver plasma membrane.

NADH oxidase activity (electron transfer from NADH to molecular oxygen) of plasma membranes purified from rat liver was characterized by a cyanide-insensitive rate of 1 to 5 nmol/min per mg protein. The activity was stimulated by growth factors (diferric transferrin and epidermal growth factor) and hormones (insulin and pituitary extract) 2- to 3-fold. In contrast, NADH oxidase was inhibited up to 80% by several agents known to inhibit growth or induce differentiation (retinoic acid, calcitriol, and the monosialoganglioside, GM3). The growth factor-responsive NADH oxidase of isolated plasma membranes was not inhibited by common inhibitors of oxidoreductases of endoplasmic reticulum or mitochondria. As well, NADH oxidase of the plasma membrane was stimulated by concentrations of detergents which strongly inhibited mitochondrial NADH oxidases and by lysolipids or fatty acids. Growth factor-responsive NADH oxidase, however, was inhibited greater than 90% by chloroquine and quinone analogues. Addition of coenzyme Q10 stimulated the activity and partially reversed the analogue inhibition. The pH optimum for NADH oxidase was 7.0 both in the absence and presence of growth factors. The Km for NADH was 5 microM and was increased in the presence of growth factors. The stoichiometry of the electron transfer reaction from NADH to oxygen was 2 to 1, indicating a 2 electron transfer. NADH oxidase was separated from NADH-ferricyanide reductase, also present at the plasma membrane, by ion exchange chromatography. Taken together, the evidence suggests that NADH oxidase of the plasma membrane is a unique oxidoreductase and may be important to the regulation of cell growth.     

Control of pyruvate carboxylase activity by the pyridine-nucleotide redox state in mitochondria from rat liver

Pyruvate carboxylation by isolated mitochondria from rat liver is inhibited by t-butylhydroperoxide in a fully reversible manner. The rate of malate formation at 10 mM pyruvate was decreased by some 80% by 30 microM t-butylhydroperoxide. The effective peroxide concentration was dependent on the mitochondrial hydrogen supply, being increased to about 120 microM in the presence of 50 microM palmitoylcarnitine. Regarding the mechanism(s) of the t-butylhydroperoxide action, pyruvate transport and intramitochondrial energy or activator supply are unlikely involved, because the effect also took place with alanine as the substrate and was not accompanied by a change in the intramitochondrial levels of adenine nucleotides and acetyl-CoA respectively. However, t-butylhydroperoxide caused a rapid fall in the 3-hydroxybutyrate/acetoacetate ratio and a marked increase in the oxidized glutathione content. Therefore, experiments were designed to disclose the participation of the respective redox couples in the expression of pyruvate carboxylase activity. From measurements of NADPH, NADH, oxidized and reduced glutathione contents of mitochondria incubated under a variety of conditions, evidence has been obtained indicating that the mitochondrial NADH supply represents an important factor in the regulation of pyruvate carboxylase activity. The results presented seemingly provide a new basis for the understanding of the functional relationship between beta-oxidation and pyruvate carboxylation.     

Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS

Potato tuber mitochondria oxidized exogenous NADH and exogenous NADPH at similar rates; the electron transfer inhibitor rotenone did not inhibit the oxidation of either substrate. Submitochondrial particles, prepared from potato tuber mitochondria, exhibited a greater capacity to oxidize NADH than NADPH; rotenone inhibited the oxidation of NADH by 29% and the oxidation of NADPH by 16%. The oxidation of both NADH and NADPH by potato mitochondria exhibited pH optima of 6.8, and although substantial NADH oxidase activity was observed at pH 8.0, little NADPH oxidase activity was detected at that pH. The oxidation of NADPH by the mitochondria was more sensitive to inhibition by EDTA than was the oxidation of NADH.     

Electron transport in Paracoccus halodenitrificans and the role of ubiquinone

The membrane-bound NADH oxidase of Paracoccus halodenitrificans was inhibited by dicoumarol, 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), and exposure to ultraviolet light (at 366 nm). When the membranes were extracted with n-pentane, NADH oxidase activity was lost. Partial restoration was achieved by adding the ubiquinone fraction extracted from the membranes. Succinate oxidation was not inhibited by dicoumarol or HQNO, but was affected by ultraviolet irradiation or n-pentane extraction. However, the addition of the ubiquinone fraction to the membranes extracted with n-pentane did not restore enzyme activity. These observations suggested that NADH and succinate were not oxidized through a common ubiquinone pool.     

The plasma membrane NADH oxidase of HeLa cells has hydroquinone oxidase activity.

The plasma membrane NADH oxidase activity partially purified from the surface of HeLa cells exhibited hydroquinone oxidase activity. The preparations completely lacked NADH:ubiquinone reductase activity. However, in the absence of NADH, reduced coenzyme Q10 (Q10H2=ubiquinol) was oxidized at a rate of 15+/-6 nmol min-1 mg protein-1 depending on degree of purification. The apparent Km for Q10H2 oxidation was 33 microM. Activities were inhibited competitively by the cancer cell-specific NADH oxidase inhibitors, capsaicin and the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984). With coenzyme Q0, where the preparations were unable to carry out either NADH:quinone reduction or reduced quinone oxidation, quinol oxidation was observed with an equal mixture of the Q0 and Q0H2 forms. With the mixture, a rate of Q0H2 oxidation of 8-17 nmol min-1 mg protein-1 was observed with an apparent Km of 0.22 mM. The rate of Q10H2 oxidation was not stimulated by addition of equal amounts of Q10 and Q10H2. However, addition of Q0 to the Q10H2 did stimulate. The oxidation of Q10H2 proceeded with what appeared to be a two-electron transfer. The oxidation of Q0H2 may involve Q0, but the mechanism was not clear. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via naturally occurring hydroquinones to acceptors at the cell surface.     

Adriamycin tolerance in human mesothelioma lines and cell surface NADH oxidase

Adriamycin tolerant human mesothelioma cell lines derived from a single tumor prior to either chemotherapy or radiation therapy and a susceptible cell line were investigated. Not only was growth resistant to low doses of adriamycin but an unusual pattern of resistance was encountered in which cells seemed to better tolerate high adriamycin doses than intermediate doses. The differential growth susceptibility of the tolerant lines compared to A549 lung carcinoma and the bimodal dose response correlated with differences in the specific activity of a plasma membrane-associated NADH oxidase (NOX). Plasma membrane fractions of high purity were isolated by aqueous two-phase partition and assayed directly. The NADH oxidase activity of the plasma membranes for the susceptible cell line was maximally inhibited by 1 microM adriamycin whereas the NADH oxidase activity of the tolerant lines was less and was maximally inhibited by 0.1 microM adriamycin with 1 and 10 microM adriamycin being less inhibitory than 0.1 microM adriamycin. The findings suggest a relationship between the growth response to adriamycin of the adriamycin tolerant mesothelioma lines and the activity of the plasma membrane-associated NADH oxidase activity of the cell surface in these cell lines.     

Overestimation of NADH-driven vascular oxidase activity due to lucigenin artifacts

Several limitations have recently been described for lucigenin, a probe frequently used to assess the activity of vascular NAD(P)H oxidase, a major superoxide source. The preferential reducing substrate of such oxidase remains unclear. We assessed whether lucigenin artifacts could affect detection of NAD(P)H oxidase activity. Initial chemiluminescence assays were performed with vascular rings or homogenates at 5, 50, or 250 microM concentrations. Results showed preferential signals with NADPH (vs. NADH) with 5 and 50 microM lucigenin, which were blocked by diphenylene iodonium (DPI), superoxide dismutase (SOD), or its cell-permeable mimetic MnTBAP. With 250 microM lucigenin, the relative signal with NADH became larger than with NADPH, and was poorly inhibited by all three antagonists above. All SOD/DPI-resistant signals were effectively blocked by the electron acceptor nitrobluetetrazolium. Spin trapping with DMPO showed an approximate doubling of DMPO-OH radical adduct signal upon addition of 5 microM lucigenin to homogenates incubated with either NADPH or NADH. With 50 or 250 microM lucigenin, much larger increases were observed with NADH, as opposed to NADPH. Furthermore, oxygen consumption measurements showed analogous results. In summary, our data suggest that: (i) Lucigenin redox-cycling is detectable in vascular tissue even at 5 microM concentrations, while at 250 microM redox-cycling becomes predominant and is markedly increased when NADH is the assayed substrate; and (ii) With 250 microM lucigenin, preferentially with NADH, signals are further overestimated by direct, oxidase-dependent, superoxide-independent two-electron transfer. Therefore, previous reports of preferential NADH affinity of the vascular oxidase may have been due to these artifacts.     

The NADH oxidase activity of the plasma membrane of synaptosomes is a major source of superoxide anion and is inhibited by peroxynitrite

Plasma membrane vesicles from adult rat brain synaptosomes (PMV) have an ascorbate-dependent NADH oxidase activity of 35-40 nmol/min/(mg protein) at saturation by NADH. NADPH is a much less efficient substrate of this oxidase activity, with a Vmax 10-fold lower than that measured for NADH. Ascorbate-dependent NADH oxidase activity accounts for more than 90% of the total NADH oxidase activity of PMV and, in the absence of NADH and in the presence of 1 mm ascorbate, PMV produce ascorbate free radical (AFR) at a rate of 4.0 +/- 0.5 nmol AFR/min/(mg protein). NADH-dependent *O2- production by PMV occurs with a rate of 35 +/- 3 nmol/min/(mg protein), and is a coreaction product of the NADH oxidase activity, because: (i) it is inhibited by more than 90% by addition of ascorbate oxidase, (ii) it is inhibited by 1 micro g/mL wheat germ agglutinin (a potent inhibitor of the plasma membrane AFR reductase activity), and (iii) the KM(NADH) of the plasma membrane NADH oxidase activity and of NADH-dependent *O2- production are identical. Treatment of PMV with repetitive micromolar ONOO- pulses produced almost complete inhibition of the ascorbate-dependent NADH oxidase and *O2- production, and at 50% inhibition addition of coenzyme Q10 almost completely reverts this inhibition. Cytochrome c stimulated 2.5-fold the plasma membrane NADH oxidase, and pretreatment of PMV with repetitive 10 microm ONOO- pulses lowers the K0.5 for cytochrome c stimulation from 6 +/- 1 (control) to 1.5 +/- 0.5 microm. Thus, the ascorbate-dependent plasma membrane NADH oxidase activity can act as a source of neuronal.O2-, which is up-regulated by cytosolic cytochrome c and down-regulated under chronic oxidative stress conditions producing ONOO-.     

Cooperative Substrate Oxidation by Mitochondria from Castor Bean Hypocotyls

Cooperative oxidation of succinate and exogenous NADH was followed in the mitochondria from five- to six-day-old castor bean (Ricinus communisL.) seedlings. Although succinate was oxidized at a much higher rate than NADH, the former inconsiderably (less than 15%) inhibited the oxidation of the latter substrate in state 4, while, in state 3 (in the presence of ATP), the two substrates did not compete and were jointly oxidized. When two substrates were oxidized by the mitochondria with the alternative CN-resistant oxidase (AO) inhibited with salicylhydroxamic acid, the rate of NADH oxidation in state 4 dropped by over 40% as compared to the initial rate. Meanwhile, the rate of succinate oxidation was not considerably affected by AO inhibition. We believe that one of the AO functions in the mitochondria is to provide for noncompeting oxidation of two (or more) substrates by employing two (or several) dehydrogenases of the respiratory chain.     

Putting together a plasma membrane NADH oxidase: A tale of three laboratories

The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism.     

Cubebin and derivatives as inhibitors of mitochondrial complex I. Proposed interaction with subunit B8

The effects on mitochondrial respiration and complex I NADH oxidase activity of cubebin and derivatives were evaluated. The compounds inhibited the state 3 glutamate/malate-supported respiration of hamster liver mitochondria with IC(50) values ranging from 12.16 to 83.96 microM. NADH oxidase reaction was evaluated in submitochondrial particles. The compounds also inhibited this activity, showing the same order of potency observed for effects on state 3 respiration, as well as a tendency towards a non-competitive type of inhibition (K(I) values ranging from 0.62 to 16.1 microM). A potential binding mode of these compounds with complex I subunit B8, assessed by docking calculations, is proposed.     

Inhibition of palmitoyl CoA of EDTA- and Mg2+-ATPase of heavy meromyosin from rabbit skeletal muscle

Palmitoyl CoA inhibited EDTA-ATPase of heavy meromyosin (HMM) prepared from rabbit skeletal muscle. The concentration for half maximum inhibition of EDTA-ATPase was about 18 microM. Myristoyl CoA, the other long chain fatty acyl CoA, also inhibited EDTA-HMM ATPase, but CoA and short chain CoA thioesters, such as butyryl CoA, acetoacetyl CoA and acetyl CoA, at 40 microM hardly inhibited EDTA-ATPase. Less than 20% inhibition of EDTA-HMM ATPase was obtained with Na-palmitate and Na-myristate at 40 microM, whereas about 90% inhibition of the enzyme occurred in the presence of 40 microM palmitoyl CoA and myristoyl CoA. Palmitoyl carnitine, as well as carnitine, failed to inhibit EDTA-HMM-ATPase. The inhibition of palmitoyl CoA of EDTA-ATPase was reversed by bovine serum albumin and spermine. Mg2+-HMM ATPase activity was enhanced by palmitoyl CoA at 2, 5, and 10 microM. About a 25% increase in Mg2+-HMM ATPase activity was obtained at 5 and 10 microM. At higher concentrations than 20 microM, the enzyme was inhibited by palmitoyl CoA and the degree of inhibition was related to the concentration of the CoA thioester. At 80 microM, the activity was about 15% of the maximum value. The efficacy of myristoyl CoA on Mg2+-ATPase was almost the same as that of palmitoyl CoA. Mg2+-ATPase activity was not enhanced by CoA, butyryl CoA, acetoacetyl CoA, Na-myristate, Na-palmitate, palmitoyl carnitine, or carnitine at 10 microM, and was hardly reduced by these substances at 40 microM. Serum albumin and spermine also canceled, to some extent, these effects of palmitoyl CoA on Mg2+-ATPase.     

Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K(Mox) values were around 4-5 microM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK(Mox) values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K(Mox) was around 1.2 microM for succinate and around 11 microM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K(Mox). However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K(Mox) increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.     

Peroxynitrite formation from the simultaneous reduction of nitrite and oxygen by xanthine oxidase

One electron reductions of oxygen and nitrite by xanthine oxidase form peroxynitrite. The nitrite and oxygen reducing activities of xanthine oxidase are regulated by oxygen with K(oxygen) 26 and 100 microM and K(nitrite) 1.0 and 1.1 mM with xanthine and NADH as donor substrates. Optimal peroxynitrite formation occurs at 70 microM oxygen with purine substrates. Kinetic parameters: V(max) approximately 50 nmol/min/mg and K(m) of 22, 36 and 70 microM for hypoxanthine, pterin and nitrite respectively. Peroxynitrite generation is inhibited by allopurinol, superoxide dismutase and diphenylene iodonium. A role for this enzyme activity can be found in the antibacterial activity of milk and circulating xanthine oxidase activity.     

The relationship between a novel NAD(P)H oxidase activity of ovoperoxidase and the CN- -resistant respiratory burst that follows fertilization of sea urchin eggs

The extracellular protein coat of the sea urchin egg is cross-linked after fertilization via dityrosyl linkages made by an exocytosed ovoperoxidase. The source of oxidant for this reaction is unknown, but eggs produce H2O2 in amounts equivalent to the cyanide-insensitive O2 uptake "respiratory burst" that follows fertilization. Several possible H2O2-forming oxidase activities, including glucose, xanthine, fatty acyl, and fatty-acyl CoA oxidases, were absent from the egg cortex. However, an NAD(P)H-O2 oxidoreductase activity was found in the egg cortex and was completely accounted for by ovoperoxidase. Homogeneous ovoperoxidase exhibits two types of NAD(P)H oxidase activity. One of these activities is similar to that of horseradish peroxidase and lactoperoxidase; it is dependent on Mn2+ ions and catalytic amounts of phenols, such as 2,4-dichlorophenol and N-acetyltyrosinamide, and is greater than 95% inhibited by 0.1 mM cyanide. A second, novel oxidase activity utilizes Ca2+ and an unidentified, heat-stable, Mr less than 1000 factor that can be extracted by ethanol from egg homogenates. This NADH oxidase activity is only 40% inhibited by 0.1 mM cyanide and is maximally stimulated by 10 mM Ca2+. It has an apparent Km for NADH of 50 microM. The stoichiometry of NADH:O2 consumption is 1.6:1, but approaches 2:1 in the presence of 20 micrograms/ml superoxide dismutase or 200 micrograms/ml catalase. This indicates that complete reduction of O2 to water occurs and that the reaction does not produce H2O2 stoichiometrically. However, nearly complete inhibition of the reaction by higher catalase concentrations suggests that H2O2 is an intermediate. The properties of this novel oxidase activity suggest that it may play such a role in vivo.