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Vanadate and molybdate stimulate the oxidation of NADH by superoxide radical
OF BIOCHEMISTRY AND BIOPHYSICS Vol. 232, No. 2, August 1, pp. 562-565, 1934
ARCHIVES
Vanadate
and Molybdate Stimulate the Oxidation of NADH by Superoxide Radical’
DOUGLAS DARR Center
IRWIN FRIDOVICH’
AND
far the Study of Aging and Human Devebpmxnt Duke University
Medical
Received January
and the Department of Biochemistry, Center, Durh.am, North Carolina 27710
16, 1934, and in revised form April
4, 1934
Vanadate or molybdate strongly accelerate the cooxidation of NADH, or of reduced nicotinamide mononucleotide, by the xanthine oxidase plus xanthine reaction. Superoxide dismutase eliminated the effect of vanadate or molybdate, while catalase was without effect. It follows that vanadate or molybdate accelerate the oxidation of dihydropyridines by 0;. A stoichiometry of 4 NADH oxidized per 0; introduced suggests a chain reaction for which a mechanism is proposed. These results provide an explanation for the reported stimulation, by vanadate, of NADH oxidation by biological membranes. The oxidation of NADH by biological membranes is markedly stimulated by vanadate (1). Inhibition of this oxidation by superoxide dismutase indicated that 0; was involved, but no mechanism for its involvement was suggested (1). A possible explanation derives from the work of Bielski and Chan (2-4), who demonstrated that the binding of NADH to lactic dehydrogenase or to glyceraldehyde-3-phosphate dehydrogenase greatly increased its reactivity towards 0;. One may similarly suppose that vanadate, or the isopolyanion formed from it (5), associate with NADH and augment its reactivity with 0;. Alternately, it is possible that 0; reacts with vanadate, generating an oxidant which then oxidizes NADH. A convenient way to explore these possibilities would be to use the xanthine oxidase reaction as a source of 0, (6-8), and then to examine the effects of vanadate on the oxidation of NADH by 02, under a i This work was supported by research grants from the Institute General Medical Sciences of the National Institutes of Health, Bethesda, Md.; the United States Army Research Office, Research Triangle Park, N. C.; and the National Institute of Aging, Bethesda, Md. ’ To whom correspondence should be addressed. 0003-9861A34 $3.00 Copyright All rights
0 19% by Academic Press. Inc. of reproduction in any form resewed.
variety of conditions. We now report that vanadate does increase the rate of oxidation of NADH by 0;. Moreover, molybdate can replace vanadate in stimulating this oxidation, and reduced nicotinamide mononucleotide can replace NADH as the reductant. MATERIALS
AND
METHODS
Xanthine oxidase, isolated from bovine cream (9), was kindly provided by R. D. Wiley and Dr. K. V. Rajagopalan. SOD’ was isolated from bovine erythrocytes as previously described (7). Bovine liver catalase, NADH, and NMNH were from the Sigma Chemical Company. Vz06 was from Alpha Products, Inc. Stock solutions (30 mM) of vanadate were prepared by dissolving VzOs in 1.0 M NaOH, neutralizing with 1.0 M HCI, and then adjusting to the desired volume with 56 rnM potassium phosphate, pH 7.0. Under these conditions one obtains a mixture of isopolyvanadates (5). Nevertheless, the concentration of these stock solutions and of dilutions thereof are expressed throughout in terms of VxO,. Ammonium molybdate was from Baker, and ita concentration is expressed as Mo,O&f. The oxidation of NADH or of NMNH was followed in terms of the decrease in absorbance at 340 nm. Reactions were carried out in 56 mM potassium
* Abbreviations used: SOD, superoxide dismutase; NMNH, reduced nicotinamide mononucleotide. 562
SUPEROXIDE
AND
VANADATE-STIMULATED
NADH
563
OXIDATION
phosphate, 0.1 mM EDTA, pH 7.8, at 25°C. Xanthine and xanthine oxidase, when present, were used at 10 and 0.01 NM, respectively. RESULTS
Vanudde or mol@date stimulate the oxidation of NADH by 0;. NADH was cooxidized by xanthine oxidase acting upon xanthine, and vanadate or molybdate, which had no effect in the absence of the xanthine oxidase reaction and which did not affect the oxidation of xanthine by xanthine oxidase, markedly accelerated this cooxidation. SOD strongly inhibited this cooxidation, while thermally denatured SOD was without effect. Catalase did not significantly inhibit the cooxidation of NADH by the xanthine oxidase reaction. These results, which are shown in Fig. 1, suggest that vanadate or molybdate increased the susceptibility of NADH to oxidation by 0;. This effect was not specific for NADH, since NMNH was similarly affected.
I
I
02
I
/
I
,
05 mu
06
07
08
/
03 04 [VANADATE]
FIG. 2. The effect of varying the concentration of vanadate. Reaction mixtures were as described in Fig. 1, and the concentration of vanadate was varied from 0 to 0.7 rnM.
tial rates of NADH oxidation were recorded. Figure 2 demonstrates that the rate of the cooxidation of NADH by the xanthine oxidase reaction increased with the concentration of vanadate, approaching a maximum at 0.5 mM vanadate, at which point a 20-fold stimulation of the basal rate was noted. The basal rate of NADH cooxidation was doubled at 30 PM vanadate. Dependence upon reactant ccmcewtra- Figure 3 illustrates the effect of varying tions. The concentrations of vanadate, the concentration of NADH and NMNH. NADH, xanthine oxidase, and SOD were Once again the rate increased linearly at independently varied while the linear inifirst and then more gradually, as a limiting rate was approached. Figure 4 shows that the oxidation of NADH under these conditions was entirely dependent upon xanthine oxidase. One might a priori have expected that the rate of NADH oxidation would be a linear function of the xanthine oxidase concentration. However, if 0; is the product of the xanthine oxidase re-
I
I
I
\’ 1
I
I
I
0
2
4
6
a
IO
12
MINUTES AT WC
1. Effects of vanadate or molybdate on the cooxidation of NADH by xanthine oxidase plus xanthine. Reaction mixtures contained 50 mM potassium phosphate, 0.1 mM EDTA, 10 pM xanthine, 0.1 mM NADH, pH 7.8, 25°C. Where indicated, vanadate or molybdate was added to 0.15 mM, xanthine oxidase to 0.01 JIM, SOD to 6.7 nrd, and catalase (40 units/3 ml) to 1.7 nrd. NADH oxidation was followed at 340 nm. FIG.
005 [NADH
010
015
020
M NMNH]
mM
025
FIG. 3. The effect of varying the concentration of NADH or NMNH. Reaction mixtures were as described in Fig. 1, and contained 0.15 mM vanadate, while the concentrations of NADH (0) and NMNH (0) were varied from 0 to 0.20 mM.
564
DARR
AND
3025 g .
z
zo8= E ; 5
IS-
E 4 a
IO-
ES 5 /gg 0
0
25 20 15 IO o” 01 [XANTHINEomAq”~
0010 XANTHINE
0020 OXIDASE (PM)
cl2
0030
FIG. 4. The effect of varying the concentration of xanthine oxidase. Reaction mixtures were as in Fig. 1, and contained 0.15 mM vanadate, while the concentration of xanthine oxidase was varied from 0 to 0.030 pM. The inset presents the data plotted as a function of the square root of the enzyme concentration.
action which was responsible for the cooxidation of NADH, then proportionality to the square root of xanthine oxidase might be anticipated. This arises because the rate of production of 0; would be a linear function of the xanthine oxidase concentration, while the spontaneous dismutation of 0, is proportional to [O;F. As previously discussed (6), the steady-state concentration of 0; in the xanthine oxidase reaction is proportional to fxanthine oxidase. The inset in Fig. 4 exhibits the affirmation of this expectation. Inhibition bg superoxide dismutase. Figure 1 demonstrated that SOD inhibited the vanadate-stimulated cooxidation of NADH by the xanthine oxidase reaction. Figure 5 presents the effect of varying the concentration of SOD. It is apparent that striking inhibition was achieved at low levels of SOD, but it is also clear that inhibition by SOD approached a limit of 90%. Xanthine oxidase is known to catalyze the oxidation of NADH, albeit at a much slower rate than its action upon xanthine (lo), and this accounts for the SOD-insensitive fraction of the vanadate-stimulated NADH oxidation. When the cooxidation of NADH, by the xanthine oxidase reaction in the presence of vanadate, was corrected for this direct oxidation of NADH, SOD was seen capable of complete inhibition. This too is shown in Fig. 5.
FRIDOVICH
Chain length. Since 02, or its conjugate acid HO:!. , can only accept or lose a single electron, the oxidation of NADH by 0% must give rise to NAD - , which can in turn reduce O2 to 0;. This provides the basis for a chain reaction in which each 0, introduced into the system is potentially capable of causing the oxidation of more than one NADH. The oxidation, by 0;) of NADH bound to lactic dehydrogenase or to glyceraldehyde-3-phosphate dehydrogenase (24) was shown to be a chain reaction. When 0.01 PM xanthine oxidase acted upon 10 PM xanthine in the presence of 0.5 mM vanadate, 0.2 mM NADH, 0.1 mM EDTA, 50 mM potassium phosphate, pH 7.8, at 25”C, we observed the oxidation of 6.3 PM NADH/ min. Under the same conditions we estimated 0; production by replacing NADH with 20 PM ferricytochrome c and following the production of ferrocytochrome c at 550 nm. We noted the reduction of 1.6 PM cytochrome c/min, and all of this was prevented by 10 pg/ml SOD. The ratio NADH oxidized/O; produced was therefore 6.3/1.6 pM/min = 4. It is clear that a chain reaction was involved. Chan and Bielski (4) noted that the NADH/O, chain length increased with decrease in pH, and it appears likely that we could similarly have observed greater chain length at pH acid to 7.8.
FIG. 5. Inhibition by superoxide dismutase. Reaction mixtures were as in Fig. 1, and contained 0.15 mM vanadate, while the concentration of SOD was varied from 0 to 6.7 nr.r. Line 1 presents total NADH oxidation while line 2 presents only the vanadate-stimulated NADH oxidation.
SUPEROXIDE
AND
VANADATE-STIMULATED
DISCUSSION
Vanadate or molybdate can accelerate the oxidation of dihydropyridines, such as NADH or NMNH, by 0;. Two general mechanisms appear possible. The first of these is that NADH bound to vanadate reacts with 0, much more rapidly than does NADH in free solution. The second is that 0; reacts with vanadate, generating an oxidant capable of the oxidation of NADH. This oxidant might be a peroxy Vuvj complex. There are ways to facilitate a decision between these two mechanisms. Thus, if binding of NADH to vanadate were an essential prerequisite for reaction with O;, then response of rate of oxidation to the concentration of the dihydropyridine should be different for NMNH than for NADH. This derives from the expectation that NADH and NMNH would not exhibit identical affinities for the vanadate polyanion. In fact, the saturation of rates of oxidation with increasing [NMNH] and [NADH] were identical (see Fig. 3). This leads to the tentative conclusion that 0, reacts with vanadate or with molybdate, yielding a species which can cause the univalent oxidation of dihydropyridines. The following reactions provide a schematic representation of this mechanism. 4
V(V) + 0;
b) V,,,,-00
-
V(IV,-00
+ NADH V,,,,-OOH
c) Vuv,-OOH d)NAD.
+O,
+ NAD .
+ H+ - VCvj+ H202 - NAD+ + 0,
The observations of Ramasarma et cd (1) may now be accommodated. Suppose that the membrane-associated NADH oxidase, which they studied, was capable of
NADH
OXIDATION
565
0; production, as is the NADPH oxidase of neutrophils (11, 12). Vanadate would then stimulate not because of a direct effect on the NADH oxidase, but rather because of its enhancement of the oxidation of NADH by 0;. In that case SOD should have inhibited the vanadate-stimulated NADH oxidation by plasma membranes, and this was observed (1). Since catalase had no significant effect when added at 13 units/ml, we conclude that H202 was not a factor in the vanadatestimulated cooxidation of NADH. This conclusion is reinforced by the observation that addition of 0.1 mM Hz02 did not influence the rate of NADH oxidation. REFERENCES 1. RAMASARMA, T., MACKELLAR, W. C., AND CRANE, F. L. (1981) Biochim. Biophys. Ada 646.88-98. 2. CHAN, P. C., AND BIELSKI, B. H. J. (1974) J. BbL them 249.1317-1319. 3. BIELSKI, B. H. J., AND CHAN, P. C. (1976) J. Biol Cbm 251, 3841-3844. 4. CHAN, P. C., AND BIELSKI, B. H. J. (1980) J. BioL Chem 255, 874-876. 5. COTTON,F. A., AND WILKINSON, G. (1972) Advanced Inorganic Chemistry, pp. 821-824, Interscience, New York. 6. MCCORD, J. M., AND FRIDOVICH, I. (1963) J. Bid Chxm. 243, 5753-5760. 7. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. Bid Chem 244, 6049-6055. 8. FRIDOVICH, I. (1970) J. Bid Chem 245,4053-4057. 9. WAUD, W. R., BRADY, F. O., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem Biophys. 169, 695-701. 10. EDMONDSON, D., MASSEY, V., PALMER, G., BEACHAM, L. M., AND ELION, G. B. (1972) J. Bid Chem 247, 1597-1604. 11. CURNUTTE, J. T., AND BABIOR, B. M. (1974) J. C%n Invest. 53,1662-1672. 12. BABIOR, B. M. (1978) New EngL J. &XL 298,659668; 721-725.
ARCHIVES
Vanadate
and Molybdate Stimulate the Oxidation of NADH by Superoxide Radical’
DOUGLAS DARR Center
IRWIN FRIDOVICH’
AND
far the Study of Aging and Human Devebpmxnt Duke University
Medical
Received January
and the Department of Biochemistry, Center, Durh.am, North Carolina 27710
16, 1934, and in revised form April
4, 1934
Vanadate or molybdate strongly accelerate the cooxidation of NADH, or of reduced nicotinamide mononucleotide, by the xanthine oxidase plus xanthine reaction. Superoxide dismutase eliminated the effect of vanadate or molybdate, while catalase was without effect. It follows that vanadate or molybdate accelerate the oxidation of dihydropyridines by 0;. A stoichiometry of 4 NADH oxidized per 0; introduced suggests a chain reaction for which a mechanism is proposed. These results provide an explanation for the reported stimulation, by vanadate, of NADH oxidation by biological membranes. The oxidation of NADH by biological membranes is markedly stimulated by vanadate (1). Inhibition of this oxidation by superoxide dismutase indicated that 0; was involved, but no mechanism for its involvement was suggested (1). A possible explanation derives from the work of Bielski and Chan (2-4), who demonstrated that the binding of NADH to lactic dehydrogenase or to glyceraldehyde-3-phosphate dehydrogenase greatly increased its reactivity towards 0;. One may similarly suppose that vanadate, or the isopolyanion formed from it (5), associate with NADH and augment its reactivity with 0;. Alternately, it is possible that 0; reacts with vanadate, generating an oxidant which then oxidizes NADH. A convenient way to explore these possibilities would be to use the xanthine oxidase reaction as a source of 0, (6-8), and then to examine the effects of vanadate on the oxidation of NADH by 02, under a i This work was supported by research grants from the Institute General Medical Sciences of the National Institutes of Health, Bethesda, Md.; the United States Army Research Office, Research Triangle Park, N. C.; and the National Institute of Aging, Bethesda, Md. ’ To whom correspondence should be addressed. 0003-9861A34 $3.00 Copyright All rights
0 19% by Academic Press. Inc. of reproduction in any form resewed.
variety of conditions. We now report that vanadate does increase the rate of oxidation of NADH by 0;. Moreover, molybdate can replace vanadate in stimulating this oxidation, and reduced nicotinamide mononucleotide can replace NADH as the reductant. MATERIALS
AND
METHODS
Xanthine oxidase, isolated from bovine cream (9), was kindly provided by R. D. Wiley and Dr. K. V. Rajagopalan. SOD’ was isolated from bovine erythrocytes as previously described (7). Bovine liver catalase, NADH, and NMNH were from the Sigma Chemical Company. Vz06 was from Alpha Products, Inc. Stock solutions (30 mM) of vanadate were prepared by dissolving VzOs in 1.0 M NaOH, neutralizing with 1.0 M HCI, and then adjusting to the desired volume with 56 rnM potassium phosphate, pH 7.0. Under these conditions one obtains a mixture of isopolyvanadates (5). Nevertheless, the concentration of these stock solutions and of dilutions thereof are expressed throughout in terms of VxO,. Ammonium molybdate was from Baker, and ita concentration is expressed as Mo,O&f. The oxidation of NADH or of NMNH was followed in terms of the decrease in absorbance at 340 nm. Reactions were carried out in 56 mM potassium
* Abbreviations used: SOD, superoxide dismutase; NMNH, reduced nicotinamide mononucleotide. 562
SUPEROXIDE
AND
VANADATE-STIMULATED
NADH
563
OXIDATION
phosphate, 0.1 mM EDTA, pH 7.8, at 25°C. Xanthine and xanthine oxidase, when present, were used at 10 and 0.01 NM, respectively. RESULTS
Vanudde or mol@date stimulate the oxidation of NADH by 0;. NADH was cooxidized by xanthine oxidase acting upon xanthine, and vanadate or molybdate, which had no effect in the absence of the xanthine oxidase reaction and which did not affect the oxidation of xanthine by xanthine oxidase, markedly accelerated this cooxidation. SOD strongly inhibited this cooxidation, while thermally denatured SOD was without effect. Catalase did not significantly inhibit the cooxidation of NADH by the xanthine oxidase reaction. These results, which are shown in Fig. 1, suggest that vanadate or molybdate increased the susceptibility of NADH to oxidation by 0;. This effect was not specific for NADH, since NMNH was similarly affected.
I
I
02
I
/
I
,
05 mu
06
07
08
/
03 04 [VANADATE]
FIG. 2. The effect of varying the concentration of vanadate. Reaction mixtures were as described in Fig. 1, and the concentration of vanadate was varied from 0 to 0.7 rnM.
tial rates of NADH oxidation were recorded. Figure 2 demonstrates that the rate of the cooxidation of NADH by the xanthine oxidase reaction increased with the concentration of vanadate, approaching a maximum at 0.5 mM vanadate, at which point a 20-fold stimulation of the basal rate was noted. The basal rate of NADH cooxidation was doubled at 30 PM vanadate. Dependence upon reactant ccmcewtra- Figure 3 illustrates the effect of varying tions. The concentrations of vanadate, the concentration of NADH and NMNH. NADH, xanthine oxidase, and SOD were Once again the rate increased linearly at independently varied while the linear inifirst and then more gradually, as a limiting rate was approached. Figure 4 shows that the oxidation of NADH under these conditions was entirely dependent upon xanthine oxidase. One might a priori have expected that the rate of NADH oxidation would be a linear function of the xanthine oxidase concentration. However, if 0; is the product of the xanthine oxidase re-
I
I
I
\’ 1
I
I
I
0
2
4
6
a
IO
12
MINUTES AT WC
1. Effects of vanadate or molybdate on the cooxidation of NADH by xanthine oxidase plus xanthine. Reaction mixtures contained 50 mM potassium phosphate, 0.1 mM EDTA, 10 pM xanthine, 0.1 mM NADH, pH 7.8, 25°C. Where indicated, vanadate or molybdate was added to 0.15 mM, xanthine oxidase to 0.01 JIM, SOD to 6.7 nrd, and catalase (40 units/3 ml) to 1.7 nrd. NADH oxidation was followed at 340 nm. FIG.
005 [NADH
010
015
020
M NMNH]
mM
025
FIG. 3. The effect of varying the concentration of NADH or NMNH. Reaction mixtures were as described in Fig. 1, and contained 0.15 mM vanadate, while the concentrations of NADH (0) and NMNH (0) were varied from 0 to 0.20 mM.
564
DARR
AND
3025 g .
z
zo8= E ; 5
IS-
E 4 a
IO-
ES 5 /gg 0
0
25 20 15 IO o” 01 [XANTHINEomAq”~
0010 XANTHINE
0020 OXIDASE (PM)
cl2
0030
FIG. 4. The effect of varying the concentration of xanthine oxidase. Reaction mixtures were as in Fig. 1, and contained 0.15 mM vanadate, while the concentration of xanthine oxidase was varied from 0 to 0.030 pM. The inset presents the data plotted as a function of the square root of the enzyme concentration.
action which was responsible for the cooxidation of NADH, then proportionality to the square root of xanthine oxidase might be anticipated. This arises because the rate of production of 0; would be a linear function of the xanthine oxidase concentration, while the spontaneous dismutation of 0, is proportional to [O;F. As previously discussed (6), the steady-state concentration of 0; in the xanthine oxidase reaction is proportional to fxanthine oxidase. The inset in Fig. 4 exhibits the affirmation of this expectation. Inhibition bg superoxide dismutase. Figure 1 demonstrated that SOD inhibited the vanadate-stimulated cooxidation of NADH by the xanthine oxidase reaction. Figure 5 presents the effect of varying the concentration of SOD. It is apparent that striking inhibition was achieved at low levels of SOD, but it is also clear that inhibition by SOD approached a limit of 90%. Xanthine oxidase is known to catalyze the oxidation of NADH, albeit at a much slower rate than its action upon xanthine (lo), and this accounts for the SOD-insensitive fraction of the vanadate-stimulated NADH oxidation. When the cooxidation of NADH, by the xanthine oxidase reaction in the presence of vanadate, was corrected for this direct oxidation of NADH, SOD was seen capable of complete inhibition. This too is shown in Fig. 5.
FRIDOVICH
Chain length. Since 02, or its conjugate acid HO:!. , can only accept or lose a single electron, the oxidation of NADH by 0% must give rise to NAD - , which can in turn reduce O2 to 0;. This provides the basis for a chain reaction in which each 0, introduced into the system is potentially capable of causing the oxidation of more than one NADH. The oxidation, by 0;) of NADH bound to lactic dehydrogenase or to glyceraldehyde-3-phosphate dehydrogenase (24) was shown to be a chain reaction. When 0.01 PM xanthine oxidase acted upon 10 PM xanthine in the presence of 0.5 mM vanadate, 0.2 mM NADH, 0.1 mM EDTA, 50 mM potassium phosphate, pH 7.8, at 25”C, we observed the oxidation of 6.3 PM NADH/ min. Under the same conditions we estimated 0; production by replacing NADH with 20 PM ferricytochrome c and following the production of ferrocytochrome c at 550 nm. We noted the reduction of 1.6 PM cytochrome c/min, and all of this was prevented by 10 pg/ml SOD. The ratio NADH oxidized/O; produced was therefore 6.3/1.6 pM/min = 4. It is clear that a chain reaction was involved. Chan and Bielski (4) noted that the NADH/O, chain length increased with decrease in pH, and it appears likely that we could similarly have observed greater chain length at pH acid to 7.8.
FIG. 5. Inhibition by superoxide dismutase. Reaction mixtures were as in Fig. 1, and contained 0.15 mM vanadate, while the concentration of SOD was varied from 0 to 6.7 nr.r. Line 1 presents total NADH oxidation while line 2 presents only the vanadate-stimulated NADH oxidation.
SUPEROXIDE
AND
VANADATE-STIMULATED
DISCUSSION
Vanadate or molybdate can accelerate the oxidation of dihydropyridines, such as NADH or NMNH, by 0;. Two general mechanisms appear possible. The first of these is that NADH bound to vanadate reacts with 0, much more rapidly than does NADH in free solution. The second is that 0; reacts with vanadate, generating an oxidant capable of the oxidation of NADH. This oxidant might be a peroxy Vuvj complex. There are ways to facilitate a decision between these two mechanisms. Thus, if binding of NADH to vanadate were an essential prerequisite for reaction with O;, then response of rate of oxidation to the concentration of the dihydropyridine should be different for NMNH than for NADH. This derives from the expectation that NADH and NMNH would not exhibit identical affinities for the vanadate polyanion. In fact, the saturation of rates of oxidation with increasing [NMNH] and [NADH] were identical (see Fig. 3). This leads to the tentative conclusion that 0, reacts with vanadate or with molybdate, yielding a species which can cause the univalent oxidation of dihydropyridines. The following reactions provide a schematic representation of this mechanism. 4
V(V) + 0;
b) V,,,,-00
-
V(IV,-00
+ NADH V,,,,-OOH
c) Vuv,-OOH d)NAD.
+O,
+ NAD .
+ H+ - VCvj+ H202 - NAD+ + 0,
The observations of Ramasarma et cd (1) may now be accommodated. Suppose that the membrane-associated NADH oxidase, which they studied, was capable of
NADH
OXIDATION
565
0; production, as is the NADPH oxidase of neutrophils (11, 12). Vanadate would then stimulate not because of a direct effect on the NADH oxidase, but rather because of its enhancement of the oxidation of NADH by 0;. In that case SOD should have inhibited the vanadate-stimulated NADH oxidation by plasma membranes, and this was observed (1). Since catalase had no significant effect when added at 13 units/ml, we conclude that H202 was not a factor in the vanadatestimulated cooxidation of NADH. This conclusion is reinforced by the observation that addition of 0.1 mM Hz02 did not influence the rate of NADH oxidation. REFERENCES 1. RAMASARMA, T., MACKELLAR, W. C., AND CRANE, F. L. (1981) Biochim. Biophys. Ada 646.88-98. 2. CHAN, P. C., AND BIELSKI, B. H. J. (1974) J. BbL them 249.1317-1319. 3. BIELSKI, B. H. J., AND CHAN, P. C. (1976) J. Biol Cbm 251, 3841-3844. 4. CHAN, P. C., AND BIELSKI, B. H. J. (1980) J. BioL Chem 255, 874-876. 5. COTTON,F. A., AND WILKINSON, G. (1972) Advanced Inorganic Chemistry, pp. 821-824, Interscience, New York. 6. MCCORD, J. M., AND FRIDOVICH, I. (1963) J. Bid Chxm. 243, 5753-5760. 7. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. Bid Chem 244, 6049-6055. 8. FRIDOVICH, I. (1970) J. Bid Chem 245,4053-4057. 9. WAUD, W. R., BRADY, F. O., WILEY, R. D., AND RAJAGOPALAN, K. V. (1975) Arch. Biochem Biophys. 169, 695-701. 10. EDMONDSON, D., MASSEY, V., PALMER, G., BEACHAM, L. M., AND ELION, G. B. (1972) J. Bid Chem 247, 1597-1604. 11. CURNUTTE, J. T., AND BABIOR, B. M. (1974) J. C%n Invest. 53,1662-1672. 12. BABIOR, B. M. (1978) New EngL J. &XL 298,659668; 721-725.