ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 278, No. 1, April, pp. 288-290, 1990
COMMUNICATION Glutathione
Reductase
Functions
as Vanadate(V)
Reductase
Xianglin Shi and N. S. Dalal’ Department
of
Chemistry,
West Virginia
University,
Morgantown,
West Virginia
26505
Received December 1,1989
The oxidation of NADPH by vanadate(V) in the presence of glutathione reductase showed typical enzymatic kinetics. The oxidation was inhibited by N-ethylmaleimide, a glutathione reductase inhibitor. Superoxide dismutase had no significant effect on the oxidation, indicating noninvolvment of the superoxide radical. The vanadate(V) reduction was found to be a one-electron transfer process. These results suggest a new pathway for vanadate(V) metabolism and a new function of glutathione reductase. 8 1990Academic PESS, IIIC.
as the decrease in absorbance at 340 nm. The spin trap, 5,5-dimethy1-pyrroline-N-oxide (DMPO), was purchased from Aldrich and used without further purification, since very weak or no spin adduct signal was obtained from the purchased sample when used alone. ESR spectra were obtained at X-band (9.7 GHz) using a Bruker ER200 ESR spectrometer. The magnetic field was calibrated with a self-tracking NMR gaussmeter (Bruker, Model ER035A) and the microwave frequency was measured with a digital frequency counter (Hewlett-Packard, Model 5340A). An ASPECT 2000 computer was used for data storage and analysis. The concentrations given in the figure legends are the final concentrations. All experiments were carried out in the phosphate buffer solution (pH 7.2) and at room temperature.
RESULTS Compounds of vanadium(V), such as NaV03, exert potent toxic effects on a wide variety of biological systems (l-3). Despite intense current activity, however, the biochemical mechanism of vanadate(V) toxicity is still not well understood (l14). It is believed that one of the important pathways involves the oxidation (by vanadate(V)) of NAD(P)H (15, 16), but, to our knowledge, there has been no report of the identification of any vanadate(V) reductase. With a view to finding if there exists any vanadate(V) reductase, we have investigated the oxidation of NADPH by vanadate(V) in the presence of glutathione reductase. Glutathione reductase (GSSG-R)’ was selected because our previous studies have shown (17) that GSSG-R can act as a reductase for chromat,e(VI), which is isoelectronic with vanadate(V). We find that GSSG-R does indeed function as a NADPH-dependent vanadate(V) reductase, thus pointing to a new pathway for the metabolism of vanadate(V), as well as to this property for GSSG-R. MATERIALS
AND
METHODS
Sodium metavanadate (NaVOB ), henceforth vanadate, was purchased from Aldrich and its solution was always freshly prepared. The GSSG-R used was from bovine intestinal mucosa and was purchased from Sigma. The oxidation of NADPH was followed photometrically ’ To whom correspondence and requests for reprint,s should be addressed. * Abbreviations used: GSSG-R, glutathione reductase; NaV03, sodium metavanadate; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide. 288
AND
DISCUSSION
When NADPH was incubated with vanadate(V) in a pH 7.2 phosphate buffer solution, little or no oxidation of NADPH was observed (Fig. la). However, a significant amount of (NADPH) oxidation was observed (Fig. lb) when the same reaction was carried out in the presence of GSSG-R. An increase in the concentration of the vanadate(V) increased the rate of NADPH oxidation (Fig. lc), while no oxidation was observed on the addition of N-ethylmaleimide, a GSSG-R inhibitor (Fig. Id). This result clearly indicated that GSSG-R was involved in the oxidation of NADPH and that the reaction was enzymatic. In order to characterize this enzymatic reaction, the kinetics of the oxidation process were investigated. A double-reciprocal plot of the initial velocity (Vi) as a function of vanadate(V) concentration [S] was made (Fig. 2a) according to the well known Michaelis-Menten equation, 1 2LL+k v, - vnm PI
1 v,,,
’
where k, is the apparent Michaelis constant and V,,,,, is the maximal velocity. It was found that k, = 0.38 mM, which is in the range of 0.01-10.0 mM for most enzymes (18). Moreover, the rate of oxidation of NADPH was found to increase linearly with increase in the amount of the enzyme (Fig. 2b), which is typical of an enzymatic reaction. For further evidence for the redox process we sought evidence for the formation of vanadium(IV), based on recent re0003-9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
GLUTATHIONE
1.01
REDIJCTASE
FUNCTIONS
I
'
10
0
20
30 Time
40
50
60
(min.)
FIG. 1. Time and concentration dependence of NADPH oxidation by vanadate(Vj catalyzed by glutathione reductase. Reaction mixtures contain (a) 0.26 mM NaVO:,, 0.17 mM NADPH; (h) same as (aj hut with 0.27 mg/ml glutathione reductase; (cj same as (a) hut with I mM NaVOiI; (d) same as (b) but with 10 mg/ml N-ethylmaleimide.
ports that vanadium(IV) reacts with H20:, to generate hydroxyl (‘OH) radicals (16, 19, 20). We thus studied the enzymatic reduction of vanadate(V) by NADPH and its subsequent reaction with H202. The reaction was followed by measuring the generated ‘OH radical concentration via electron spin resonance (ESR) spin trapping, using DMPO as the spin trap. An aqueous solution of DMPO, H20,, and vanadate(V) with NADPH or with GSSG-R did not give any detectable ESR signal (Figs. 3a and 3b, respectively). A reaction mixture of DMPO, H202, vanadate(V), NADPH, and GSSG-R generated an ESR spectrum (Fig. 3c), consisting of a 1:2:2:1 quartet with nitrogen and hydrogen hyperfine couplings aN = a,, = 14.9 G. On the basis of these splitting constants and the 1:2:2:1 quartet lineshape, the spectrum in Fig. 3c was assigned to the DMPOOH adduct. As a confirmatory test of the ‘OH radical formation, formate was added as a secondary trap (21). The spec-
AS VANADATE(Vj
289
REDUCTASE
trum in Fig. 3d shows that addition of formate results in an appearance of the DMPO-COOadduct (indicated by the arrowheads) due to the reaction of ‘OH with formate, confirming the ‘OH radical formation in the original reaction. Addition of the GSSG-R inhibitor, N-ethylmaleimide, to a reaction mixture consisting of DMPO, Hz02, vanadate(V), NADPH, and GSSG-R significantly inhibited the ‘OH radical formation (Fig. 3e). The above results support our premise that GSSG-R is involved in the oxidation of NADPH by vanadate(V) and it functions as a vanadate(V) reductase. The biochemical mechanism underlying the vanadate(V) reductase behavior of GSSG-R is not yet clear. In this context we noted that recent studies have shown that vanadate(V) stimulates the oxidation of NAD(P)H by cell membranes (410) and this has been attributed to a vanadate(V)-dependent NAD(P)H oxidase or to an NAD(P)H dehydrogenase (4-10). However, it has been reported recently that the vanadate(V) stimulation of NADPH oxidation by biological membranes involves oxidation by 0; (produced by some membrane-associated oxidase or dehydrogenase) plus vanadate(V), and not directly by any enzyme (3, 11). To examine if 0; plays any role in the reductase behavior of GSSG-R, we utilized SOD as an 0, inhibitor. It was found that SOD had no appreciable effect on the oxidation of NADPH or the production of ‘OH radicals in the above reactions, showing that 0; does not play a significant role in the GSSG-R-catalyzed oxidation of NADPH by vanadate(V). In conclusion, the present study establishes an important new property of GSSG-R, its function as a vanadate(V) reductase, thus suggesting a new biochemical pathway for the metabolism (and hence toxicity) of vanadium. This finding is of further interest because the main function of this enzyme is generally thought to be a catalyst in the reduction of some specific disulfide bonds, with its main substrates being the oxidized glutathione (GSSG), mixed disulfide between glutathione and y-glutamylcysteine (22), and that between glutathione and coenzyme A (23). It is surprising that GSSG-R catalyzed the reduction of vanadate(V) and chromate (17) which have no obvious similarity to a disulfide linkage. Thus further inves-
a
0.10
[vanadate
1-l
(mM-‘)
glutathione
0.20
reductase
(mg)
FIG. 2. (a) Determination of the Michaelis-Menten constant (k,). The reciprocals of the initial velocities are plotted as a function of the reciprocal of vanadate(V) concentrations according to the Michaelis-Menten equation (see text). The intercept gives (-l/k,), from which we obtained h,, = 0.46 mM. (h) The effect of enzyme concentration on enzyme activity. The oxidation of NADPH increases linearly when the amount of enzyme is increased.
290
SHI AND
a
DMPO+V5++H202+
-NADPH LA
DMPO+V5++H202+ -GSSG-R
>J~,+N
ADPH
P
W+HCOONa
e
-%---4c)+N-Ethylmaleimide 10G -H
FIG. 3. ESR spectra of reaction mixtures containing (a) 0.1 M DMPO, 1 mM H202, 0.2 mM NaVOs, 0.5 mM NADPH; (b) 0.1 M DMPO, 1 mM H202, 0.2 mM NaVOs, 0.27 mm/ml glutathione reductase; (c) same as (b) but with 0.5 mM NADPH; (d) same as (c) but with 0.25 M HCOONa; (e) same as (c) but with 10 mg/ml N-ethylmaleimide. The spectrum in (a) was assigned to the DMPO-OH adduct and that of(d) was assigned to a mixture of DMPO-OH and DMPO-COOadducts (as indicated by the arrowheads).
DALAL 4 Erdmann, E., Krawietz, W., Philipp, G., Hackbarth, I.. Schmitz, W., Scholz, H., and Crane, F. L. (1979) Nature (London) 282, 335-336. 5. Erdmann, E., Krawietz, W., Hackbarth, I., Schmitz, W., and Scholz, H. (1981) Nature (London) 294,288. 6. Coulombe, R. A., Briskin, D., Keller, R. J., Thornley, W. R., and Scharma, R. P. (1987) Arch. Biochem. Biophys. 255,267-273. 7. Rau, M., Patole, M. S., Vijaya, S., Kurup, C. K. R., and Ramasarma, T. (1987) CellBiochem. 75,151-159. 8. Datole, M. S., Kurup, C. K. R., and Ramasarma, T. (1986) Biothem. Biophys. Res. Commun. 141,171-175. 9. Datole, M. S., Kurup, C. K. P., and Ramasarma, T. (1987) Mol. Cell Biochem. 75, 161-167. 10. Reif, D. W., Coulombe, R. A., and Aust, S. D. (1989) Arch. Biothem. Biophys. 270,137-143. 11. Liochev, S., and Fridovich, I. (1986) Arch. Biochem. Biophys. 250, 139-145. 12. Darr, D., and Fridovich, 562-565.
I. (1984) Arch. Biochem. Biophys. 232,
13. Darr, D., and Fridovich, 220-227.
I. (1985) Arch. Biochem. Biophys.
14. Liochev,
S., and Fridovich,
243,
I. (1985) J. Free Radical Biol. Med. 1,
287292. 15. Keller, R. J., Coulombe, R. A., Sharma, R. P., Grover, T. A., and Piette, L. H. (1989) Arch. Biochem. Biophys. 271,40-48. 16. Keller, R. J., Coulombe, R. A., Sharma, R. P., Grover, T. A., and Piette, L. H. (1989) Free Radical Biol. Med. 6, 15-22. 17. Shi, X., and Dalal, N. S. (1989) Biochem. Biophys. Res. Commun.
163,627-634. tigations on the mechanism of this enzyme’s function as metalion reductase at the molecular level would be highly interesting since the results could have implications for other biochemical reactions also.
REFERENCES 1. Chasteen, N. D. (1983) Struct. Bonding 53, 107-137. 2. Boyd, D. W., and Kustin, K. (1986) Adu. Inorg. Riochem. 6,311365. 3. Liochev, S. I., and Fridovich, I. (1989) Free Radical Biol. Med. 6, 617-622.
18. Karlson, P. (1967) Kurzes Lehrbuch Thieme, Stuttgart.
der Biochemie,
6th ed., p. 69,
19. Ozawa, T., and Hanaki, A. (1989) Chem. Pharm. Bull. 37, 14071409. 20. Keller, R. J., Sharma, R. P., Grover, T. A., and Piette, L. H. (1988) Arch. Biochem. Biophys. 265,524-533. 21. Buettner, G. R. (1987) Free Radical Biol. Med. 3, 259-303. 22. Griffith, 0. W., Novogrodsky, A., and Meister, A. (1980) Proc. N&l. Acad. Sci. USA 78,7492-7496. 23. Carlberg, I., and Mannervik, 484,268-274.
B. (1977) Biochim.
Biophys.
Acta
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 278, No. 1, April, pp. 288-290, 1990
COMMUNICATION Glutathione
Reductase
Functions
as Vanadate(V)
Reductase
Xianglin Shi and N. S. Dalal’ Department
of
Chemistry,
West Virginia
University,
Morgantown,
West Virginia
26505
Received December 1,1989
The oxidation of NADPH by vanadate(V) in the presence of glutathione reductase showed typical enzymatic kinetics. The oxidation was inhibited by N-ethylmaleimide, a glutathione reductase inhibitor. Superoxide dismutase had no significant effect on the oxidation, indicating noninvolvment of the superoxide radical. The vanadate(V) reduction was found to be a one-electron transfer process. These results suggest a new pathway for vanadate(V) metabolism and a new function of glutathione reductase. 8 1990Academic PESS, IIIC.
as the decrease in absorbance at 340 nm. The spin trap, 5,5-dimethy1-pyrroline-N-oxide (DMPO), was purchased from Aldrich and used without further purification, since very weak or no spin adduct signal was obtained from the purchased sample when used alone. ESR spectra were obtained at X-band (9.7 GHz) using a Bruker ER200 ESR spectrometer. The magnetic field was calibrated with a self-tracking NMR gaussmeter (Bruker, Model ER035A) and the microwave frequency was measured with a digital frequency counter (Hewlett-Packard, Model 5340A). An ASPECT 2000 computer was used for data storage and analysis. The concentrations given in the figure legends are the final concentrations. All experiments were carried out in the phosphate buffer solution (pH 7.2) and at room temperature.
RESULTS Compounds of vanadium(V), such as NaV03, exert potent toxic effects on a wide variety of biological systems (l-3). Despite intense current activity, however, the biochemical mechanism of vanadate(V) toxicity is still not well understood (l14). It is believed that one of the important pathways involves the oxidation (by vanadate(V)) of NAD(P)H (15, 16), but, to our knowledge, there has been no report of the identification of any vanadate(V) reductase. With a view to finding if there exists any vanadate(V) reductase, we have investigated the oxidation of NADPH by vanadate(V) in the presence of glutathione reductase. Glutathione reductase (GSSG-R)’ was selected because our previous studies have shown (17) that GSSG-R can act as a reductase for chromat,e(VI), which is isoelectronic with vanadate(V). We find that GSSG-R does indeed function as a NADPH-dependent vanadate(V) reductase, thus pointing to a new pathway for the metabolism of vanadate(V), as well as to this property for GSSG-R. MATERIALS
AND
METHODS
Sodium metavanadate (NaVOB ), henceforth vanadate, was purchased from Aldrich and its solution was always freshly prepared. The GSSG-R used was from bovine intestinal mucosa and was purchased from Sigma. The oxidation of NADPH was followed photometrically ’ To whom correspondence and requests for reprint,s should be addressed. * Abbreviations used: GSSG-R, glutathione reductase; NaV03, sodium metavanadate; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide. 288
AND
DISCUSSION
When NADPH was incubated with vanadate(V) in a pH 7.2 phosphate buffer solution, little or no oxidation of NADPH was observed (Fig. la). However, a significant amount of (NADPH) oxidation was observed (Fig. lb) when the same reaction was carried out in the presence of GSSG-R. An increase in the concentration of the vanadate(V) increased the rate of NADPH oxidation (Fig. lc), while no oxidation was observed on the addition of N-ethylmaleimide, a GSSG-R inhibitor (Fig. Id). This result clearly indicated that GSSG-R was involved in the oxidation of NADPH and that the reaction was enzymatic. In order to characterize this enzymatic reaction, the kinetics of the oxidation process were investigated. A double-reciprocal plot of the initial velocity (Vi) as a function of vanadate(V) concentration [S] was made (Fig. 2a) according to the well known Michaelis-Menten equation, 1 2LL+k v, - vnm PI
1 v,,,
’
where k, is the apparent Michaelis constant and V,,,,, is the maximal velocity. It was found that k, = 0.38 mM, which is in the range of 0.01-10.0 mM for most enzymes (18). Moreover, the rate of oxidation of NADPH was found to increase linearly with increase in the amount of the enzyme (Fig. 2b), which is typical of an enzymatic reaction. For further evidence for the redox process we sought evidence for the formation of vanadium(IV), based on recent re0003-9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
GLUTATHIONE
1.01
REDIJCTASE
FUNCTIONS
I
'
10
0
20
30 Time
40
50
60
(min.)
FIG. 1. Time and concentration dependence of NADPH oxidation by vanadate(Vj catalyzed by glutathione reductase. Reaction mixtures contain (a) 0.26 mM NaVO:,, 0.17 mM NADPH; (h) same as (aj hut with 0.27 mg/ml glutathione reductase; (cj same as (a) hut with I mM NaVOiI; (d) same as (b) but with 10 mg/ml N-ethylmaleimide.
ports that vanadium(IV) reacts with H20:, to generate hydroxyl (‘OH) radicals (16, 19, 20). We thus studied the enzymatic reduction of vanadate(V) by NADPH and its subsequent reaction with H202. The reaction was followed by measuring the generated ‘OH radical concentration via electron spin resonance (ESR) spin trapping, using DMPO as the spin trap. An aqueous solution of DMPO, H20,, and vanadate(V) with NADPH or with GSSG-R did not give any detectable ESR signal (Figs. 3a and 3b, respectively). A reaction mixture of DMPO, H202, vanadate(V), NADPH, and GSSG-R generated an ESR spectrum (Fig. 3c), consisting of a 1:2:2:1 quartet with nitrogen and hydrogen hyperfine couplings aN = a,, = 14.9 G. On the basis of these splitting constants and the 1:2:2:1 quartet lineshape, the spectrum in Fig. 3c was assigned to the DMPOOH adduct. As a confirmatory test of the ‘OH radical formation, formate was added as a secondary trap (21). The spec-
AS VANADATE(Vj
289
REDUCTASE
trum in Fig. 3d shows that addition of formate results in an appearance of the DMPO-COOadduct (indicated by the arrowheads) due to the reaction of ‘OH with formate, confirming the ‘OH radical formation in the original reaction. Addition of the GSSG-R inhibitor, N-ethylmaleimide, to a reaction mixture consisting of DMPO, Hz02, vanadate(V), NADPH, and GSSG-R significantly inhibited the ‘OH radical formation (Fig. 3e). The above results support our premise that GSSG-R is involved in the oxidation of NADPH by vanadate(V) and it functions as a vanadate(V) reductase. The biochemical mechanism underlying the vanadate(V) reductase behavior of GSSG-R is not yet clear. In this context we noted that recent studies have shown that vanadate(V) stimulates the oxidation of NAD(P)H by cell membranes (410) and this has been attributed to a vanadate(V)-dependent NAD(P)H oxidase or to an NAD(P)H dehydrogenase (4-10). However, it has been reported recently that the vanadate(V) stimulation of NADPH oxidation by biological membranes involves oxidation by 0; (produced by some membrane-associated oxidase or dehydrogenase) plus vanadate(V), and not directly by any enzyme (3, 11). To examine if 0; plays any role in the reductase behavior of GSSG-R, we utilized SOD as an 0, inhibitor. It was found that SOD had no appreciable effect on the oxidation of NADPH or the production of ‘OH radicals in the above reactions, showing that 0; does not play a significant role in the GSSG-R-catalyzed oxidation of NADPH by vanadate(V). In conclusion, the present study establishes an important new property of GSSG-R, its function as a vanadate(V) reductase, thus suggesting a new biochemical pathway for the metabolism (and hence toxicity) of vanadium. This finding is of further interest because the main function of this enzyme is generally thought to be a catalyst in the reduction of some specific disulfide bonds, with its main substrates being the oxidized glutathione (GSSG), mixed disulfide between glutathione and y-glutamylcysteine (22), and that between glutathione and coenzyme A (23). It is surprising that GSSG-R catalyzed the reduction of vanadate(V) and chromate (17) which have no obvious similarity to a disulfide linkage. Thus further inves-
a
0.10
[vanadate
1-l
(mM-‘)
glutathione
0.20
reductase
(mg)
FIG. 2. (a) Determination of the Michaelis-Menten constant (k,). The reciprocals of the initial velocities are plotted as a function of the reciprocal of vanadate(V) concentrations according to the Michaelis-Menten equation (see text). The intercept gives (-l/k,), from which we obtained h,, = 0.46 mM. (h) The effect of enzyme concentration on enzyme activity. The oxidation of NADPH increases linearly when the amount of enzyme is increased.
290
SHI AND
a
DMPO+V5++H202+
-NADPH LA
DMPO+V5++H202+ -GSSG-R
>J~,+N
ADPH
P
W+HCOONa
e
-%---4c)+N-Ethylmaleimide 10G -H
FIG. 3. ESR spectra of reaction mixtures containing (a) 0.1 M DMPO, 1 mM H202, 0.2 mM NaVOs, 0.5 mM NADPH; (b) 0.1 M DMPO, 1 mM H202, 0.2 mM NaVOs, 0.27 mm/ml glutathione reductase; (c) same as (b) but with 0.5 mM NADPH; (d) same as (c) but with 0.25 M HCOONa; (e) same as (c) but with 10 mg/ml N-ethylmaleimide. The spectrum in (a) was assigned to the DMPO-OH adduct and that of(d) was assigned to a mixture of DMPO-OH and DMPO-COOadducts (as indicated by the arrowheads).
DALAL 4 Erdmann, E., Krawietz, W., Philipp, G., Hackbarth, I.. Schmitz, W., Scholz, H., and Crane, F. L. (1979) Nature (London) 282, 335-336. 5. Erdmann, E., Krawietz, W., Hackbarth, I., Schmitz, W., and Scholz, H. (1981) Nature (London) 294,288. 6. Coulombe, R. A., Briskin, D., Keller, R. J., Thornley, W. R., and Scharma, R. P. (1987) Arch. Biochem. Biophys. 255,267-273. 7. Rau, M., Patole, M. S., Vijaya, S., Kurup, C. K. R., and Ramasarma, T. (1987) CellBiochem. 75,151-159. 8. Datole, M. S., Kurup, C. K. R., and Ramasarma, T. (1986) Biothem. Biophys. Res. Commun. 141,171-175. 9. Datole, M. S., Kurup, C. K. P., and Ramasarma, T. (1987) Mol. Cell Biochem. 75, 161-167. 10. Reif, D. W., Coulombe, R. A., and Aust, S. D. (1989) Arch. Biothem. Biophys. 270,137-143. 11. Liochev, S., and Fridovich, I. (1986) Arch. Biochem. Biophys. 250, 139-145. 12. Darr, D., and Fridovich, 562-565.
I. (1984) Arch. Biochem. Biophys. 232,
13. Darr, D., and Fridovich, 220-227.
I. (1985) Arch. Biochem. Biophys.
14. Liochev,
S., and Fridovich,
243,
I. (1985) J. Free Radical Biol. Med. 1,
287292. 15. Keller, R. J., Coulombe, R. A., Sharma, R. P., Grover, T. A., and Piette, L. H. (1989) Arch. Biochem. Biophys. 271,40-48. 16. Keller, R. J., Coulombe, R. A., Sharma, R. P., Grover, T. A., and Piette, L. H. (1989) Free Radical Biol. Med. 6, 15-22. 17. Shi, X., and Dalal, N. S. (1989) Biochem. Biophys. Res. Commun.
163,627-634. tigations on the mechanism of this enzyme’s function as metalion reductase at the molecular level would be highly interesting since the results could have implications for other biochemical reactions also.
REFERENCES 1. Chasteen, N. D. (1983) Struct. Bonding 53, 107-137. 2. Boyd, D. W., and Kustin, K. (1986) Adu. Inorg. Riochem. 6,311365. 3. Liochev, S. I., and Fridovich, I. (1989) Free Radical Biol. Med. 6, 617-622.
18. Karlson, P. (1967) Kurzes Lehrbuch Thieme, Stuttgart.
der Biochemie,
6th ed., p. 69,
19. Ozawa, T., and Hanaki, A. (1989) Chem. Pharm. Bull. 37, 14071409. 20. Keller, R. J., Sharma, R. P., Grover, T. A., and Piette, L. H. (1988) Arch. Biochem. Biophys. 265,524-533. 21. Buettner, G. R. (1987) Free Radical Biol. Med. 3, 259-303. 22. Griffith, 0. W., Novogrodsky, A., and Meister, A. (1980) Proc. N&l. Acad. Sci. USA 78,7492-7496. 23. Carlberg, I., and Mannervik, 484,268-274.
B. (1977) Biochim.
Biophys.
Acta
