(Received for publication, October 1, 1996, and in revised form, November 20, 1996)
From the Human glutathione transferases (GSTs) were shown
to catalyze the reductive glutathione conjugation of aminochrome
(2,3-dihydroindole-5,6-dione). The class Mu enzyme GST M2-2 displayed
the highest specific activity (148 µmol/min/mg), whereas GSTs A1-1,
A2-2, M1-1, M3-3, and P1-1 had markedly lower activities (<1
µmol/min/mg). The product of the conjugation, with a UV spectrum
exhibiting absorption peaks at 277 and 295 nm, was
4-S-glutathionyl-5,6-dihydroxyindoline as determined by NMR
spectroscopy. In contrast to reduced forms of aminochrome
(leucoaminochrome and o-semiquinone),
4-S-glutathionyl-5,6-dihydroxyindoline was stable in the
presence of molecular oxygen, superoxide radicals, and hydrogen
peroxide. However, the strongly oxidizing complex of Mn3+
and pyrophosphate oxidizes
4-S-glutathionyl-5,6-dihydroxyindoline to
4-S-glutathionylaminochrome, a new quinone derivative
with an absorption peak at 620 nm. GST M2-2 (and to a lower
degree, GST M1-1) prevents the formation of reactive oxygen
species linked to one-electron reduction of aminochrome catalyzed by
NADPH-cytochrome P450 reductase. The results suggest that the reductive
conjugation of aminochrome catalyzed by GSTs, in particular GST M2-2,
is an important cellular antioxidant activity preventing the formation of o-semiquinone and thereby the generation of reactive
oxygen species.
Dopamine, like other catecholamines, can be oxidized to the
corresponding o-quinone (1-4). The formation of
catecholamine o-quinones is followed by cyclization
involving the amino group of their side chains. The reduction of
aminochrome, dopachrome, noradrenochrome, and adrenochrome is
accompanied by their subsequent reoxidation by oxygen, which gives rise
to reactive chemical species (5-8). The oxidative conversion of
dopamine into aminochrome (2,3-dihydroindole-5,6-dione) and its
subsequent reduction and reoxidation by oxygen are believed to be the
cause of neurodegenerative processes in the dopaminergic system (9).
Degeneration of the dopaminergic neurons in the nigrostriatal system as
a consequence of aminochrome activation to produce reactive chemical
species has been proposed to contribute to the development of
Parkinson's disease (9, 10). In addition,
aminochrome-dependent neurodegeneration of the dopaminergic
system in the mesolimbic system has been suggested as a contributing
process in the development of schizophrenia (for reviews, see Refs. 11
and 12).
Glutathione transferases (GSTs)1 catalyze
the detoxication of a variety of xenobiotics including carcinogens,
environmental contaminants, anticancer agents, antibiotics, and
products of oxidative processes (for review, see Ref. 13). However, the ability of GSTs to catalyze conjugation of quinones is still largely unexplored. Quinones are a widespread group of xenobiotics that may
give rise to cytotoxicity, mutagenesis, and carcinogenesis (14-16).
The toxic effects of quinones are exploited in certain anticancer
agents. On the other hand, endogenous quinones such as vitamin
K1 and ubiquinone serve important physiological functions. In addition to their well established roles, ubiquinone has been proposed to be an important antioxidant in biological membranes. The
protective function is provided in the reduced hydroquinone state (17),
generated by the two-electron reduction catalyzed by
NAD(P)H:quinone oxidoreductase (18). To study the possibility that GSTs catalyze the conjugation and detoxication of quinones, aminochrome has been chosen as a representative of endogenous quinones
of obvious pathophysiological significance. This work shows that human
GSTs catalyze the formation of a glutathione conjugate of aminochrome,
which is more resistant to redox cycling than the parent compound.
Thus, glutathione conjugation appears to be an important mechanism for
protection against oxidative stress.
Chemicals
Dopamine, NADH, NADPH, and GSH were purchased from Sigma.
Animals
Male Sprague-Dawley rats with body weights of ~100 g were
used. The animals were kept on standard laboratory diet and starved for
24 h before being sacrificed. The animals used for
NADPH-cytochrome P450 reductase purification were injected
intraperitoneally with 8 mg of sodium phenobarbital once daily for 4 days.
Preparation of Enzymes
Human glutathione transferases M1-1 (allelic variant b), M2-2,
M3-3, A1-1, A2-2, and P1-1 (for nomenclature, see Ref. 19) were
obtained by heterologous expression in Escherichia coli and purified by affinity chromatography on S-hexylglutathione
immobilized on Sepharose 4B (20). NADPH-cytochrome P450 reductase was
purified from liver microsomes from phenobarbital-treated rats
according to Yasukochi and Masters (21). Protein determination was
performed according to the method of Bradford (22).
Synthesis of Aminochrome and Related Compounds
Oxidation of dopamine to aminochrome was performed as described
previously (4). The oxidizing agent was the
Mn3+-pyrophosphate complex prepared as described by
Archibald and Fridovich (23). Oxidation of
4-S-glutathionyl-5,6-dihydroxyindoline to
4-S-glutathionyl-5,6-dihydroxyindole was performed as
described by Mason (24). Three-hundred microliters of 12 M
HCl was added to 1 ml of 300 µM
4-S-glutathionyl-5,6-dihydroxyindoline solution in 25 mM sodium phosphate, pH 6.5, to decrease the pH to 1.3. The
reaction mixture was incubated for 30 min at room temperature. The
absorption spectra before and after oxidation of the indoline ring to
indole were recorded.
Assay Conditions
The standard
assay system for measuring conjugation of aminochrome with GSH
catalyzed by GSTs contained 0.1 M sodium phosphate, pH 6.5, 1 mM GSH, and 300 µM aminochrome at 30 °C.
The reaction was started by the addition of GST. The reaction was
recorded by monitoring the decrease in aminochrome absorption at
475 nm. The reaction rate was calculated by using the extinction
coefficient of aminochrome of 3058 M The assay system of 1 ml contained 50 mM
Tris-Cl, pH 6.5, 0.25 M sucrose, 30 µM
aminochrome, 1 mM (when present) GSH, 60 µg (when
present) of GST M1-1 or 8 µg (when present) of GST M2-2, and 250 µM NADPH. The formation of aminochrome (4) was allowed to
proceed for 2 min in the cuvette before the addition of GSH and
NADPH. The reaction was started by the addition of GST and 5 µg
of purified NADPH-cytochrome P450 reductase at 30 °C. The reaction
was monitored by recording NADPH oxidation at 340 nm (using an
extinction coefficient of 6220 M NMR Studies of the Reaction Product
For the structural analysis 300 µM aminochrome and
1 mM GSH were incubated with 100 µg of GST M1-1 in 100 ml
of 0.1 M sodium phosphate, pH 6.5, for 30 min. The aqueous
solution was diluted with 1-butanol and concentrated to 25 ml under
reduced pressure. More 1-butanol was added, and the concentration
procedure was repeated until all traces of water were removed. The
resulting solution was concentrated to 0.5 ml. The residue was mixed
with diethyl ether (10 ml), and acetic anhydride (0.5 ml) was added, followed by triethylamine (1 ml) and 4-dimethylaminopyridine (catalytic amount). The resultant mixture gave a clear solution after 10 min and
was stirred for a further 2 h, during which time considerable darkening occurred. The reaction mixture was poured into saturated sodium hydrogen carbonate solution (50 ml), extracted with
dichloromethane (5 × 50 ml), dried with sodium sulfate, and
concentrated. The residue was purified on a silica gel column (0.5 × 20 cm) using a stepwise gradient of ethyl acetate in pentane
(0-100%) followed by ethanol in ethyl acetate (0-30%) as eluent.
The major fraction was isolated and analyzed in CDCl3
solution by NMR spectroscopy. NMR spectra were recorded at 399.78 MHz
using a JEOL JNM400 spectrometer. Standard pulse sequences were used
for double quantum-filtered COSY and NOESY (nuclear Overhauser
enhancement spectroscopy) spectra; for the latter, mixing times were
varied between 100 and 600 ms.
GSH reacts
nonenzymatically with aminochrome, forming a conjugate at pH 6.5 and
30 °C (Table I). The enzymatic conjugation of
aminochrome with GSH was studied with six different human isoenzymes, GSTs A1-1, A2-2, P1-1, M1-1, M2-2, and M3-3. All these GSTs were found
to catalyze the conjugation of aminochrome, although major differences
in the rates were found. Class Mu GSTs exhibited the highest specific
activity, in particular GST M2-2 (148 µmol/min/mg of enzyme), while
other GSTs had much lower specific activities (Table I). The activity
of GST M2-2 with aminochrome was comparable to that with
1-chloro-2,4-dinitrobenzene, which usually is by far the most active
substrate for GSTs. The conjugation of aminochrome was recorded by
following the decrease in the absorbance at 475 nm, which is the
typical quinone absorption peak (Fig. 1, A
and B). A linear correlation between initial velocity and
amount of enzyme was demonstrated with GSTs M1-1 and M2-2 (Fig.
2).
Glutathione conjugation of aminochrome catalyzed by various human
GSTs
The decrease in
the absorbance at 475 nm in the visible region caused by conjugation of
aminochrome with GSH was also accompanied by a decrease in the
absorbance of aminochrome at 298 nm in the ultraviolet region (Fig. 1).
The decrease in aminochrome absorbance at 475 nm suggests that GSTs
M1-1 and M2-2 catalyze a conjugation, which can be regarded as a
two-electron reduction of aminochrome. The absorption spectrum of the
glutathione conjugate exhibited two peaks at 277 and 295 nm. However,
to determine whether the decrease in the absorbance at 475 was due to
the reduction of aminochrome or was a consequence of the addition of
glutathione to the carbonyl groups, a potent oxidizing agent was used
to oxidize leucoaminochrome-GSH to the corresponding
o-quinone. The addition of the
Mn3+-pyrophosphate complex to leucoaminochrome-GSH resulted
in its oxidation to aminochrome-GSH with a new absorption peak in the visible region at 620 nm, supporting the view that the addition of
glutathione does not affect the o-quinone structure (Fig.
3A). In the ultraviolet region, a new peak at
310 nm was also observed (data not shown).
NMR studies were performed to determine the structure of the
glutathione conjugate. However, direct NMR spectroscopic observation of
the product was not possible since concentration of the reaction solution resulted in polymerization. Azeotropic removal of the water
with 1-butanol under reduced pressure followed by acylation of the
concentrate afforded the crude tetraacetate (Fig.
4A). NMR spectroscopy of the product showed
coupled doublets at 6.64 and 7.4 ppm, consistent with oxidation of the
dihydroxyindoline followed by isomerization to the corresponding
indole, probably occurring during the workup procedure. The structure
of the product was confirmed by the presence of nuclear Overhauser
effect correlations between H-7 and two acetate groups and between H-3
and the CH2 of the cysteine residue (Fig. 4B).
We found that the structure of the acylated conjugate was
4-S-[(acetylglutamoyl)-N-cysteinyl-N-glycinyl]-5,6-diacetoxyindole: 1H NMR
An important question was whether the glutathione conjugate could be
oxidized by molecular oxygen like reduced forms of aminochrome (o-semiquinone and o-hydroquinone). Fig.
3B shows the absorption spectra of the aminochrome
(spectrum a) and
4-S-glutathionyl-5,6-dihydroxyindoline at 15, 30, and 60 min
(spectra b-d) in the visible region. However, no increase
in the absorbance at 620 nm, where the o-quinone has an
absorption maximum, was observed even after 60 min (spectrum d), suggesting that
4-S-glutathionyl-5,6-dihydroxyindoline is stable in the
presence of oxygen. These results were in agreement with the fact that
no oxygen consumption was measurable during conjugation of aminochrome
to 4-S-glutathionyl-5,6-dihydroxyindoline or after
completion of the reaction (data not shown).
Superoxide radicals (O One-electron reduction of
aminochrome catalyzed by enzymes such as NADPH-cytochrome P450
reductase has been postulated to be responsible for the formation of
reactive oxygen species that promote neurodegenerative processes in the
dopaminergic system (9). Therefore, it is of interest to know whether
GST M2-2 and other GSTs may compete with NADPH-cytochrome P450
reductase in the metabolism of aminochrome. Fig. 5 shows
that the reduction of aminochrome by NADPH-cytochrome P450 reductase is
accompanied by a constant rate of NADPH oxidation (21 nmol of
NADPH/min) recorded at 340 nm. The addition of 1 mM GSH to
the incubation mixture resulted in 31% inhibition of autoxidation.
However, the addition of GST M1-1 (60 µg/ml) to the incubation
mixture resulted in a strong inhibition (94%) of NADPH oxidation. The
inhibitory effect of GST M2-2 (8 µg/ml) was even more marked since
the addition of a smaller amount of this enzyme resulted in 98%
inhibition of NADPH oxidation. A strong inhibition of oxygen
consumption was also observed when GST M1-1 was added to the incubation
mixture. It is of significance that no increase in the
o-quinone absorbance peak at 620 nm was observed when GSH
and/or glutathione transferase was added to the incubation mixture
(data not shown).
Aminochrome is the product of the oxidation of dopamine to
o-quinone followed by cyclization of its aminoethyl chain at
physiological pH. It has been proposed that the formation of
aminochrome and the subsequent generation of reactive oxygen species
are involved in the neurodegenerative processes that occur in
dopaminergic neurons in the nigrostriatal system in Parkinson's
disease (9, 10) and in the mesolimbic system in schizophrenia (11,
12).
The results presented in this paper demonstrate that human GSTs
catalyze the reductive conjugation of aminochrome to
4-S-glutathionyl-5,6-dihydroxyindoline (Scheme
1). The NMR studies showed that the structure of the
acetylated derivative produced for analysis was
4-S-[(acetylglutamoyl)-N-cysteinyl-N-glycinyl]-5,6-diacetoxyindole. We conclude that the structure of the product of the GST-catalyzed reaction is 4-S-glutathionyl-5,6-dihydroxyindoline since the
spectrum of the corresponding oxidized (nonacetylated) compound,
4-S-glutathionyl-5,6-dihydroxyindole, is not the same as the
conjugate (4-S-glutathionyl-5,6-dihydroxyindoline) enzymatically produced. The two peaks of
4-S-glutathionylleucoaminochrome at 278 and 295 nm are
shifted to 295 and 307 nm in
4-S-glutathionyl-5,6-dihydroxyindole due to oxidation of the
dihydroxyindoline followed by acid-catalyzed isomerization to indole
during the workup and acylation procedures required for NMR
analysis.
Scheme 1.
GSTs are known to have broad and overlapping substrate specificities,
but only a few of their numerous substrates are so markedly preferentially used by one isoenzyme as aminochrome. With this substrate, GST M2-2 has a specific activity >200-fold above that of
any other GST (Table I). Another example is
2-cyano-1,3-dimethyl-1-nitrosoguanidine, for which GST M2-2 is also the
most active enzyme by almost 2 orders of magnitude (26). The finding
that aminochrome is an excellent GST substrate extends the number of
physiologically occurring compounds that serve as substrates for GSTs.
Aminochrome is a product of oxidative metabolism, like many other
pathophysiologically relevant GST substrates such as alkenals and
hydroperoxides generated by biological oxidations and radical reactions
(27).
NADPH-cytochrome P450 reductase and NAD(P)H:quinone oxidoreductase
catalyze one- and two-electron reduction of aminochrome to
o-semiquinone and leucoaminochrome
(o-hydroquinone), respectively. These products are oxidized
by O2, yielding reactive oxygen species. However, a major
difference in the rate of oxidation of o-semiquinone and
o-hydroquinone forms has been observed (4, 9). The
autoxidation of o-semiquinone is too fast to be recorded by
conventional steady-state kinetics, whereas the autoxidation of the
o-hydroquinone can readily be monitored by the decrease in
the quinone absorption peak at 475 nm. The lability of
o-semiquinone and leucoaminochrome in the presence of
oxygen contrasts with the chemical stability of 4-S-glutathionyl-5,6-dihydroxyindoline since no oxidation
was observed during conjugation or after the completion of the
reaction. The reactivity of
4-S-glutathionyl-5,6-dihydroxyindoline was found to be
completely different from that of leucoaminochrome and
o-semiquinone. Molecular oxygen (O2) is mainly
responsible for oxidation of o-semiquinone (91% of the
total oxygen-dependent autoxidation) (9), while superoxide
radicals are mainly responsible for oxidation of the leucoaminochrome
(89% of the total oxygen-dependent autoxidation) (4, 9).
However, superoxide radicals, hydrogen peroxide, and molecular oxygen
are not able to oxidize
4-S-glutathionyl-5,6-dihydroxyindoline under the conditions
investigated.
It seems reasonable to consider glutathione conjugation of aminochrome
as a reaction terminating any possible redox cycling since it prevents
the formation of reactive oxygen species during one- or two-electron
reduction of aminochrome. It is obvious that a very small amount of
aminochrome can give rise to a large production of reactive oxygen
species due the redox cycling properties of aminochrome, emphasizing
the importance of the glutathione conjugation. Our results show that
GST M2-2, as well as GST M1-1, by a competing conjugation reaction
prevents autoxidation otherwise elicited by the reduction of
aminochrome catalyzed by NADPH-cytochrome P450 reductase (Fig. 5).
One-electron reduction of aminochrome catalyzed by NADPH-cytochrome
P450 reductase has been postulated to be the mechanism of formation of
reactive oxygen species, which can cause neurodegeneration that in the
nigrostriatal system may result in Parkinson's disease or in the
mesolimbic system in schizophrenia (9).
The glutathione conjugation catalyzed by class Mu GSTs seems to be the
most important of the reported antioxidant reactions related to
aminochrome metabolism since this reaction prevents the redox cycling
between oxidized and reduced forms of aminochrome as a
consequence of the chemical stability of
4-S-glutathionyl-5,6-dihydroxyindoline. NAD(P)H:quinone oxidoreductase has also been postulated to
provide an important cellular defense against formation of reactive
oxygen species since the reaction catalyzed by NAD(P)H:quinone
oxidoreductase competes with one-electron donors such as
NADPH-cytochrome P450 reductase for the reduction of aminochrome to
leucoaminochrome (4). However, the antioxidant role of NAD(P)H:quinone
oxidoreductase is dependent on the presence of superoxide dismutase,
catalase, or GSH peroxidase, which inhibit leucoaminochrome
autoxidation, or on the action of conjugation enzymes such as
sulfotransferase or catechol ortho-methyltransferase.
It seems plausible that human subjects with low expression or lack of
the genes coding for NAD(P)H:quinone oxidoreductase, GST M1-1,
and/or GST M2-2 will be at higher risk for neurodegeneration of the
dopaminergic nigrostriatal or mesolimbic system, which may result in
the development of Parkinson's disease and schizophrenia,
respectively.
We conclude that GST M2-2 efficiently catalyzes the conjugation of
aminochrome, thus preventing the formation of reactive oxygen species.
Other GSTs, such as the class Mu enzymes GST M1-1 and GST M3-3, also
promote this conjugation reaction, albeit with much lower catalytic
activities. We cannot disregard the possibility that other enzymes not
yet tested also play a role in detoxication of aminochrome. An example
is GST M5-5, which has been cloned from a cDNA library from the
frontal cortex of human brain (28). To establish the physiological
relevance of the GST activity to neurodegenerative diseases such as
Parkinson's disease and schizophrenia, it is necessary to explore the
regional distribution of GST M2-2 and other class Mu GSTs in the brain
and in the dopaminergic system.
Division of Biochemistry,
Department of Biochemistry, and
the ** Division of Organic Pharmaceutical Chemistry,
1
cm
1 (4).
1
cm
1) (4). Oxygen consumption was monitored with an oxygen
electrode from Hansatech D. W. (King's Lynn, United Kingdom).
Glutathione Conjugation of Aminochrome
GST
CDNBa
Aminochrome
µmol/min/mg
µmol/min/mg
A1-1
46 ± 3
0.037 ± 0.002
A2-2
32 ± 5
0.031 ± 0.002
M1-1
96
± 20
0.763 ± 0.090
M2-2
138 ± 5
148 ± 13
M3-3
3.4 ± 0.8
0.143 ± 0.022
P1-1
41
± 10
0.041 ± 0.023
a
CDNB, 1-chloro-2,4-dinitrobenzene.
Fig. 1.
Reductive conjugation of aminochrome with
glutathione catalyzed by GSTs M1-1 and M2-2. A, catalytic
effect of GST M1-1 on the reaction between 130 µM
aminochrome and 1 mM GSH in 0.1 M sodium
phosphate, pH 6.5. Spectra were recorded after 0 (spectrum a), 1 (spectrum b), 5 (spectrum c), and 10 (spectrum d) min. B, catalytic effect of GST M2-2
on 160 µM aminochrome and 1 mM GSH. Spectra
were recorded after 0 (spectrum a) and 1 (spectrum
b) min.
[View Larger Version of this Image (17K GIF file)]
Fig. 2.
Formation of
4-S-glutathionyl-5,6-dihydroxyindoline as a function of the
amount of GSTs M1-1 (A) and M2-2 (B). The incubation conditions are described under "Experimental
Procedures."
[View Larger Version of this Image (16K GIF file)]
Fig. 3.
Oxidation of
4-S-glutathionyl-5,6-dihydroxyindoline by the
Mn3+-pyrophosphate complex (A) and stability of
the conjugate under aerobic conditions (B). A,
102 µM aminochrome (spectrum a) was reduced
with GST M1-1 (60 µg) to
4-S-glutathionyl-5,6-dihydroxyindoline (spectrum
b) in the presence of 1 mM GSH.
4-S-Glutathionyl-5,6-dihydroxyindoline was oxidized to
4-S-glutathionylaminochrome by the addition of 1 mM Mn3+-pyrophosphate complex. B, 80 µM aminochrome (spectrum a) was reduced with
500 µM GSH in the presence of 60 µg of GST M1-1, and
the absorption spectra were registered 10 (spectrum b), 30 (spectrum c), and 60 (spectrum d) min after
reduction.
[View Larger Version of this Image (17K GIF file)]
(ppm) 1.88 (2H, dt, J = 7.0, 8.0 Hz,
-CH2-Glu), 2.20-2.30 (14H, m,
-CH2-Glu, CH3-acetyl), 3.08 (2H, m,
CH2-Cys), 3.40-3.44 (2H, m (AB), CH2-Gly),
3.76 (1H, t, J = 8.0 Hz, CH-Glu), 4.16 (1H, m, CH-Cys),
6.64 (1H, d, J = 3.0 Hz, H-3), 7.10, (1H, s, H-7), and
7.45 (1H, d, J = 3.0 Hz, H-2). The data obtained were consistent with the data for the nonacylated compound previously isolated (25). Therefore, we conclude that the structure of the
nonacylated conjugate is
4-S-glutathionyl-5,6-dihydroxyindole. However,
4-S-glutathionyl-5,6-dihydroxyindole has been reported to
have two peaks in the ultraviolet region at 295 and 307 nm (25), while
the product of glutathione conjugation of aminochrome exhibited two
different peaks at 277 and 295 nm. The possibility that the two peaks
of the conjugate at 277 and 295 nm change to 295 and 307 nm as a
consequence of oxidation of the indoline ring to indole during
acylation of the product before NMR analysis was studied. Therefore,
the spectrum of the conjugate product was compared with the spectrum
obtained after oxidation of indoline to indole under acid catalysis
according to Mason (24). The two peaks of the conjugate at 277 and 295 nm did change after acid catalysis to 295 and 307 nm, a result in
agreement with the data reported by D'Ischia et al. (25)
(data not shown). Therefore, we conclude that the structure of the
conjugate is 4-S-glutathionyl-5,6-dihydroxyindoline, which
was subsequently oxidized to the indole derivative during the workup
and acylation procedures.
Fig. 4.
Analysis of the aminochrome-GSH conjugate.
A, acetylation of
4-S-glutathionyl-5,6-dihydroxyindoline to block the free hydroxyl and amino groups accompanied by oxidation and isomerization of
indoline to indole. Acetylation was performed as described under
"Experimental Procedures." B, nuclear Overhauser effect correlations used for structural assignment of the indole-glutathione conjugate by NMR analysis.
[View Larger Version of this Image (17K GIF file)]
2) have been reported to play a major
role in the reoxidation of leucoaminochrome produced by the
NAD(P)H:quinone oxidoreductase-catalyzed reduction of aminochrome (9).
Therefore, we have studied the possibility that superoxide radicals may
catalyze the autoxidation of
4-S-glutathionyl-5,6-dihydroxyindoline by using xanthine
oxidase and hypoxanthine to produce superoxide radicals. However, no
oxidation of 4-S-glutathionyl-5,6-dihydroxyindoline in the
presence of superoxide radicals was observed. The possibility that
other biological oxidizing agents such as H2O2
may oxidize the conjugate was also studied. However, no autoxidation of
4-S-glutathionyl-5,6-dihydroxyindoline was observed in the
presence of H2O2 (data not shown).
Fig. 5.
Glutathione transferases M1-1 (A)
and M2-2 (B) compete with NADPH-cytochrome P450 reductase
in the reduction of aminochrome. The incubations conditions are
described under "Experimental Procedures." The values are the
means ± S.D. of three experiments.
[View Larger Version of this Image (20K GIF file)]
*
This work was supported by the Tore Nilson Foundation of
Medical Research, the Swedish Natural Science Research Council, and the
Swedish Cancer Society. The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Div. of Biochemistry,
Dept. of Pharmaceutical Bioscience, BMC, Box 578, S-751 23 Uppsala,
Sweden. Tel.: 46-18-174281; Fax: 46-18-558778; E-mail: Juan.SeguraAguilar{at}farmbio.uu.se.
1
The abbreviation used is: GSTs, glutathione
transferases.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.