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Vol. 56, Issue 2, 272-278, August 1999
Divisions of Immunology (S.C., K.W., D.Z.) and Biology (M.S.), Beckman Research Institute of the City of Hope, Duarte, California; Department of Medical Oncology, Charing Cross Hospital, London, England (R.K.); and Department of Chemical Biology, College of Pharmacy, Rutgers University, Piscataway, New Jersey (C.S.Y.)
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Summary |
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 Top
 Summary
 Introduction
Experimental Procedures
Results
Discussion
References
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The molecular basis of the interaction of DT-diaphorase with a
cytotoxic nitrobenzamide CB1954
[5-(aziridin-1-yl)-2,4-dinitrobenzamide] and five inhibitors was
investigated with wild-type DT-diaphorase (human and rat) and
five mutants [three rat mutants (rY128D, rG150V, rH194D) and two human
mutants (hY155F, hH161Q)]. hY155F and hH161Q were generated to
evaluate a hypothesis that Tyr155 and His161 participate in the
obligatory two-electron transfer reaction of the enzyme. The catalytic
properties of hY155F and hH161Q were compared with a naturally
occurring mutant, hP187S. Pro187 to Ser mutation disturbs the structure
of the central parallel 
-sheet, resulting in a reduction of the
binding affinity of the flavin-adenine dinucleotide prosthetic
group. With NADH as the electron donor and menadione as the electron
acceptor, the kcat values for the wild-type
human DT-diaphorase, hY155F, hH161Q, and hP187S were measured as
66 ± 1, 23 ± 0, 5 ± 0 and 8 ± 2 × 103 min
1, respectively. Because hY155F still
has significant catalytic activity, the hydroxyl group on Tyr155 may
not be as important as proposed. Interestingly, hY155F was found to be
3.3 times more active than the human wild-type DT-diaphorase in the
reduction of CB1954. Computer modeling based on our results suggests
that CB1954 is situated in the active site, with the aziridinyl group pointing toward Tyr155 and the amide group placed near a hydrophobic pocket next to Tyr128. Dicoumarol, Cibacron blue, chrysin,
7,8-dihydroxyflavone, and phenindone are competitive inhibitors of the
enzyme with respect to nicotinamide coenzymes. The binding orientations
of dicoumarol, flavones, and phenindone in the active site of
DT-diaphorase were predicted by results from our inhibitor-binding
studies and computer modeling based on published X-ray structures. Our
studies generated results that explain why dicoumarol is a potent
inhibitor and binds differently from flavones and phenindone in the
active site of DT-diaphorase.
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Introduction |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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DT-diaphorase
(EC 1.6.99.2), also called NAD(P)H: (quinone-acceptor) oxidoreductase,
is a flavoprotein that catalyzes two-electron reduction of quinones and
quinonoid compounds to hydroquinones, with either NADH or NADPH as the
electron donor (Ernster, 1987
). This enzyme is a dimer enzyme, with one
flavin-adenine dinucleotide (FAD) prosthetic group per active site. The
two identical subunits are in a head-to-tail arrangement. Thus, each
active site is made up of parts of both subunits. DT-diaphorase can
function physiologically as one of several vitamin K reductases in the
vitamin K cycling involved in the hepatic posttranslational
modification of vitamin K hydroquinone-dependent blood coagulation
factors (Wallin et al., 1978). Oral anticoagulants such as dicoumarol
and warfarin (Hollander and Ernster, 1975; Lind et al., 1979) have been
found to be potent competitive inhibitors with respect to nicotinamide coenzymes of the enzyme. Flavones isolated from the Chinese herb Scutellariae radix (Huang Qin), which has
anticoagulating properties, were first shown by Liu et al. (1990) to be
potent inhibitors of DT-diaphorase. This enzyme has been shown to be
capable of the reduction in vitamin K3
(menadione) but not vitamin K1 (Preusch and
Smalley, 1990). The reduction in vitamin K1 by
microsomal preparations could not be inhibited by 10 µM dicoumarol.
Whereas Cibacron blue (Prochaska, 1988), phenindone (Hollander and
Ernster, 1975), and flavones (Chen et al., 1993) were all shown to be
competitive inhibitors with respect to nicotinamide coenzymes, they
were found to inhibit DT-diaphorase in a synergistic fashion when used
together with dicoumarol. These results suggest that these inhibitors
bind differently in the active site of DT-diaphorase, although they are
all competitive inhibitors with respect to nicotinamide coenzymes. However, the molecular basis of the interaction of these
inhibitors/anticoagulants to DT-diaphorase is not yet established.
DT-diaphorase is also known to reductively activate cytotoxic antitumor
quinones such as mitomycins, anthracyclines, and
aziridinyl-benzoquinones (Siegel et al., 1990a,b; Walton et al., 1991),
as well as nitrobenzamides such as CB1954
[5-(aziridin-1-yl)-2,4-dinitrobenzamide] (Boland et al., 1991).
Enzymatic reduction of these antitumor compounds gives rise to reactive
intermediates that can then undergo nucleophilic additions with DNA and
other macromolecules, suggesting a possible mechanism for their
cytotoxicity (Lin et al., 1972). Although these compounds are
substrates of DT-diaphorase, their binding characteristics in the
active site are not yet known.
Several DT-diaphorase mutants have been generated in our laboratories
to study the structure-function relationship of the enzyme (Chen et
al., 1992, 1997; Ma et al., 1992a,b; Cui et al., 1995). In addition,
the X-ray structure of rat DT-diaphorase has been reported by Li et al.
(1995). In this article, we report on the reactivities of prodrugs such
as CB1954 with the wild-type DT-diaphorase and five mutants. In
addition, the inhibition profiles of five inhibitors (i.e., dicoumarol,
Cibacron blue, chrysin, 7,8-dihydroxyflavone, and phenindone) on the
wild-type and mutant forms of the enzyme were determined. By analyzing
these results and reviewing the X-ray structure of the enzyme,
information regarding the molecular basis of the interaction of CB1954
and five inhibitors with DT-diaphorase was obtained. The results are
important to further develop prodrugs for cancer treatment and oral
anticoagulants to treat heart diseases.
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Experimental Procedures |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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Materials.
Dihydronicotinamide riboside (NRH) was prepared
by alkaline phosphatase treatment of dihydronicotinamide mononucleotide
with a reported method (Friedlos et al., 1992). Alkaline phosphatase was used to remove the phosphate group from dihydronicotinamide mononucleotide, and NRH was purified by reversed-phase HPLC. CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] was synthesized at the Institute of Cancer Research and kindly supplied by P. Burke (Charing Cross Hospital, London, UK).
; Chen et al., 1997).
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Mutant Preparations.
A polymerase chain reaction
(PCR)-based mutagenesis method described by Nelson and Long
(1989) was used to generate the three human mutants (hP187S, hY155F,
and hH161Q). The desired PCR product was resolved on 1% agarose gel
and then extracted with the QIAquick Gel Extraction Kit (Qiagen, Inc.,
Chatsworth, CA). The gel-purified PCR product was cloned into PCRII
vector from the TA cloning kit (Invitrogen Co., San Diego, CA).
Mutant clones were then selected by dideoxy sequencing. The resulting
mutant constructs were religated into the Escherichia coli
expression vector pKK233-2 (Pharmacia, Piscataway, NJ), through the
NcoI and HindIII restriction sites. For the
hP187S mutant, the cloned cDNA was ligated into the PKK-(His)6 vector,
which was prepared as described previously (Wu et al., 1998).
Purification of Mutant Proteins.
The transformed cells were
cultured following a previously described procedure by Chen et al.
(1992). The cells were sonicated and centrifuged with the reported
procedure. The supernatant from the 90-min centrifugation at
105,000g was applied to a 50-ml Affi-Gel Blue
(Bio-Rad, Richmond, CA) column, and the column was washed according to
the published method, except for the final elution step. The mutant was
eluted from the column with a buffer containing 50 mM Tris buffer (pH
7.5), 0.25 M sucrose, 0.5 M KCl, 10 mM NADH, and 5 µM FAD. The active
fractions were pooled and concentrated by centrifugation with a
centriplus concentrator unit (Amicon, Beverly, MA) with the
molecular-weight cutoff at 30,000.
.
Enzyme Assay.
NADH (NRH)-menadione reductase activity was
determined spectrophotometrically by measuring the reduction of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) at 610 nm
[
(610 nm) = 11.3 × 103
M1 · cm1]
at 25°C. The assay mixture (1 ml) contained 50 mM potassium phosphate, pH 7.5, 500 µM NADH (or NRH), 10 µM menadione, and 0.3 mg/ml MTT. In the assay, menadione was used as the electron acceptor,
and MTT was included to continuously reoxidize the menadiol formed. In
addition, the 2,6-dichlorophenolindophenol reductase activity of the
preparation was determined following the procedure by Benson et al.
(1980). The reduction in CB1954 was analyzed by HPLC. The enzyme
preparations were incubated with NADH or NRH (500 µM) and CB1954 at
different concentrations (0.1-2.0 mM), in sodium phosphate buffer (10 mM; pH 7) at 37°C. At various times, aliquots (10 µl) were injected
onto a Partisil 10 SCX (250 × 4.7 mm) HPLC column and eluted
isocratically (1.5 ml/min) with 130 mM unbuffered sodium phosphate. The
eluent was continuously monitored for absorption at 340 and 260 nm, and
the spectra of the eluting components were recorded with a diode array
detector (Waters 996). This separation system could resolve all of the
expected reduction products (Boland et al., 1991), and reduction of
CB1954 was monitored by either the decrease in its peak area or an
increase in the area of the peak corresponding to the reduction
product, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. All of the
assays were initiated with the addition of the enzyme and were
performed in duplicate.
Inhibition Studies. The enzyme was assayed in the presence of various inhibitors at different concentrations. Inhibitors except dicoumarol were dissolved in ethanol, and the maximal volume of ethanol was maintained at 10 µl/ml assay mixture. The activity of the enzyme was not affected by ethanol in amounts up to 10 µl/ml. Dicoumarol was dissolved in 15 mM NaOH. These experiments were performed in triplicate. The Ki values for inhibitors have been derived from Dixon plots (1/v versus [I]). These inhibitors have been shown to be competitive inhibitors of DT-diaphorase with respect to NADH, and the Km value of NADH for each enzyme preparation was determined and used in this calculation. During the enzyme assay, the concentration of NADH was 200 µM.
Molecular Modeling.
Crystallographic coordinates for rat
DT-diaphorase with bound FAD, duroquinone, and Cibacron blue (file
1QRD; Li et al., 1995) were obtained from the Protein Data Bank
(Berstein et al., 1977). For modeling purposes, the physiological dimer
form was used (MacroMolecular file 1qrd__1.mmol). The coordinates were
used without further refinement.
) were obtained from the Cambridge Structural Database (File
DNEIBA; Allen et al., 1983) and used without further refinement. All
other prodrugs or inhibitors were built via fragment libraries supplied
with the modeling software. The initial structures, most of which are
semirigid, were first energy minimized to an root-mean-square
force of less than 0.001 with the consistent valence force field
(Dauber-Osguthorpe et al., 1988). The resulting structures were then
subjected to 10 ps of molecular dynamics at 300 K, followed by
conjugate gradient minimization to an RMS force of less than 0.001 to
complete the refinement cycle. This cycle was repeated 10 times, and
the lowest energy conformer was selected from the resulting ensemble of structures.
The energy-minimized substrate and inhibitor molecules were then
individually placed with the active site of the enzyme model such that
overlap with the bound substrate (or inhibitor) was optimal. The active
site was then solvated with an 8-Å layer of pre-equilibrated water.
Each compound was again energy minimized but this time in the context
of the active site (defined as residues within 10 [Angst] of the
substrate or inhibitor). This optimized the position of each inhibitor
within the pocket, which was assumed to be rigid. Finally, the
minimizations were repeated without constraints so that active-site
residues and solvent could move to better accommodate the bound
prodrugs or inhibitors (induced fit theory of molecular interactions;
Jorgensen, 1991). No predictive tests of the docking models were
carried out. The models simply attempt to rationalize the binding data.
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Results |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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Catalytic Properties of DT-Diaphorase Mutants.
We have
reported previously that the catalytic properties of human and rat
forms of DT-diaphorase are different (Chen et al., 1997). The
NADH-menadione reductase activity and the NADH-CB1954 reductase
activity of the human enzyme were found to be 46 and 14% of those of
the rat enzyme, respectively. Previous mutagenesis studies revealed
that residue 104 (Tyr in the rat enzyme and Gln in the human enzyme) is
an important residue responsible for the different CB1954 reductase
activities between the rat and human enzymes. The presence of Gln at
position 104 instead of Tyr in the human DT-diaphorase allows the
flavin prosthetic group to move deeper into the protein (Chen et al.,
1997; Fig. 2B). Such a change may modify
the rate of electron transfer between FAD and a substrate such as
CB1954.
|
). Gly150 and His161 are thought to
interact with oxygens O4'N and
O2'N of the ribose adjacent to nicotinamide,
respectively. His194 may form hydrogen bonds between its N atom and
two oxygens of the pyrophosphate. Cui et al. (1995) performed
mutagenesis studies at Tyr128, Gly150, and His194. The catalytic
properties of the three rat mutants used in this study, i.e., rY128D,
rG150V, and rH194D, have been published (Cui et al., 1995). With
2,6-dichlorophenolindophenol as the electron acceptor, the activities
of rY128D and rG150V were determined to be 16 and 23% of the wild-type
rat enzyme, and the Km values of these
mutants for NADH were found to be 2.7 and 3.3 times that of the
wild-type enzyme (Ma et al., 1992b). Furthermore, rY128D was found to
be significantly less sensitive to dicoumarol than the wild-type enzyme
(Ma et al., 1992b). The Km value of rH194D
for NADPH was found to be much larger than that of the wild-type enzyme
(Cui et al., 1995). These mutagenesis experiments confirm that Tyr128,
Gly150, and His194 are important residues in the nicotinamide coenzyme
binding site of DT-diaphorase.
We have prepared the human mutants hH161Q and hY155F. As mentioned
above, X-ray structural analysis suggested a direct interaction of
His161 with nicotinamide coenzymes (Li et al., 1995). In addition, Tyr155 and His161 have also been thought to be involved in the charge
relay mechanism to facilitate the obligatory two-electron transfer
reaction (Li et al., 1995). The imidazole of His161 is close to the
nicotinamide ring, so a positive charge is thought to move over a very
short distance from the imidazole ring of His161 to the nicotinamide.
This process is reversed when the hydride is transferred to the
quinone. Therefore, it is not surprising that the
kcat value of hH161Q is only 8% that of
the wild-type human DT-diaphorase (Table
1). The hydroxyl group of Tyr155 is believed to form a hydrogen bond with O2F of the flavine ring (Li et al., 1995) and to participate in the two-electron transfer reaction by interacting with His161. The importance of the hydroxyl group of Tyr155 is questioned, because hY155F still has 35% of the
wild-type activity (Table 1). As discussed below, the CB1954 reductase
activity of hY155F is three times that of the wild-type enzyme. In
Table 1, the catalytic properties of these human mutants are compared
with those of a naturally occurring mutant, hP187S. The
Km values of these three human mutants for
NADH and menadione are similar to those of the wild-type human enzyme,
except for hP187S, the Km for menadione of
which is slightly higher (Table 1). hP187S was also found to be
significantly less active than the wild-type human enzyme. Previous
studies found that a low activity for hP187S results from a weaker
affinity for FAD than the wild-type enzyme (Wu et al., 1998). Pro187 is
not positioned in the active site, but the mutation disturbs the
structure of the central parallel -sheet, resulting in a reduction
in the binding affinity of the FAD prosthetic group (Fig. 2A). Similar Km values for both NADH and menadione and
kcat values lower than the wild-type human
enzyme again support the hypothesis that the His161 to Gln mutation
mainly affects the catalytic process rather than the binding affinities
of NADH and menadione.
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Inhibition Studies. The inhibitor-binding constants (Ki) of five inhibitors (Cibacron blue, chrysin, 7,8-dihydroxyflavone, phenindone, and dicoumarol) for the wild-type (human and rat) enzyme and five mutants [three rat mutants (rY128D, rG150V, rH194D) and two human mutants (hY155F, hH161Q)] were determined and are shown in Table 4. Each of the five mutations modified the binding of five inhibitors to a different extent.
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Discussion |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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Binding Nature of CB1954.
Considering the fact that CB1954 is
a substrate of DT-diaphorase, and its size is only slightly bigger than
duroquinone, the compound is thought to be situated where duroquinone
is found in the X-ray structure, with the ring parallel to the flavin
ring. This allows for an efficient electron transfer during the
reductive activation. Based on four docking simulations and results
from mutant kinetic analysis, we have predicted the preferred
orientation of CB1954 within the active site. We propose that CB1954
situates in the active site, with the aziridinyl group pointing toward Tyr-155 and the amide group placed near a hydrophobic pocket next to
Tyr128 (Fig. 2B). Because the Km values of
rY128D, rG150V, and rH194D for CB1954 are similar to that of the
wild-type rat DT-diaphorase and the Vmax
values of these mutants are lower than that of the wild-type rat
enzyme, we feel that these mutations reduce the rate of the electron
transfer reaction rather than modify the binding affinity of CB1954. On
the other hand, the Km value of hY155F for
CB1954 is found to be significantly lower than that of the wild-type
human enzyme. In addition, the Vmax value
of hY155F is 3.3 times higher than that the wild-type human enzyme.
These results suggest that the binding affinity and the reduction
efficiency of CB1954 are improved, with a Phe at position 155. We
therefore predict that the aziridinyl group of CB1954 is in this area.
The amide group is thought to be near a hydrophobic pocket next to
Tyr128, similar to the amide group of the nicotinamide coenzyme in
which the carbonyl makes hydrogen bonds to Tyr126 and Tyr128 (Li et
al., 1995). Experiments performed in our laboratories have revealed
that the amide group of CB1954 can be replaced with a hydrophobic side
chain, and such derivatives are better substrates of the human
DT-diaphorase (R.K., K.W. and S.C., unpublished results).
). Results shown
in Table 3 indicate that the Gly150 to Val mutation affects the binding
of NADH to a greater extent than the binding of NRH, suggesting that
the orientations of the ribose moiety of these two nicotinamide coenzymes in the active site of DT-diaphorase are not exactly the same.
It is also possible that the Gly150 to Val mutation reduces the size of
the active site pocket, preventing the effective binding of NADH, which
is a significantly larger molecule than NRH. On the other hand, the
relative activities of NADH and NRH for rY128D are similar, which
suggests that the binding orientations of the nicotinamide group of the
coenzymes within the active site of this mutant are probably not much
different. The two subunits of the enzyme are in a head-to-tail
arrangement, and each active site is made up of parts from both
subunits. As shown in structural models, Tyr128 is from a different
subunit than Gly150, Tyr155, His161, and His194.
Binding Nature of Inhibitors. The Ki values of Cibacron blue, chrysin, 7,8-dihydroxyflavone, phenindone, and dicoumarol to rY128D, rG150V, and hH161Q were significantly greater than those for the wild-type human enzymes, indicating that Tyr128, Gly150, and His161 are situated in the binding pockets of these inhibitors. On the other hand, Tyr155 and His194 are probably not in direct contact with the inhibitors, because the binding of inhibitors are not affected by Tyr155 to Phe and His194 to Asp mutations.
The X-ray structure of Cibacron blue bound to rat DT-diaphorase has been published (Li et al., 1995) and was used to help interpret the
results of our inhibition studies. The binding of Cibacron blue is
significantly reduced by the Tyr128 to Asp mutation and the Gly150 to
Val mutation. The Ki values of Cibacron
blue for rY128D and rG150V are 110 and 23 times that for the wild-type rat DT-diaphorase, respectively. The X-ray structural analysis revealed
that the inhibitor's trizaine ring is sandwiched between these two residues.
As indicated in Fig. 1, dicoumarol is structurally similar to a dimer
of the substrate menadione. To facilitate the discussion of the
results, the two halves of the molecule dicoumarol are named rings A
and B. The Ki values of dicoumarol for
rG150V and rY128F are 437 and 70 times that for the wild-type rat
enzyme, respectively, and the Ki value for
hH161Q is 140 times that for the wild-type human enzyme. Results
obtained from inhibition studies with dicoumarol indicate that the
strong binding of dicoumarol (Ki = 2 and
0.5 nM for the rat and human NQO1, respectively) can be explained by a
- interaction of one ring (ring A, as indicated in Fig. 1)
with the isoalloxazine ring of FAD and a - interaction of ring B
with the phenol ring of Tyr128, thus explaining why the binding
affinity of dicoumarol is greatly reduced for rY128D (Ki = 140 nM for rY128D; computer models,
see Fig. 2C). Like the triazine ring of Cibacron blue, we predict that
ring B of dicoumarol is sandwiched between Gly150 and Tyr128, thus
explaining the great increase in the Ki
value (i.e., 970 nM) of dicoumarol for rG150V. Because the
Ki value for hH161Q is also large (i.e., 70 nM) compared with the wild-type human enzyme, His161 is also thought to
be part of the dicoumarol binding site. Specifically, our model
suggests that the hydroxyl group of ring A packs against this residue.
Rings A and C of chrysin are thought to be near Gly150 and His161,
explaining why the Ki values of chrysin for
rG150V and hH161Q are 60 and 82 times those of both rat and human
wild-type DT-diaphorase (see Fig. 2D). The results of our inhibition
studies also lead us to conclude that chrysin (i.e.,
5,7-dihydroxyflavone; Ki = 100 nM for both
rat and human DT-diaphorase) and 7,8-dihydroxyflavone (Ki = 60 and 30 nM for rat and human
enzyme, respectively) bind to the active site, with the 7-hydroxyl
group (in chrysin) placed near His161, thus explaining the large
increase in the Ki value for hH161Q
(Ki = 8200 and 4000 nM for chrysin and
7,8-dihydroxyflaovone, respectively). Because the
Ki value of 7,8-dihydroxyflavone for rG150V
(16,700 nM) is much larger than that of chrysin (6000 nM), the
8-hydroxyl group is thought to pack against Gly150. Therefore, we
propose that flavone binds to the active site of DT-diaphorase with the
C-8 ring carbon near Gly150 and the 4-keto group pointing away from
Gly150 toward the solvent.
Interestingly, the binding of phenindone to the human enzyme, like
Cibacron blue, is significantly poorer than the rat enzyme (Table 4).
It is felt that phenindone binds to the active site in an orientation
similar to that of 7,8-dihydroxyflavone, with the fused-ring system
involved in a - interaction with Tyr128 and a packing interaction
with Gly150 (Fig. 2E). This binding orientation explains why the
Ki values of phenindone for rY128D and
rG150V are significantly higher than that of the wild-type rat
DT-diaphorase (see Table 4). Because there is no side chain on ring A,
the His161 to Gln mutation modifies the binding of phenindone
moderately (Ki = 500 and 1200 nM for the
wild-type human enzyme and hH161Q, respectively). Interestingly,
phenindone binds to rH194D significantly better than the wild-type rat
enzyme. Computer-modeling analysis suggests that this residue sits
above the bound inhibitor, near one of the keto groups.
In summary, by evaluating results obtained from a combined approach
involving site-directed mutagenesis of DT-diaphorase, enzyme kinetic
analysis, inhibitor-binding studies, and computer modeling based on the
published X-ray structure of the rat form of the enzyme, we have
predicted the binding orientations of the prodrug CB1954 and four
inhibitors, dicoumarol, chrysin, 7,8-dihydroxyflavone, and phenindone,
in the active site of DT-diaphorase. Our findings may facilitate the
design of better oral anticoagulants for treatment of recurring heart
attack and novel prodrugs for treatment of cancer.
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Footnotes |
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Received January 28, 1999; Accepted May 6, 1999
1 This article is dedicated to the memory of Dr. Lars Ernster, who passed away recently. DT-diaphorase was originally isolated in Dr. Ernster's laboratory in 1958.
2 Current address: Enzacta Ltd., Building 115, Porton Down Science Park, Salisbury SP4 0JQ, UK.
The research was supported by American Heart Association Grant 95007330 and a Seed Grant from the City of Hope. S.C. and M.S. are members of the City of Hope Cancer Center (CA 33572). D.Z. was supported by the Student Research Program, American Heart Association, Western States and Greater Los Angeles Affiliates.
Send reprint requests to: Dr. Shiuan Chen, Division of Immunology and Biology, Beckman Research Institute of the City of Hope, 1450 E. Duarte Road, Duarte, CA 91010. E-mail: schen{at}smtplink.coh.org
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Abbreviations |
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FAD, flavin-adenine dinucleotide; CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide; PCR, polymerase chain reaction; NRH, dihydronicotinamide riboside; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.
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References |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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 B. N Ames, I. Elson-Schwab, and E. A Silver High-dose vitamin therapy stimulates variant enzymes with decreased coenzyme binding affinity (increased Km): relevance to genetic disease and polymorphisms Am. J. Clinical Nutrition, April 1, 2002; 75(4): 616 - 658. [Abstract] [Full Text] [PDF]
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M. Stiborova, E. Frei, B. Sopko, M. Wiessler, and H. H. Schmeiser Carcinogenic aristolochic acids upon activation by DT-diaphorase form adducts found in DNA of patients with Chinese herbs nephropathy Carcinogenesis, April 1, 2002; 23(4): 617 - 625. [Abstract] [Full Text] [PDF]
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 L. B. Sánchez, H. Elmendorf, T. E. Nash, and M. Müller NAD(P)H:menadione oxidoreductase of the amitochondriate eukaryote Giardia lamblia: a simpler homologue of the vertebrate enzyme Microbiology, March 1, 2001; 147(3): 561 - 570. [Abstract] [Full Text]
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D. Siegel, A. Anwar, S. L. Winski, J. K. Kepa, K. L. Zolman, and D. Ross Rapid Polyubiquitination and Proteasomal Degradation of a Mutant Form of NAD(P)H:Quinone Oxidoreductase 1 Mol. Pharmacol., February 1, 2001; 59(2): 263 - 268. [Abstract] [Full Text]
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M. Faig, M. A. Bianchet, P. Talalay, S. Chen, S. Winski, D. Ross, and L. M. Amzel Structures of recombinant human and mouse NAD(P)H:quinone oxidoreductases: Species comparison and structural changes with substrate binding and release PNAS, March 28, 2000; 97(7): 3177 - 3182. [Abstract] [Full Text] [PDF]
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