Mecha-nistic and kinetic analyses indicate that DS reacts with the active site cysteine residue of NAT1, leading to the irreversible inhibition of the enzyme, with a half-maxi-mal inhibi
Trang 1N-acetyltransferase 1 inhibition by disulfiram
Florence Malka, Julien Dairou, Nilusha Ragunathan, Jean-Marie Dupret and
Fernando Rodrigues-Lima
Universite´ Paris Diderot-Paris 7, Unite´ de Biologie Fonctionnelle et Adaptative (BFA), CNRS Equipe d’Accueil Conventione´e (EAC) 7059, Laboratoire des Re´ponses Mole´culaires et Cellulaires aux Xe´nobiotiques, 75013, Paris, France
Introduction
Disulfiram (DS) or tetraethylthiuram disulfide (TTD)
(Antabuse) has been used clinically in the treatment
of chronic alcoholism since 1948 [1] (Fig 1) DS acts
by irreversibly inhibiting the hepatic aldehyde
dehydro-genase, leading to the accumulation of acetaldehydes
after alcohol ingestion [2] The combined intake of DS
and ethanol provokes unpleasant reactions (nausea
and vomiting), which are the basis of the therapeutic
use of DS Several studies have shown that DS inhibits
hepatic aldehyde dehydrogenase through covalent
modification of an active site cysteine residue [3] New
potential therapeutic uses, in particular for human
can-cers and fungal infections, have been reported recently
for DS [4], indicating that the clinical applications of this drug are broader than previously thought Never-theless, the mechanisms underlying these effects of DS remain poorly understood
DS is known to react with protein thiols to form mixed disulfides or covalent adducts [3] However, many thiol-containing proteins do not react with DS, indicating that covalent modification by this drug shows marked specificity [5] In addition to aldehyde dehydrogenase, other enzymes have been reported to
be targeted by DS [6–8] In particular, certain xeno-biotic-metabolizing enzymes (XMEs), such as cytochrome-P450 enzymes (CYP2E1) and glutathione
Keywords
arylamine N-acetyltransferase; cancer; drug
target; inhibition; kinetics
Correspondence
F Rodrigues-Lima, Universite´ Paris
Diderot-Paris 7, Unit of Functional and
Adaptative Biology (BFA) – CNRS EAC
7059, 75013 Paris, France
Fax: +33 1 57 27 83 29
Tel: +33 1 57 27 83 32
E-mail: fernando.rodrigues-lima@
univ-paris-diderot.fr
(Received 20 April 2009, revised 28 May
2009, accepted 1 July 2009)
doi:10.1111/j.1742-4658.2009.07189.x
Disulfiram has been used for decades to treat alcoholism Its therapeutic effect is thought to be mediated by the irreversible inhibition of aldehyde dehydrogenase Recent reports have indicated new therapeutic uses of disulfiram, in particular in human cancers Although the biochemical mech-anisms that underlie these effects remain largely unknown, certain enzymes involved in cancer processes have been reported to be targeted by disulfi-ram Arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine drugs and carcinogens In addi-tion to its role in xenobiotic metabolism, several studies have suggested that NAT1 is involved in other physiological and⁄ or pathological pro-cesses, such as folate metabolism or cancer progression In this report, we provide evidence that human NAT1 is a new enzymatic target of disulfi-ram We found that disulfiram at clinically relevant concentrations impairs the activity of endogenous NAT1 in human cancer cells Further mechanis-tic and kinemechanis-tic studies indicated that disulfiram reacts irreversibly with the active site cysteine residue of NAT1, leading to its rapid inhibition (IC50= 3.3 ± 0.1 lm and ki= 6· 104 m)1Æmin)1)
Abbreviations
AcCoA, acetyl-coenzyme A; DS, disulfiram; GSH, reduced glutathione; GST, glutathione S-transferase; IC50,half-maximal inhibitory
concentration; NAT, arylamine N-acetyltransferase; NAT1, arylamine N-acetyltransferase 1; PAS, p-aminosalicylic acid; PNPA,
p-nitrophenylacetate; TTD, tetraethylthiuram disulfide; XME, xenobiotic-metabolizing enzyme.
Trang 2S-transferase (GST), are impaired by DS with
subse-quent effects on xenobiotic metabolism in vivo [9–11]
Arylamine N-acetyltransferases (NATs) are phase 2
XMEs that catalyse the transfer of an acetyl group
from acetyl-coenzyme A (AcCoA) to the nitrogen or
oxygen atom of primary arylamines, hydrazines and
their N-hydroxylated metabolites [12] NATs plays a
key role in the detoxification and⁄ or activation of
numerous drugs and carcinogens [13] In humans, two
functional isoforms, NAT1 and NAT2, have been
described [14] Although their protein sequences are
similar (81% identical), their kinetic selectivity and
efficiency for aromatic substrates and tissue
distribu-tion differ markedly (NAT2 is found mainly in the
liver and intestinal epithelium, whereas NAT1 shows
widespread expression) [15,16] Both NAT isoforms
are affected by genetic polymorphisms, which can be
a potential source of pharmacological and⁄ or
patho-logical susceptibility [17] In addition to the genetic
mechanisms that govern NAT expression and activity,
recent data have shown that NAT enzymes can be
affected by environmental chemicals, such as drugs or
pollutants [18] More specifically, human NAT1 has
been shown to be impaired by oxidants [19–21] and
by certain therapeutic drugs, such as cisplatin [22]
and acetaminophen [23] Several recent studies have
indicated that NAT1 may contribute to increased
can-cer risk and carcinogenesis [24–26], suggesting that
this XME could be relevant for cancer treatment
[27,28]
The identification and characterization of the
molec-ular targets of DS are of prime importance in order to
understand the pharmacological and⁄ or toxicological
effects of this therapeutic compound In this study, we
show that NAT1 is inhibited by therapeutic
concentra-tions of DS in vitro and in human cancer cells
Mecha-nistic and kinetic analyses indicate that DS reacts with
the active site cysteine residue of NAT1, leading to the
irreversible inhibition of the enzyme, with a
half-maxi-mal inhibitory concentration (IC50) of 3.3 ± 0.1 lm
and a second-order inhibition rate constant (ki) of
6· 104m)1Æmin)1 Our work shows that NAT1 is a
new molecular target of DS
Results
DS impairs the activity of NAT1 in human cancer cells at clinically relevant concentrations
DS is well known to inhibit the activity of liver alde-hyde dehydrogenase [2] However, recent reports have shown that DS may also react with other pro-teins⁄ enzymes, in particular in cancer cells [4,7,8,29] The exposure of human lung cancer cells NCI-H292 to therapeutically relevant concentrations of DS ( 30 lm) [4] led to the dose-dependent inhibition of NAT1 (Fig 2) Similar results were obtained with another human lung cancer cell line (A549) (data not shown) These data suggest that NAT1 may be a new cellular target of DS
Therapeutically relevant concentrations
of DS inhibit recombinant human NAT1 in an irreversible and dose-dependent manner
To investigate the molecular mechanisms underlying the DS-dependent inhibition of NAT1, we carried out further biochemical and kinetic analyses using recom-binant purified NAT1 To test whether DS inhibits NAT1 directly, the recombinant enzyme was incubated with different clinically relevant concentrations of the drug and its residual activity was measured As shown
in Figure 3, NAT1 was inhibited in a dose-dependent manner by DS Complete inhibition was observed with
0%
0 15 30 45 20%
40%
60%
80%
100%
120%
*
*
*
[DS] (µ M )
Fig 2 Inhibition of endogenous NAT1 in human cancer cells by clinically relevant concentrations of DS NCI-H292 cells were exposed to different concentrations of DS in NaCl ⁄ P i for 30 min at
37 C NAT1 activity was measured by HPLC in total cell extracts Extracts from untreated cells were used as controls Enzyme activi-ties were normalized with respect to protein concentration Error bars indicate the standard deviations (*P < 0.05) Similar results were obtained with A549 cells (data not shown) An activity of 100% corresponds to a specific activity towards 2-aminofluorene of
10 ± 1 nmolÆmin)1Æmg)1.
N
S
S
S
Disulfiram (DS) or tetraethylthiuram disulfide (TTD)
Fig 1 Chemical structure of DS.
Trang 3concentrations as low as 8 lm and the IC50value was
estimated to be equal to 3.3 ± 0.1 lm
To test whether the DS-dependent inhibition of
NAT1 was irreversible, dialysis experiments were
car-ried out as described by Butcher et al [30] DS-treated
NAT1 showed a residual activity of only 12 ± 1%
and 3 ± 1% for dialysed and undialysed proteins,
respectively [100% activity corresponded to a specific
activity towards p-aminosalicylic acid (PAS) of
98 ± 7 nmolÆmin)1Æmg)1], suggesting the irreversible
inhibition of NAT1 by DS Dialysis had no significant
effect on NAT1 activity, as also reported by Butcher
et al.[30]
We then investigated whether the DS-dependent
inhibition of NAT1 could be reversed by reducing
agents As shown in Table 1, after inhibition by DS,
NAT1 activity could not be restored by physiological
concentrations of reduced glutathione (GSH) or by
high concentrations of the non-physiological reductant dithiothreitol These data suggest that the DS-depen-dent inhibition of NAT1 is unlikely to be a result of the formation of a mixed disulfide, but rather of the formation of a stable DS adduct
DS reacts with NAT1 thiol groups at therapeutic concentrations
DS is a drug that reacts with thiols, and most of its effects are probably a result of its affinity for sulfhy-dryl groups in target proteins [3–5] To test whether therapeutic concentrations of DS react with the NAT1 cysteine residue, we incubated the purified enzyme with
DS in the concentration range shown to inhibit NAT1 (Fig 3) Free unmodified cysteine residues in the enzyme were labelled with fluorescein-conjugated iodoacetamide and detected in western blots, as described previously [31] As shown in Figure 4, DS reacted with NAT1 cysteine residues in a dose-depen-dent manner, as indicated by the disappearance of the fluorescein signal This dose-dependent modification was correlated with the dose-dependent inhibition of
0%
0 2 3 4 5
20%
40%
60%
80%
100%
120%
[DS] (µ M )
*
*
*
*
Fig 3 Dose-dependent inhibition of recombinant human NAT1 by
DS NAT1 was incubated with clinically relevant concentrations of
DS in 25 m M Tris ⁄ HCl, pH 7.5, 1 m M EDTA for 30 min at 37 C.
NAT1 activity was then determined Errors bars indicate standard
deviations (*P < 0.05) The results are presented as a percentage
of control activity (100% activity corresponds to a specific activity
towards PAS of 98 ± 7 nmolÆmin)1Æmg)1).
Table 1 Effects of reducing agents on DS-dependent inhibition of
NAT1.
a
100% activity corresponded to a specific activity towards PAS of
98 ± 7 nmolÆmin)1Æmg)1.
Fig 4 Detection of the DS-dependent modification of NAT1 cyste-ine residues NAT1 was incubated with DS in 25 m M Tris ⁄ HCl, pH 7.5, 1 m M EDTA for 30 min at 37 C The reaction mixture was incubated with fluorescein-conjugated iodoacetamide for 10 min at
37 C Samples were then subjected to SDS-PAGE under reducing conditions, followed by western blotting using an anti-fluorescein IgG (anti-fluorescein) or an anti-6 · His-tag IgG (anti-6 · Histag) For the control (Ct), NAT1 was not treated with DS Quantification of the signals was carried out using IMAGE J software (http://rsbweb nih.gov/ij/) The fluorescein intensity was normalized with respect
to the anti-6 · His-tag signal.
Trang 4NAT1 by DS (Fig 2), suggesting that the processes
are linked
DS-dependent inhibition of NAT1 involves
interactions with the active site
Among the five cysteine residues present in the
human NAT1 protein, one is localized in the enzyme
active site and is required for catalysis [32] To test
whether the DS-dependent inhibition of NAT1 is a
result of reaction at the active site cysteine residue,
we carried out protection experiments in the presence
of the physiological acetyl donor AcCoA, as
described by Liu et al [33] This approach has been
largely used to identify whether the inhibition of
NAT enzymes by chemical compounds involves
inter-actions with the active site [33,34] The incubation of
NAT1 with DS (8 lm) caused 93 ± 2% inhibition of
the enzyme, whereas the presence of AcCoA at 1 and
2 mm decreased the extent of inhibition to 35 ± 5%
and 2 ± 4%, respectively (100% activity
corre-sponded to a specific activity towards PAS of
98 ± 7 nmolÆmin)1Æmg)1) These results suggest that
the DS-dependent inhibition of NAT1 is caused
by irreversible reaction with the active site cysteine
residue
Kinetic analysis of the DS-dependent inhibition
of NAT1
We further analysed the inhibition of NAT1 by DS by
carrying out time course inhibition of the enzyme at
different DS concentrations As shown in Figure 5A,
incubation of the enzyme with DS led to a
monoexpo-nential time-dependent loss of activity, indicating that
the inhibition reaction obeyed apparent first-order
kinetics The apparent first-order inhibition constants
(kobs) were calculated for each concentration of DS
The plot of kobsas a function of DS concentration
fit-ted well to a line passing at the origin (r2= 0.99)
(Fig 5B), indicating that the inhibition of NAT1 by
DS occurs through a single-step bimolecular reaction
The second-order rate constant for the inhibition of
NAT1 by DS (ki) was deduced from the slope of kobs
as a function of the DS concentration [DS], and was
6· 104m)1Æmin)1 The order of the reaction of DS
with NAT1 (n) can be deduced from the equation
kobs= ki[DS]n, and can be calculated by plotting ln
kobs as a function of ln [DS] (Fig 5C) Linear
regres-sion of these data indicated that they fitted well to a
line (r2= 0.99) with a slope (n) equal to 0.99,
suggest-ing that NAT1 inhibition by DS occurs through a
1 : 1 stoichiometry
0.0
0.2 0.4 0.6 0.8 1.0
Time (min)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
–1.6 –1.2
–0.8
–0.4
kobs
kobs
ln ([DS])
A
B
C
Fig 5 Kinetic analysis of the DS-induced inhibition of NAT1 activ-ity (A) NAT1 was incubated with various concentrations of DS in
25 m M Tris ⁄ HCl, pH 7.5, 1 m M EDTA at 37 C At various short-time intervals, aliquots were removed and assayed for residual activity Plots of the relative residual activity as a function of time are shown and the data were found to fit well to a monoexponential time-dependent process The error bars indicate standard deviations An activity of 100% corresponds to a specific activity towards PAS of
98 ± 7 nmolÆmin)1Æmg)1 (B) The apparent first-order inhibition con-stant (kobs) was calculated for each DS concentration and plotted The second-order inhibition constant (k i ) was determined from the slope and was 61 · 10 3
M )1Æmin)1 The error bars indicate standard
deviations (C) To determine the stoichiometry of the reaction of DS with NAT1, the natural logarithm (ln) of k obs was plotted as a func-tion of ln [DS] The slope was 0.99, indicating a 1 : 1 stoichiometry The error bars indicate standard deviations.
Trang 5Overall, our results suggest that DS-dependent
inhi-bition of NAT1 is caused by an irreversible single step
This inhibition occurs in a competitive manner
through the modification of the active site catalytic
cysteine residue of NAT1 by DS
Discussion
DS is an inhibitor of aldehyde dehydrogenase and is
currently being used clinically for the treatment of
alcoholism Recent data suggest that DS could have
new therapeutic uses, particularly in cancer [4]
Inter-estingly, DS was found to inhibit enzymes that have
been associated with cancer progression, such as DNA
topoisomerases, matrix metalloproteinases and
protea-some [6–8] The biological effects of DS are now
con-sidered to involve different cellular pathways [4]
Therefore, the deciphering of new targets of DS may
help us to understand the therapeutic and toxicological
effects of this drug In this article, we provide evidence
that the human XME NAT1 is a new target of DS
We found that endogenous NAT1 expressed by two
human cancer cell lines was readily inhibited by
short-time exposure (30 min) to clinically relevant
concentrations of DS [4] Interestingly, several studies
have associated NAT1 activity with an increased risk
of cancer [13] In addition, recent data support the
suggestion that NAT1 may play a role in breast cancer
progression [16,27] NAT1 expression has also been
shown to be increased by androgens in human prostate
cancer cells, which may have pathological implications
[25] Overall, these studies suggest that NAT1 is a
can-cer-associated XME that could be targeted for cancer
treatment [16,27]
Mechanistic and kinetic analyses were carried out to
better understand the molecular basis for the
DS-dependent inhibition of NAT1 activity in cells Our
data indicate that recombinant NAT1 was irreversibly
inhibited by low clinically relevant concentrations of
DS ( 30 lm) with an IC50 value equal to
3.3 ± 0.1 lm Recombinant aldehyde dehydrogenase
and DNA topoisomerases have been reported to be
inhibited in vitro in a similar manner with IC50values
close to 35 lm [6,35] IC50 values ranging from five to
hundreds of micromoles have been reported for
differ-ent GST isoforms [10] Kinetic analysis has also shown
that the DS-dependent inhibition of NAT1 occurs
rapidly with a second-order rate constant of
6· 104 m)1Æmin)1 Overall, these data suggest that DS
has an inhibitory potency against NAT1, similar to
that of known enzymatic DS targets
DS is known to react with thiol groups, leading to
the formation of mixed disulfides or covalent adducts
on target proteins [3] The inhibition of target enzymes, such as aldehyde dehydrogenase, by DS often occurs through the modification of an active site cysteine residue [3] Accordingly, our data (iodoaceta-mide labelling, AcCoA protection assay and stoichi-ometric analysis) indicate that the DS-dependent inhibition of NAT1 is probably a result of the modifi-cation of the cysteine residue present in the enzyme active site Reducing agents, such as GSH or dith-iothreitol, used at high concentrations (up to 5 mm), were unable to restore NAT1 activity This suggests that the DS-dependent inhibition of NAT1 is unlikely
to depend on the formation of a mixed disulfide which is readily reduced by 1 mm dithiothreitol at neutral pH, with subsequent recovery of enzymatic activity, as observed for aldehyde dehydrogenase [3] Indeed, contrary to aldehyde dehydrogenase, NAT1 does not possess two vicinal thiols in its active site [36] and cannot thus be inhibited by the DS-depen-dent formation of an intramolecular mixed disulfide [3] Therefore, the DS-dependent inhibition of NAT1
is probably the result of the formation of a stable DS adduct that could be inaccessible to displacement by thiol reagents [37] Cisplatin, an anticancer drug, has been reported recently to irreversibly inhibit NAT1 (ki= 700 m)1Æmin)1) in vivo and in vitro This inhibi-tion also occurs through the formainhibi-tion of an adduct with the NAT1 active site cysteine, which cannot be reduced by reducing agents [22] Interestingly, the reaction of DS with NAT1 occurs 87 times faster (ki= 6.104 m)1Æmin)1) than the reaction of cisplatin with the enzyme, thus supporting the suggestion that NAT1 could be an in vivo target of DS The human NAT2 isoform shares a similar structure and mecha-nism of action to human NAT1 [38] This isoform is thus likely to be inhibited by DS through the modifi-cation of its catalytic cysteine Further studies are needed, however, to address whether DS reacts with NAT1 and NAT2 in a similar manner NAT2 metab-olizes several aromatic amine drugs, such as isoniazid The inhibition of NAT2 by DS could lead to drug–drug interactions as defects in NAT2 activity are associated with isoniazid hepatotoxicity [39]
DS has been used for decades to treat alcoholism, and its therapeutic activity is thought to be mediated through the irreversible inhibition of aldehyde dehy-drogenase However, DS has been shown recently to have new potential therapeutic applications [4] Accordingly, the biochemical mechanisms and cellular pathways that underlie the action of DS have also begun to emerge with the identification of new pro-tein targets of this drug [6–8] Among them, XMEs such as CYP 2E1 and certain GST isoforms have
Trang 6been shown to be inhibited by DS, with subsequent
effects in vivo on xenobiotic metabolism [9–11] Our
data clearly indicate that the XME NAT1 could be a
new target of DS In addition to its role in
xenobi-otic metabolism, there is increasing evidence to
sug-gest that NAT1 may also be involved in other
physiological and⁄ or pathological processes, such as
folate metabolism [40,41] and cancer progression
[26,27] The overexpression of NAT1 in normal
lumi-nal epithelial breast cells induced two of the hallmark
traits of cancer, i.e enhanced growth and resistance
to certain therapeutic cytotoxic drugs used in cancer
treatment (etoposide) [42] Recent studies from
Minchin et al [25] have shown that NAT1 is induced
by androgens in human prostate cancer cells, with
possible implications for cancer risk The increasing
evidence for an association of NAT1 with
carcino-genesis suggests that its inhibition could be used in
cancer therapy The synthesis of small molecules that
inhibit NAT1 in breast cancer cells has been reported
recently [28] The molecular mechanisms that underlie
the anti-cancer activity of DS remain poorly
under-stood The DS-dependent inhibition of proteasome
and inactivation of ATF⁄ CREB transcription factor
have been suggested to mediate DS anti-tumoral
activity [8,29] However, the anti-cancer effects of DS
are probably a result of multiple mechanisms that
could act synergistically [4] The DS-dependent
impairment of NAT1 could be one of these
mecha-nisms
Experimental procedures
Materials
PAS, p-nitrophenylacetate (PNPA), AcCoA, DS (or TTD),
GSH and fluorescein-conjugated iodoacetamide were
pur-chased from Sigma (St-Quentin-Fallavier, France) Cell
culture reagents were from Invitrogen (Cergy-Pontoise,
France) Anti-fluorescein Fab¢ fragments conjugated to
peroxidase, monoclonal antibodies directed against His tag
and anti-mouse IgG were obtained from Roche (Meylan,
France) The Bradford protein assay kit was purchased
from Bio-Rad (Marne la Coquette, France) All other
reagents were obtained from Sigma, or Euromedex
(Souffelweyersheim, France)
Cell culture and exposure to DS
Human A549 lung carcinoma cells [43] and human
NCI-H292 pulmonary mucoepidermoid carcinoma cells [44] were
grown in DMEM⁄ F12 medium supplemented with 10%
(v⁄ v) fetal bovine serum, penicillin (100 UÆmL)1) and
strep-tomycin (100 lgÆmL)1) Cell monolayers (100 mm petri dishes) were washed with NaCl⁄ Piand exposed to different concentrations of DS in 10 mL of NaCl⁄ Pi for 30 min at
37C Controls were performed in the absence of DS On exposure, cells were washed with NaCl⁄ Piand resuspended
in NaCl⁄ Pi containing 0.2% Triton X-100 supplemented with protease inhibitors Cells were sonicated and centri-fuged for 15 min at 13 000 g The supernatants were removed, their protein concentration determined and assayed for NAT1 activity using 2-aminofluorene
Expression and purification of recombinant human NAT1
Human NAT1 was expressed as a 6· His-tagged protein in Escherichia coli BL21 (DE3) cells transformed with a pET28a-based plasmid, as described previously [31] On purification on nickel-agarose beads, recombinant NAT1 was reduced by incubation with 10 mm dithiothreitol for
10 min at 4C and dialysed against 25 mm Tris ⁄ HCl, pH 7.5 Purity was assessed by SDS-PAGE and protein concen-trations were determined using the Bradford reagent follow-ing the manufacturer’s instructions with bovine serum albumin as a standard
Enzyme assays
Recombinant NAT1 enzyme activity was determined spec-trophotometrically using PNPA as the acetyl donor and PAS as the arylamine substrate [45] Briefly, treated or untreated samples containing NAT1 enzyme were assayed
in a reaction mixture containing PAS (final concentration,
500 lm) in 25 mm Tris⁄ HCl, pH 7.5, 1 mm EDTA Reac-tions were started by the addition of PNPA (final concen-tration, 2 mm) In all reaction mixtures, the final concentration of NAT1 was 115 nm The reaction mixtures was incubated for up to 15 min at 37C, and the reaction was then quenched by the addition of SDS (final concentra-tion, 2%) P-Nitrophenol, generated by the NAT1-medi-ated hydrolysis of PNPA in the presence of PAS, was quantified by measuring the absorbance at 410 nm [Biotek (Colmar, France) microplate reader] For the controls, we omitted the enzyme or PAS All enzyme reactions were per-formed in triplicate in conditions in which the initial rates were linear Enzymes activities are shown as percentages of control NAT1 activity
NAT1 activity was measured in cell extracts using reverse-phase HPLC, as described previously [14] Samples (25 lL) were mixed with the aromatic amine substrate 2-aminofluorene (final concentration, 1 mm) in assay buffer (25 mm Tris⁄ HCl, pH 7.5) at 37 C AcCoA (final concen-tration, 1 mm) was added to start the reaction, and the samples were incubated at 37C for various periods of time (up to 20 min) The reaction was quenched by the addition
of 200 lL of ice-cold aqueous perchlorate (15% w⁄ v), and
Trang 7proteins were recovered by centrifugation for 5 min at
12 000 g; 20 lL of the supernatant was injected onto a C18
reverse-phase HPLC column All assays were performed
under initial reaction rate conditions Enzyme activities
were normalized according to the protein concentration of
cellular extracts determined using a Bio-Rad protein assay
kit
Reaction of recombinant NAT1 with DS
We assessed the effect of DS on NAT1 enzyme activity by
the incubation of purified NAT1 samples (final
concentra-tion of 75 lgÆmL)1 with a specific activity towards PAS of
98 ± 7 nmolÆmin)1Æmg)1) with various concentrations of
DS (up to a final concentration of 8 lm) in 25 mm
Tris⁄ HCl, pH 7.5, 1 mm EDTA for 30 min Mixtures were
then assayed for NAT1 activity as described above
To test whether the reaction of DS with NAT1 was
irre-versible, the recombinant enzyme was incubated with DS
(final concentration, 8 lm), and the mixture was dialysed
against 25 mm Tris⁄ HCl, pH 7.5, 1 mm EDTA, for 4 h at
4C Control assays were performed with untreated NAT1
and gave 100% NAT1 activity
To test whether the DS-dependent inhibition of NAT1
activity could be reversed by reducing agents, NAT1 (final
concentration, 75 lgÆmL)1; specific activity towards PAS of
98 nmolÆmin)1Æmg)1) was first inhibited by DS (final
con-centration, 8 lm) for 20 min at 37C The mixture was
then incubated for 10 min at 37C with various
concentra-tions of GSH or dithiothreitol (final concentraconcentra-tions up to
5 mm) A NAT1 enzyme assay was then carried out Assays
performed in these conditions, but without DS, gave 100%
NAT1 activity
In substrate (AcCoA) protection experiments, NAT1
(final concentration, 75 lgÆmL)1; specific activity towards
PAS of 98 ± 7 nmolÆmin)1Æmg)1) was incubated with DS
(final concentration, 8 lm) in the presence of various
con-centrations of AcCoA (final concon-centrations up to 2 mm)
for 30 min at 37C in 25 mm Tris ⁄ HCl, pH 7.5, 1 mm
EDTA Samples were then assayed Assays performed in
these conditions with AcCoA alone gave 100% NAT1
activity
For the kinetic analysis of DS-mediated NAT1
inhibi-tion, NAT1 (final concentrainhibi-tion, 75 lgÆmL)1; specific
activ-ity towards PAS of 98 ± 775 nmolÆmin)1Æmg)1) was
incubated with different concentrations of DS (final
concen-tration up to 8 lm) at 37C in 25 mm Tris ⁄ HCl, pH 7.5,
1 mm EDTA At various time intervals, aliquots were
removed and assayed for residual activity The equation for
the rate of inhibition of recombinant NAT1 by DS can be
represented as – d[NAT1]⁄ dt = ki[NAT1][DS], where
[NAT1] is the concentration of active enzyme and kiis the
second-order inhibition rate constant The apparent
first-order inhibition rate constants (kobs= ki[DS]) can be
calculated for each DS concentration from the slope of the
natural logarithm of the percentage residual activity plotted against time The second-order rate constant was deter-mined from the slope of kobs plotted against DS concen-tration
Fluorescein-conjugated iodoacetamide labelling
of NAT1 cysteine residues
Purified NAT1 (final concentration, 75 lgÆmL)1; specific activity towards PAS of 98 ± 7 nmolÆmin)1Æmg)1) was pre-incubated with or without (control, Ct) various concentra-tions of DS (up to a final concentration of 8 lm) in 25 mm Tris⁄ HCl, pH 7.5, 1 mm EDTA for 30 min at 37 C Sam-ples were incubated with fluorescein-conjugated iodoaceta-mide (final concentration, 20 lm) for 10 min at 37C Samples were then analysed by SDS-PAGE under reducing conditions, followed by western blotting, using anti-fluores-cein Fab¢ fragments conjugated to peroxidase Samples were also analysed by western blotting with monoclonal antibodies directed against 6· His tag
Protein determination, SDS-PAGE and western blotting
Protein concentrations were determined using a Bradford assay (Bio-Rad) Samples were combined with reducing SDS sample buffer and separated by SDS-PAGE Gels were stained with Coomassie Brilliant Blue R-250 To detect proteins labelled by fluorescein-conjugated iodoaceta-mide in western blots, anti-fluorescein Fab¢ fragments con-jugated to horseradish peroxidase (1 : 50 000) were used
To control protein loading, the same membrane was stripped by incubation for 1 h at 37C with stripping buf-fer (20 mm Tris⁄ HCl, pH 7.5, 20% SDS, 2 mm dithiothrei-tol) and probed with anti-monoclonal anti-His IgG (1 : 10 000)
Statistical analysis
The data are the means ± standard deviation of at least two independent experiments performed in triplicate, unless otherwise stated One-way analysis of variance (anova) was performed and followed by Student’s t-test (unpaired and paired) between two groups using statview 5.0 (SAS Institute Inc., Cary, NC, USA)
Acknowledgements
This work was supported by Agence Franc¸aise de Se´curite´ Sanitaire de l’Environnement et du Travail (AFSSET), Association pour la Recherche sur le Can-cer (ARC), Leg Poix (Chancellerie des Universite´s de Paris), Association Franc¸aise contre les Myopathies
Trang 8(AFM) and Universite´ Paris Diderot-Paris 7 We
thank Emile Petit for growing the cells
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