Báo cáo khoa học: Caenorhabditis elegans expresses a functional ArsA pptx

Caenorhabditis elegans expresses a functional ArsA
Yuen-Yi Tseng, Chan-Wei Yu and Vivian Hsiu-Chuan Liao
Department of Bioenvironmental Systems Engineering, National Taiwan University, Taipei, Taiwan
Arsenic is a potent toxin and carcinogen. The two bio-
logically relevant oxidation states of inorganic arsenic
are arsenite [As(III)] and arsenate [As(V)]. In general,
As(III) is more hazardous to organisms than As(V). In
bacteria, high-level resistance to arsenic is conferred by
the ars operon. The arsenic-resistant ars operon in
Escherichia coli has both plasmid [1] and chromosomal
[2] determinants. The well-characterized plasmid-borne
ars operon of E. coli is composed of two regulatory
(arsR and arsD ) and three structural (arsA, arsB, and
arsC) genes [3,4]. The well-characterized ArsAB pump
can extrude As(III) and antimonite [Sb(III)] from cells,
thereby lowering the intracellular concentration of
these toxic metalloids and producing resistance [5].
ArsA is the catalytic subunit of the anion pump that
hydrolyzes ATP in the presence of As(III) or Sb(III)
oxyanions [6]. ATP hydrolysis is coupled to the extru-
sion of As(III) and Sb(III) via the ArsB transporter,
which serves as both a membrane anchor for the ArsA
portion and a toxic oxyanion translocating pathway
[7].
Homologs of bacterial ArsA ATPase have been
found in nearly every organism studied [8]. Although
the function of bacterial ArsA has been identified,
the ubiquity of the ArsA ATPase-dependent pathway
in other organisms remains to be delineated. Here, we
describe the identification and biochemical characteri-
zation of the Caenorhabditis elegans homolog of the

Keywords
antimonite; arsenite; ASNA-1; ATPase;
Caenorhabditis elegans
Correspondence
V. H C. Liao, Department of
Bioenvironmental Systems Engineering,
National Taiwan University, no. 1 Roosevelt
Road, Sec. 4, Taipei 106, Taiwan
Fax: +886 2 3366 3462
Tel: +886 2 3366 5239
E-mail:
(Received 12 January 2007, revised 12
March 2007, accepted 15 March 2007)
doi:10.1111/j.1742-4658.2007.05791.x
Because arsenic is the most prevalent environmental toxin, it is imperative
that we understand the mechanisms of metalloid detoxification. In prokary-
otes, arsenic detoxification is accomplished by chromosomal and plasmid-
borne operon-encoded efflux systems. Bacterial ArsA ATPase is the
catalytic component of an oxyanion pump that is responsible for resistance
to arsenite (As(III)) and antimonite (Sb(III)). Here, we describe the identifi-
cation of a Caenorhabditis elegans homolog (asna-1) that encodes the
ATPase component of the Escherichia coli As(III) and Sb(III) transporter.
We evaluated the responses of wild-type and asna-1-mutant nematodes to
various metal ions and found that asna-1-mutant nematodes are more sen-
sitive to As(III) and Sb(III) toxicity than are wild-type animals. These
results provide evidence that ASNA-1 is required for C. elegans’ defense
against As(III) and Sb(III) toxicity. A purified maltose-binding protein
(MBP)–ASNA-1 fusion protein was biochemically characterized, and its
properties compared with those of ArsAs. The ATPase activity of the
ASNA-1 protein was dependent on the presence of As(III) or Sb(III).

As(III) stimulated ATPase activity by 2 ± 0.2-fold, whereas Sb(III) stimu-
lated it by 4.6 ± 0.15-fold. The results indicate that As(III)- and Sb(III)-
stimulated ArsA ATPase activities are not restricted to bacteria, but extend
to animals, by demonstrating that the asna-1 gene from the nematode,
C. elegans, encodes a functional ArsA ATPase whose activity is stimulated
by As(III) and Sb(III) and which is critical for As(III) and Sb(III) tolerance
in the intact organism.
Abbreviations
MBP, maltose-binding protein; NGM, nematode growth medium.
2566 FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS
bacterial ArsA protein, designated ASNA-1, a puta-
tive arsenite-translocating ATPase in C. elegans. Our
results provide evidence that As(III)- and Sb(III)-sti-
mulated ArsA ATPase activities are not restricted to
bacteria, but extend to animals by demonstrating that
the asna-1 gene of the nematode, C. elegans, encodes a
functional ArsA ATPase whose activity is stimulated
by As(III) and Sb(III).
Results
Sequence analysis of ASNA-1
Using E. coli ArsA sequence as a probe in a BLAST
analysis, we identified the putative ArsA homolog in
C. elegans, which we designated ASNA-1. The predic-
ted version of this gene has been previously reported
in the C. elegans genome database (Wormbase: http://
www.wormbase.org/) as ZK637.5 (GenBank accession
no. NM_066564). The asna-1 locus was physically
mapped to the gene cluster region of chromosome III
on the cosmid, ZK637. The predicted mRNA contains
an ORF of 1029 bp encoding a protein of 342 amino

acids and exhibits 33% identity to E. coli ArsA
ATPase. A BLAST search showed that eukaryotic
ArsAs from Saccharomyces cerevisiae (Arr4p), human
(hASNA-I), mouse (Asna1), and Drosophila melano-
gaster exhibit 46–56% identity to C. elegans ASNA-1.
Also, ASNA-1 contains a conserved Walker A motif,
or P loop (GKGGVGKT), and a signal transduction
domain (DTAPTGHT).
Toxicity tests of metals ions
To investigate whether ASNA-1 is required for As(III)
and Sb(III) tolerance in the intact organism, wild-type
and asna-1 deletion mutant nematodes were exposed
to a range of As(III) and Sb(III) ion concentrations
and the number of dead worms was scored over 24 h
(Fig. 1). The proportion of worms surviving at each
As(III) or Sb(III) concentration after 24 h exposure
varied considerably between wild-type and asna-1-
mutant worms (Fig. 1). The results showed that
asna-1-mutant nematodes were more sensitive to both
As(III) and Sb(III) toxicity than were the wild-type
strain. The toxic effect of As(III) and Sb(III) exposure
for wild-type and asna-1-mutant worms was further
investigated for time dependence. Survival of wild-type
and asna-1-mutant worms subjected to As(III)- and
Sb(III)-induced toxicity was time dependent. As shown
in Fig. 2, the mortality rate for both wild-type and
asna-1-mutant worms increased as the incubation times
with As(III) and Sb(III) increased. Also, the Kaplan–
Meier survival curve showed that asna-1-mutant nema-
todes were significantly less resistant (P<0.0001) to

both As(III) and Sb(III) toxicity than the wild-type
worms (Fig. 2).
To further explore the protective role of ASNA-1
against other metal ions, asna-1-mutant worms were
exposed to Pb(II), Cu(II), Al(III), Cr(VI), and Zn(II).
The exposure concentration for these metals was based
on previously reported lethal concentration (LC
50
) val-
ues for N2 worms [9]. The survival of asna-1-mutant
worms treated with the aforementioned metal ions
after 24 h exposure was not significantly different from
that of N2 worms (data not shown). Together, the
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0
20
40
60
80
100
[As(III)] m
M

Survival rate (%)
Survival rate (%)
A
0
5 10
15
20
25
0
20
40
60
80
100
[Sb(III)] m
M
B
Fig. 1. C. elegans As(III) and Sb(III) toxicity assay. Wild-type and
asna-1-mutant worms were incubated at 20 °C and the number of
dead worms was scored over 24 h. The number of dead worms
was determined as described in Experimental procedures. (A) Pro-
portion of worms surviving a range of As(III) concentrations. (B)
Proportion of worms surviving a range of Sb(III) concentrations.
Values are presented as the percentage of worms still alive at a
particular metal ion concentration after 24 h exposure at 20 °C;
(d) wild-type worms; (j) asna-1-mutant worms. Data is represen-
ted as mean ± SEM, n ¼ 6.
Y Y. Tseng et al. Caenorhabditis elegans ASNA-1
FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS 2567
toxicity results provide evidence that ASNA-1 is

required for C. elegans’ defense against As(III) and
Sb(III) toxicity, but not defense against other metals.
Effects of As(III) and Sb(III), enzyme
concentrations, and temperature on ATPase
activity
To biochemically characterize the C. elegans ASNA-1,
we designed an expression plasmid, maltose-binding
protein (MBP)–ASNA-1, to produce a plasmid MBP–
ASNA-1 fusion protein in E. coli. ASNA-1 with the
MBP tag was purified from E. coli cytosol using a
column of amylose resin, as described in Experimental
procedures. Approximately 2 mg of purified ASNA-1
protein was obtained per 500 mL of cells. Purified
recombinant ASNA-1 proteins were analyzed for their
ability to catalyze metalloid-stimulated ATPase activ-
ity. The specific activity of the MBP–ASNA-1 fusion
protein in the presence and absence of Sb(III) or
As(III) was determined. MBP protein without ASNA-
1 was also expressed and purified, and its ATPase
activity was analyzed. A very low ATPase activity of
2.4 ± 0.2 nmolÆmin
)1
Æmg
)1
was detected for MBP pro-
tein lacking the ASNA-1 portion. In the absence of
Sb(III) or As(III), a basal oxyanion-independent
ATPase activity of 5.4 ± 1.3 nmolÆmin
)1
Æmg

)1
was
measured using six different MBP–ASNA-1 fusion
protein preparations. Our results showed that As(III)
and Sb(III) stimulated ATPase activity of the MBP–
ASNA-1 fusion protein, and that the reaction was
initiated by the addition of MgCl
2
. Although the meas-
ured ATPase activity varied slightly between different
protein preparations, the activity increased consistently
in the presence of Sb(III) or As(III). Stimulation of
ATPase activity by Sb(III) and As(III) was not addit-
ive. When Sb(III) and As(III) were added in a ratio of
1:10, the ATPase activity was  18% less than in the
presence of Sb(III) alone, indicating that the two ani-
ons bind to the same site on the ASNA-1 protein.
Moreover, the effects of other metal ions, including
Pb(II), Al(III), Cd(II), Cr(VI), Co(II), Cu(II), Fe(II),
Hg(II), Mn(II), Ni(II), and Zn(II), on the ATPase
activity of the ASNA-1 fusion protein were examined.
The metal ions tested neither stimulated nor inhibited
the ATPase activity of the ASNA-1 fusion protein
(data not shown).
The reaction rate was proportional to the amount of
enzyme added, as shown in Fig. 3. Moreover, the reac-
tion rate increased with increasing amounts of Sb(III)
in the reaction (Fig. 3). Increasing the assay tempera-
ture from 25 to 37 °C did not increase basal
ATPase activity, but produced a two-fold increase in

Sb(III)-stimulated ATPase activity. Based on these
observations, the ATPase assay in this study was rou-
tinely performed with 15 lg protein at 37 °C, and
experiments were usually completed within 30 min of
recovering the protein preparation.
Affinity of ASNA-1 protein for substrates
To examine the affinity of the ASNA-1 protein for
ATP, the apparent K
m
for ATP was determined at
pH 7.4 in the absence and presence of a saturating
concentration of Sb(III) (0.5 mm) using six indepen-
dently prepared MBP–ASNA-1 fusion proteins.
A V
max
of 6.21 ± 0.24 nmolÆmin
)1
Æmg
)1
was observed
0 5
10
15
20
25
30 35
40
0
20
40

60
80
100
120
Hours
Survival rate (%)Survival rate (%)
A
0
5
10
15
20
25
30
35
40
0
20
40
60
80
100
120
Hours
B
Fig. 2. Time-dependent lethality of worms exposed to As(III) and
Sb(III). Wild-type and asna-1-mutant worms were exposed to As(III)
and Sb(III) at 0.25 m
M. Worms were incubated at 20 °C and the
number of dead worms was scored at different time points ranging

from 1 to 36 h (± 10 min). The number of dead worms was deter-
mined as described in Experimental procedures. Survival data were
subjected to Kaplan–Meier survival curve analysis. (A) Proportion of
wild-type (solid line) and asna-1 mutant (dash line) worms surviving
with As(III) treatment. (B) Proportion of wild-type (solid line) and
asna-1-mutant (dash line) worms surviving with Sb(III) treatment.
Caenorhabditis elegans ASNA-1 Y Y. Tseng et al.
2568 FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS
for the basal ATPase activity, whereas V
max
for the
Sb(III)-stimulated ATPase activity was 28.87 ± 3.31
nmolÆmin
)1
Æmg
)1
(Fig. 4A). Similar K
m
values of
0.21 ± 0.03 and 0.26 ± 0.09 mm were obtained in the
absence and presence of Sb(III), respectively. Sb(III)-
induced stimulation of ATPase activity was thus due
to a 4.6-fold maximal increase in V
max
rather than to
increased affinity of the MBP–ASNA-1 fusion protein
for ATP. In addition, similar K
m
and V
max

values were
observed in the presence of either 2.5 or 5.0 mm
MgCl
2
(Fig. 4B).
Half-maximal stimulatory concentrations of Sb(III)
and As(III) on ATPase activity were determined at
ATP saturation (Fig. 5). Apparent K
m
values for
Sb(III) and As(III) were found to be 0.04 ± 0.00
and 3.54 ± 0.33 mm, respectively. The V
max
for
Sb(III)-stimulated ATPase activity was 19.62 ± 0.16
nmolÆmin
)1
Æmg
)1
, whereas the As(III)-stimulated activ-
ity had a V
max
of 11.67 ± 0.48 nmolÆmin
)1
Æmg
)1
.
Discussion
Our findings represent the first demonstration of ArsA
protein-mediated detoxification of the metalloids,

As(III) and Sb(III), in an animal. Moreover, we show
that As(III)- and Sb(III)-stimulated ArsA ATPase
activities are not restricted to bacteria, but extend to
animals. This was shown by demonstrating that the
asna-1 gene of C. elegans encodes a functional ArsA
whose activity is stimulated by As(III) and Sb(III),
and which is critical for As(III) and Sb(III) tolerance
in the intact organism. Although the function of bac-
terial ArsA has been identified, the ubiquity of the
ArsA ATPase-dependent pathway in other animals
remains to be delineated. E. coli ArsA hydrolyzes ATP
in the presence of As(III) or Sb(III), and is the cata-
lytic component of an oxyanion pump that provides
resistance to As(III) or Sb(III). Several eukaryotic
ArsA homologs have been identified and studied.
However, they showed no biochemical functions sim-
ilar to that of bacterial ArsA. Mammalian ArsA
homologs have been identified in humans [10] and
A
0.0
0.2
0.4 0.6
0.8
1.0
0
5
10
15
20
25

30
[ATP] m
M
0.0
0.2
0.4 0.6
0.8
1.0
[ATP] m
M
ATPase Activity (nmol/min/mg)ATPase Activity (nmol/min/mg)
0
5
10
15
20
25
30
B
Fig. 4. Affinity of the ASNA-1 protein for ATP. The ATPase activity
of purified ASNA-1 protein (15 lg) was measured over a range of
ATP concentrations in the absence (s) and presence (h) of 0.5 m
M
Sb(III) containing 5.0 mM MgCl
2
(A) and in the presence of 0.5 mM
Sb(III) containing either 2.5 mM MgCl
2
(n) or 5.0 mM MgCl
2

(j) (B).
The reaction was initiated by the addition of MgCl
2
after 10 min
incubation of ASNA-1 protein at 37 °C. Solid and dashed lines indi-
cate fitting of data to the Michaelis–Menten equation by a nonlinear
regression using
PRISM 4.0 software.
0
5
10
15
20
0.0
0.1
0.2
0.3
0.4
ASNA-1 (µg)
ATPase Activity (nmol/min)
Fig. 3. Effects of enzyme concentrations on ATPase activity.
ATPase activity was measured in the presence of the indicated
amounts of the ASNA-1 protein as described in Experimental proce-
dures. The reaction was initiated by the addition of 5 m
M MgCl
2
after 10 min incubation of the ASNA-1 protein at 37 °C with or
without Sb(III) in assay buffer. At each protein concentration, the
activity was measured with no oxyanion (s), or 0.1 m
M (n)or

0.5 m
M (h) Sb(III).
Y Y. Tseng et al. Caenorhabditis elegans ASNA-1
FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS 2569
mouse [8]. Biochemical analysis of the hASNA-I
human homolog showed that it has a low level of
ATPase activity, which was simulated 1.6-fold in the
presence of As(III), but not in the presence of Sb(III)
[11]. The mouse homolog (Asna1) has been shown to
be an unlikely component of an As(III) pump in mam-
mals [12]. The ArsA homolog (Arr4p) in S. cerevisiae
exhibited a low level of ATPase activity but was not
stimulated by As(III), Sb(III), or any other metals [13].
Therefore, it is noteworthy that, although human
hASNA-I, mouse Asna1, and yeast Arr4p cDNA were
isolated using homology to the bacterial ArsA, the
protein they encodes is not an As(III)- and Sb(III)-
stimulated ATPase, indicating that these eukaryotic
ArsA homologs are biochemically distinct from that of
bacterial ArsA. Moreover, the protective roles of these
eukaryotic ArsA homologs against As(III) and Sb(III)
remain to be studied.
In both mouse [12] and C. elegans, deletion of the
gene encoding the ATPase is lethal, suggesting involve-
ment of the asna-1 gene in embryonic or larval devel-
opment. After submission of this article, Kao et al.
reported that worms lacking asna-1 gene activity arrest
at the L
1
stage, even in the presence of abundant food

[14]. ASNA-1 functions nonautonomously to regulate
growth [14]. They further showed that ASNA-1 posi-
tively regulates insulin secretion in C. elegans and
mammalian cells [14]. However, the precise mechanism
by which ASNA-1 protects against As(III) and Sb(III)
toxicity remains to be further elucidated. Several
observations, however, show the unique features of
ASNA-1. First, ATPase activity of the ASNA-1 pro-
tein was stimulated only by As(III) and Sb(III). Other
metal ions neither stimulated nor inhibited the ATPase
activity of ASNA-1. Second, although a few ABC
transporters, namely one member of the multidrug
resistance-associated protein (MRP-1) and two mem-
bers of the P-glycoprotein subfamily (PGP-1 and PGP-
3), have been shown to contribute to heavy metal tol-
erance, including As(III) tolerance in C. elegans,no
increased sensitivity to Sb(III) was found in the dele-
tion mutants compared with wild-type worms [15].
Therefore, it is unlikely that the ASNA-1 protein is a
component of a eukaryotic ABC transporter.
Possibly the most interesting characteristic of the
ASNA-1 ATPase is the fact that it was activated by
the binding of As(III) or Sb(III), the same ions that
are transported by the ArsAB pump in E. coli. At this
point, there is no conclusive biochemical evidence that
the ions which activate the hydrolytic activity of
ASNA-1 are the same ions that are transported across
the membrane in C. elegans. In bacteria, the ArsAB
pump confers an evolutionary advantage to organisms
exposed to high levels of metalloid salts, reflecting the

fact that ATP-driven pumps are capable of forming
higher concentration gradients than carrier proteins.
Thus, the ArsAB system reduces the intracellular con-
centration of metalloid ions to lower levels than can be
realized by ArsB alone, providing evolutionary pres-
sure to acquire an arsA gene. However, it is interesting
to note that no eukaryotic ArsB orthologs have been
identified to date. Therefore, C. elegans may contain a
novel transporter that is possibly unrelated to ArsB
but with a similar function. Isolation of the metalloid
transporter is necessary before the role of ASNA-1 as
a component of a nematode efflux pump for As(III)
0.0
0.5
1.0
1.5
2.0
2.5
0
5
10
15
20
25
[Sb(III)] mM
A
0
2 4
6
8 10

0
2
4
6
8
10
[As(III)] mM
ATPase Activity (nmol/min/mg)ATPase Activity (nmol/min/mg)
B
Fig. 5. Affinity of the ASNA-1 protein for Sb(III) and As(III). The
half-maximal stimulatory concentrations for Sb(III) (A) and As(III) (B)
were determined over a range of concentrations using 15 lgof
purified ASNA-1 protein in the presence of 1 m
M ATP. The reaction
was initiated by the addition of 5 m
M MgCl
2
after 10 min incubation
of the ASNA-1 protein at 37 °C in assay buffer. The solid line indi-
cates fitting of the data to the Michaelis–Menten equation by non-
linear regression using
PRISM 4.0 software.
Caenorhabditis elegans ASNA-1 Y Y. Tseng et al.
2570 FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS
and Sb(III) detoxification can be examined, and this is
the subject of a future study.
Experimental procedures
General methods
All C. elegans, bacterial strains, and plasmids were supplied
by the Caenorhabditis Genetics Center (University of Min-

nesota, MN), which is funded by the NIH National Center
for Research Resources. The exception was the cDNA
clone yk747c10, which was kindly provided by Y. Kohara
(National Institute of Genetics, Mishima, Japan). The
following strains were used: wild-type C. elegans N2
(var. Bristol); asna-1 mutant: [tag-205(ok938)III ⁄ hT2
[bli-4(e937)let-?(q782)qIs48](I;III)], which is a homozygous
lethal deletion chromosome balanced by bli-4- and GFP-
marked translocation. C. elegans was grown in Petri dishes
on nematode growth medium (NGM) at 20 °C using the
E. coli OP50 strain as the food source.
Metal ions toxicity analyses
One hundred and twenty L
3
-stage hermaphrodites of wild-
type (N2) or asna-1 mutant (non-GFP ok938 homozygotes)
were transferred from NGM plates to Costar 24-well tissue
culture plates containing 1 mL of K medium (53 mm NaCl,
32 mm KCl) [9] with appropriate concentrations of metal
ions per well. Wild-type and asna-1-mutant worms were
exposed to 0, 0.25, 0.5, 1.5, and 3.0 mm nominal concentra-
tions of As(III) or 0, 0.25, 5, 10, and 20 mm of Sb(III). For
other metal ions, wild-type and asna-1-mutant worms were
exposed to 0.39 mm of Pb(II), 0.16 mm of Cu(II), 0.37 mm
of Al(III), 0.54 mm of Cr(VI), and 1.48 mm Zn(II). Worms
were incubated at 20 ° C and the number of dead worms
was scored at different time points ranging from 1 to 36 h
(± 10 min). The number of dead worms was determined
by the absence of touch-provoked movement when probed
with a platinum wire. Tests were performed between three

and six times for each metal ion.
Construction of the recombinant MBP–ASNA-1
fusion protein expressed in E. coli
Translational fusion was constructed by directional cloning
of the PCR-amplified cDNA of ASNA-1 into multiple clo-
ning sites of the pMAL-c2X vector (New England Biolabs,
Hertfordshire, UK). Plasmid yk747c10, kindly provided by
Y. Kohara (National Institute of Genetics, Mishima,
Japan), was used as a template for PCR to generate the
DNA fragment. PCR primers were designed with either
PstI (forward primer) or HindIII (reverse primer) recogni-
tion sequence extensions (underlined). Sequences of PCR
primers were as follows: forward 5¢-CCG
CTGCAGGAA
AAAACGCTAAAATGGA-3¢ and reverse 5¢-CGC
AAG
CTTAGAACAAATTAGTTTAGT-3¢. The amplified PCR
product was purified using a QIAquick PCR purification
kit (Qiagen, Hilden, Germany). Purified PCR-amplified
DNA fragment was digested with both PstI and HindIII
and then ligated with the pMAL-c2X expression vector that
had been similarly digested with both enzymes. The result-
ing construct, MBP–ASNA-1, contained the asna-1 gene
cloned inframe with the sequence for an N-terminal MBP
tag. The correct reading frame and DNA sequence were
verified by DNA sequencing. The MBP–ASNA-1 plasmid
was used to transform BL21 cells.
Purification of the MBP–ASNA-1 protein
For expression, cells of the BL21 E. coli strain harboring
the MBP–ASNA-1 plasmid were grown overnight in 5 mL

of Luria–Bertani medium containing 0.2% glucose and
100 lgÆmL
)1
ampicillin at 37 °C. This culture was then
diluted in 500 mL of the same medium and incubated
with shaking at 37 ° C until D
600
¼ 0.5 was reached. Iso-
propyl thio-b-d-galactoside was added to a final concen-
tration of 0.1 mm, and the culture was incubated for an
additional 2 h and harvested by centrifugation. Cell pellets
were washed once with column buffer (20 mm Tris ⁄ HCl
pH 7.4, 200 mm NaCl, 1 mm EDTA, 1 mm sodium azide,
and 10 mm b-mercaptoethanol). Lysozyme was added to a
final concentration of 1 mgÆmL
)1
and incubated on ice for
30 min. Cells were suspended in 10 mL of column buffer
containing 1 mgÆmL
)1
lysozyme. Cells were sonicated on
ice using six 15-s bursts at 200–300 W with 15 s cooling
off between each burst. The lysate was centrifuged at
10 000 g for 30 min at 4 °C, and then preincubated for
1 h with 2 mL of amylose resin (New England Biolabs).
The supernatant and amylose resin solution were loaded
onto a column at a flow rate of 0.5 mLÆmin
)1
. The col-
umn was washed with column buffer at a flow rate of

1.0 mLÆmin
)1
. Finally, ASNA-1 was eluted with 15 mL of
column buffer containing 10 mm maltose. ASNA-1-con-
taining fractions were identified by SDS ⁄ PAGE. Purified
ASNA-1 was either quickly frozen and stored at )80 °C
or kept in small aliquots at 4 °C. The concentration of
purified ASNA-1 was determined by the Bradford assay
(BioRad, Hercules, CA).
ATPase activity assays
ATPase activity was estimated colorimetrically from the
release of inorganic phosphate as described by Gawronski
and Benson [16]. Freshly purified MBP–ASNA-1 protein
maintained at 4 °C was used for biochemical characteriza-
tion throughout this work. ATPase activity was measured
spectrophotometrically at room temperature from a
released inorganic phosphate concentration of 650 nm.
Y Y. Tseng et al. Caenorhabditis elegans ASNA-1
FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS 2571
The reaction was allowed to equilibrate for 15 min at
room temperature before reading the absorbance at
650 nm. The assay mixture contained ATP with or
without Sb(III) or As(III), and was prewarmed to 37 °C.
Fifteen micrograms of MBP–ASNA-1 was preincubated at
37 °C in the reaction mixture for 10 min before the reac-
tion was initiated by the addition of MgCl
2
. The concen-
tration of purified MBP–ASNA-1 protein was determined
using the Bradford assay.

Data analysis
Survival data were subjected to Kaplan–Meier survival
curve analysis using prism 4.0 (GraphPad Software, San
Diego, CA). A log-rank test was performed comparing
wild-type with asan-1-mutant worms. Kinetic parameters
were calculated with prism 4.0 using the nonlinear regres-
sion of the Michaelis–Menten equation. Experiments were
performed between three and nine times for standard error
analyses.
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