Báo cáo khoa học: Purification and partial characterization of seven glutathione S -transferase isoforms from the clam Ruditapes decussatus pptx

Purification and partial characterization of seven glutathione
S
-transferase isoforms from the clam
Ruditapes decussatus
Pascal Hoarau, Ginette Garello, Mauricette Gnassia-Barelli, Miche
`
le Romeo and Jean-Pierre Girard
UMR 1112 INRA-UNSA, Laboratoire Re
´
ponse des Organismes aux Stress Environnementaux, Faculte
´
des Sciences,
Universite
´
de Nice-Sophia Antipolis, Nice, France
This paper deals with the purification and the partial char-
acterization of glutathione S-transferase (GST) isoforms
from the clam Ruditapes decussatus. For the first step of
purification, two affinity columns, reduced glutathione
(GSH)–agarose and S-hexyl GSH–agarose, were mounted
in series. Four affinity fractions were thus recovered. Further
purification was performed using anion exchange chroma-
tography. Seven fractions, which present a GST activity with
1-chloro-2,4-dinitrobenzene (CDNB) as substrate, were
collected and analyzed by RP-HPLC. Seven distinct GST
isoforms were purified, six of them were homodimers, the
last one was a heterodimer consisting of the subunits 3 and 6.
Kinetic parameters were studied. Results showed that
isoforms have distinct affinity and V
max
for GSH and CDNB

as substrates. The catalytic activity of the heterodimer
isoform appeared to be a combination of the ability of each
subunit. The immunological properties of each purified
isoform were investigated using three antisera anti-pi, anti-
mu and anti-alpha mammalian GST classes. Three isoforms
(3-3,6-6and3-6)seemtobecloselyrelatedtothepi-class
GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera
to pi and alpha classes and the isoform 5-5 cross-reacted with
the antisera to mu and pi classes. Subunit 4 was recognized
by the three antisera used, and its N-terminal amino acid
analysis showed high identity (53%) with a conserved
sequence of an alpha/ml/pi GST from Fasciola hepatica.
Keywords: clam; glutathione S-transferase; immunology;
kinetics; N-terminal analysis.
The glutathione S-transferases (GSTs; EC 2.5.1.18) bind
lipophilic nonsubstrate ligands and compounds such as
heme and bilirubin [1], and are implicated in the biosynthe-
sis of prostaglandins [2]. Apart from their metabolic
activities, they are involved in the detoxication of electro-
philic and genotoxic compounds by both catalytic activity
and direct binding. In fact, the effect of these reactions is to
convert a reactive (lipophilic) molecule into a water-soluble
(nonreactive conjugate) which can be excreted. These
mechanisms play an important role in cellular protection
against the toxicity of endogenous compounds and of a
variety of xenobiotics [3].
The diversity of compounds metabolized by GSTs results
from both the relatively nonspecific nature of the hydrophilic
substrate binding site and the existence of numerous GST
isoforms. GST activities are often assayed using 1-chloro-

2,4-dinitrobenzene (CDNB), a relatively nonspecific GST
reference substrate [4]. GST-CDNB activity reflects the
integration of GST isoenzyme activities. However, the use
of GST substrates such as 1,2-dichloro-4-nitrobenzene,
ethacrynic acid (ETHA), nitrobutyl chloride and D5-andro-
stene-3,17-dione in conjunction with CDNB allows for a
more complete biochemical characterization of GST iso-
zyme activities [5].
GSTs are a multigene family of enzymes (isoforms),
which have been grouped into seven classes based upon
sequence homology and ability to catalyze the conjugation
of glutathione to a broad range of electrophilic substrates in
animal organisms. These classes are named alpha, mu, pi,
theta, sigma, kappa, zeta and omega [1,6,7]. Some bacteria
express a beta class GST [8] and higher invertebrates express
a delta class GST [9]. The GST family is found in most
aerobic eukaryotes and in some prokaryotes.
In vertebrate organisms, particularly in rats (where 13
different isoforms have been found in the liver [10]) and in
fish [11], GST isoenzymes are extensively studied and are
distinct with regards to their subunit structure, isoelectric
point, kinetics and immunological properties. In compari-
son with what is known of vertebrates, relatively little
information is available concerning GSTs from marine
invertebrate organisms. Fitzpatrick and Sheehan [12]
reported the purification of four GST isoforms in the
digestive gland and gills of the blue mussel Mytilus edulis.
In previous work [13], the differential induction of GSTs
was studied in the clam Ruditapes decussatus exposed to
organic compounds. The present paper describes the

purification of clam GST isoforms by affinity chromato-
graphy using reduced glutathione (GSH)–agarose and
S-hexyl GSH–agarose columns. The fraction recovered
were then submitted to anion exchange chromatography,
leading to the distinction of isoforms. Their characterization
was performed by studying the kinetic parameters (V
max
,
K
m
with the commonly used substrate CDNB and GSH)
and by performing immunoblotting reactions. RP-HPLC
analysis was used to identify the different constitutive
Correspondence to P. Hoarau, UMR 1112 INRA-UNSA, Laboratoire
Re
´
ponse des Organismes aux Stress Environnementaux, Faculte
´
des
Sciences, Universite
´
de Nice-Sophia Antipolis, Parc Valrose,
06108 Nice Cedex 2, France. Tel.: +33 04 92 07 68 96,
E-mail:
Abbreviations:GST,glutathioneS-transferase; GSH, reduced gluta-
thione; CDNB, 1-chloro-2, 4-dinitrobenzene; ETHA, ethacrynic acid.
Enzyme: glutathione S-transferases (GST; EC 2.5.1.18).
(Received 14 May 2002, revised 11 July 2002, accepted 25 July 2002)
Eur. J. Biochem. 269, 4359–4366 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03141.x
subunits of the purified isoforms. N-terminal amino acid

analysis was carried out on a particular subunit in order to
evaluate to which class of GST isoforms this subunit may
belong.
MATERIALS AND METHODS
Animal maintenance
Animals were collected from the lagoon of Thau (N.W.
Mediterranean sea) and placed on ice for transport to the
laboratory. They were then transferred to a 200-L aquarium
filled with natural aerated sea water, in a closed circuit, at
18 °C with a photoperiod of 12 : 12 h for 2 days.
Preparation of the postmitochondrial fraction
All procedures were carried out at 0–4 °C. A pool of 12
animals was homogenized in a buffer (20% w/v, 10 m
M
Hepes, 250 m
M
sucrose, 1 m
M
phenylmethanesulfonyl
fluoride, 1 m
M
dithiothreitol pH 7.4) using an ultra-Turrax
homogenizer. The homogenate was centrifuged at 9000 g
for 20 min to remove cell debris, nuclei and mitochondria.
All analyses were performed on the supernatants which
were used immediately or stored at )80 °C.
Enzyme assays
GST activity was measured spectrophotometrically at
37 °C using two substrates, CDNB and ETHA, according
to the method of Habig et al. [14]. Specific activity was

expressed as lmol productÆmin
)1
Æmg
)1
protein. The
protein concentration was determined according to
Bradford [15].
Purification of GSTs
All procedures were performed at 0–4 °C. Fifty milliliters
of the supernatant were dialyzed with a cut-off of 10 kDa
against a solution of 20 m
M
Tris/HCl, 0.2
M
NaCl, 1 m
M
dithiothreitol pH 7.9 to eliminate GSH. The supernatants
were passed through two affinity columns (Sigma) moun-
ted in series. The first was a GSH–agarose column (C1)
and the second a S-hexyl GSH–agarose column (C2).
Each column was then separately eluted with two
solutions. The first solution (E1) was 200 m
M
Tris/HCl
containing 0.2 m
M
GSH pH 9, and the second (E2) was
200 m
M
Tris/HCl containing 5 m

M
GSH pH 9. Four
fractions were thus obtained, they were called: C1E1,
C1E2, C2E1 and C2E2. The fractions were then dialyzed
(cut-off 10 kDa) against 20 m
M
Tris/HCl, 1 m
M
dithio-
threitol pH 9.
GSTs from the two fractions of each column (C1 and C2)
were fractionated further by anion exchange chromatogra-
phy using a 5-mL Q cartridge column (Bio-Rad). The
column was equilibrated with 20 m
M
Tris/HCl, 1 m
M
dithiothreitol pH 9, at a flow rate of 1.0 mLÆmin
)1
.The
absorbance was measured at 280 nm during the experiment.
The elution conditions are shown in Table 1. One-milliliter
fractions were collected during the experiment and their
activity with CDNB as a substrate was measured. Further
analyses were performed only in the seven fractions where
CDNB activity was measurable.
Kinetic parameters
The K
m
and V

max
determinations with CDNB as a substrate
for GST assays were performed in triplicate at 25 °Cwith
varying concentrations of CDNB (0.04–5.12 m
M
)anda
constant GSH concentration (50 m
M
). The K
m
and V
max
with GSH as a substrate were performed with varying
concentrations of GSH (0.026–15.6 m
M
) and a constant
CDNB concentration (5 m
M
) according to the method of
Gallagher et al. [4]. Kinetic constants were calculated using
nonlinear regression iterative program that gave a best fit of
the experimentally measured activities to the Michaelis–
Menten equation. k
cat
was calculated as follows according
to the Michaelis–Menten equation for double substrate
enzymes such as GST:
V
i
¼ k

cat
E
0
Á
GSH½
K
mGSH
þ GSH½

Á
CDNB½
K
mCDNB
þ CDNB½

where V
i
is the initial velocity and E
0
is the initial
concentration of enzyme.
SDS/PAGE
The seven anion exchange chromatography purified GSTs
were submitted to reducing and nonreducing SDS/PAGE,
using a Bio-Rad Mini-Protean II electrophoresis unit, with
a 15% and 10% resolving gel, respectively, and a 4%
stacking gel [16].
Western blotting
Purified GSTs from ion exchange chromatography were
separated by SDS/PAGE and then transferred electropho-

retically to Immobilon-P membrane (Millipore). Nonspe-
cific binding sites were saturated by incubation in 50 m
M
Tris/HCl, 150 m
M
NaCl, 0.1% (v/v) Tween 20 (buffer A),
containing 5% (w/v) nonfat dried milk for 1 h at room
temperature. The membrane was then incubated with
primary antibody diluted with buffer A containing 1%
Table 1. Elution conditions of the four affinity fractions during anion exchange chromatography.
Fraction
NaCl
(m
M
)
Time
(min)
NaCl
(m
M
)
Time
(min)
NaCl
(m
M
)
Time
(min)
NaCl

(m
M
)
Time
(min)
C1E1 0–450 3 450–640 12 650–750 5
C1E2 0–350 3 350–400 11 400–450 1 450–500 5
C2E1 0–200 3 200–550 12 550–750 5
C2E2 0–350 3 350–450 12 450–750 5
4360 P. Hoarau et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(w/v) dried milk (buffer B) overnight under gentle agitation
at 4 °C.
The membrane was then rinsed three times with buffer B
for 5 min and incubated with peroxidase-conjugated goat
anti-rabbit IgG from Bio-Rad diluted 1 : 2500 with buffer B
for 1 h at room temperature. The proteins which cross-
reacted with the antibody were detected by luminescence
(ECL, Amersham). Three antisera were used as primary
antibody: a rabbit anti-(pi class GST) (polyclonal antibody;
Chemicon) diluted 1 : 2000, a rabbit anti-(mu class GST)
(polyclonal antibody; Interchim) diluted 1 : 500 and a
rabbit anti-(alpha class GST) (polyclonal antibody; Inter-
chim) diluted 1 : 2000.
RP-HPLC
HPLC analysis of each of the four affinity fractions and of
all the anion exchange chromatography-purified GST
isoforms was performed using a reverse phase column
(250 · 4 mm) Licrosorb RP 18 Merck, particle size 5 lm, a
Merck model L 500 and a D 2000 integrator. The mobile
phase linear gradient (0–50% B over 40 min) of 0.05% (v/v)

trifluoroacetic acid in water (solvent A) and 0.05% (v/v)
trifluoroacetic acid in acetonitrile (solvent B) was used at a
flow rate of 0.8 mLÆmin
)1
. Peptide detection was performed
at 214 nm [17]. Samples were injected in a 500-lL loop valve
with bracketing, a method which improved the resolution
and increased the peak heights according to Guinn and
Hendricx [18].
N-Terminal amino acid sequencing
One of the seven anion exchange purified isoforms, which is
recognized by the three antisera used, was chosen for
N-terminal amino acid sequencing. For this purpose,
30 pmol were applied to 15% SDS/PAGE and then
transferred to a poly(vinylidene difluoride) membrane
(Immobilon-P Millipore). This analysis was carried out by
the Laboratory of Protein Microsequencing of Pasteur
Institute (Paris, France).
Statistical analyses
Statistical comparison of the results was made using a
nonparametric Mann–Whitney test with n ¼ 3ineachcase.
RESULTS
Purification of GSTs and HPLC separation of subunits
The GST activities with CDNB and ETHA in each affinity
purified fraction are shown in Fig. 1. The highest activities
were found with CDNB (Fig. 1A), whereas the activities
measured with ETHA were % 100 times lower than with
CDNB. The GST/CDNB activity showed significant
differences (% twofold; Fig. 1A) according to the affinity
column used for the first elution carried out with 0.2 m

M
GSH. No differences were observed between C1 and C2 in
the second elution performed with 5 m
M
GSH and the
measured activity appeared to be much lower than with
0.2 m
M
GSH. The GST/ETHA activity showed significant
differences (% fivefold; Fig. 1B) according to the column
used only in the case of the second elution. Quantitatively
75% of the total CDNB activity loaded on the two columns
were recovered in the four fractions.
RP-HPLC analysis was performed on each of the four
affinity fractions (Fig. 2). As all GST subunits were eluted
before 20 min, results were thus presented within this
period. Six subunits were identified and numbered from 1 to
6 as a function of their retention time. In the C1E1 affinity
fraction, three peptides were eluted at 12.06, 14.09 and
16.40 min after the elution gradient starts. In the C2E1
fraction, two peptides were detected at 14.83 and 16.40 min.
In the C1E2 fraction, three peptides were eluted at 7.2, 9.02
and 14.83 min and in the C2E2 fraction, only one peptide
was eluted at 14.83 min. The first affinity column (C1)
allowed binding of the six subunits while the second column
(C2) bound only subunits 5 and 6 (Fig. 2).
Proteins of each of the four affinity fractions were also
separated using anion exchange chromatography. GST
isoforms 1-1, 2-2 were isolated from the C1E2 affinity
fraction; GST isoforms 3-3, 4-4 and 3-6 from the C1E1 frac-

tion; and GST isoforms 5-5 and 6-6 from the C2E1 fraction.
The purified fractions, which showed a significant GST/
CDNB activity, were further analyzed by RP-HPLC
(Fig. 3) and by SDS/PAGE in nondenaturing conditions
and denaturing conditions (data not shown). Results
showed that six GST isoforms (1-1, 2-2, 3-3, 4-4, 5-5, and
6-6) appeared to be composed of two identical subunits.
They are thus homodimers with native apparent molecular
weight ranging from 44 to 52 kDa. The seventh purified
isoform (3-6) is an heterodimer consisting of two subunits:
subunits 3 and 6, as shown by the retention time (Fig. 3).
Activities and kinetics specificities
Table 2 summarizes the retention time (already given in
Fig. 3), the apparent molecular mass and the GST-specific
enzymatic activities of purified GST isoforms of R. decus-
satus. All purified isoforms catalyzed the conjugation of
GSH with CDNB, whereas isoforms 1-1, 2-2 and 4-4 had
zero or low activity with ETHA as a substrate. Table 2
shows four groups according to their specific activities. The
first group, composed of isoforms 1-1 and 2-2, had low
Fig. 1. GST activities using (A) CDNB and (B) ETHA as substrates in
the two eluted fractions (E1 and E2) from the two affinity columns. C1,
GSH–agarose column; C2, S-hexyl GSH–agarose. Values are given as
means ± SD.
Ó FEBS 2002 Purification of R. decussatus GST isoforms (Eur. J. Biochem. 269) 4361
activity with CDNB (< 100 lmolÆmin
)1
Æmg
)1
) and no

activity with ETHA; the second group, represented only by
the 4-4 isoform, had high activity with CDNB (% 300
lmolÆmin
)1
Æmg
)1
) and low activity with ETHA (< 5
lmolÆmin
)1
Æmg
)1
)l the third group of isoforms (5-5, 6-6
and3-6) showedhighactivitywithboth CDNB(> 300 lmolÆ
min
)1
Æmg
)1
) and ETHA (> 15 lmolÆmin
)1
Æmg
)1
); and the
fourth group (isoform 3-3) showed the highest activity with
ETHA and an intermediate activity with CDNB.
In previous work [13], it was observed that GST isoforms
induced by pesticides showed great activity with ETHA as a
substrate. Thus, among the purified isoforms, those pre-
senting high GST activity with this substrate were chosen to
determine the kinetic parameters. Table 3 displays the
kinetic constants of the purified isoforms (3-3, 5-5, 6-6 and

3-6). No statistical differences in V
max
(around 200 lmolÆ
min
)1
Æmg
)1
) were observed in the case of CDNB or GSH
for any of the isoforms studied. On the contrary, the
isoforms presented statistically different affinities for the two
substrates GSH and CDNB, except in the case of the
affinity for GSH of GST3-6 and GST 6-6. The 3-3 isoform
showed the highest affinity for CDNB (K
m
¼ 0.17 m
M
)
compared to the isoforms 3-6, 5-5 and 6-6 (in decreasing
order). GSH K
m
values displayed a wide range from
0.37 m
M
(5-5 isoform) to 4.82 m
M
(3-3 isoform). The
comparison of k
cat
demonstrated that all purified isoforms
had rapid and distinct turnover in a narrow range (214.3–

250.2Æs
)1
). Differences were observed when comparing the
efficiencies (expressed by the ratio k
cat
/K
m
)forthetwo
substrates. The GST 3-3 isoform showed the best efficiency
for CDNB followed by GSTs 3-6, 5-5 and 6-6 (in decreasing
order). For GSH, the GST 5-5 presented the best efficiency
compared to isoforms 6-6, 3-6 and 3-3 (in decreasing order).
The 3-6 isoform, with kinetic parameters between those of
GST isoforms 3-3 and 6-6, seemed to have the properties of
each of its subunits.
Immunoblotting
Fig. 4A shows Western blotting analysis of the seven
purified isoforms from R. decussatus performed with rabbit
anti-(pi class GST) serum. Cross-reactivity was observed
with all the purified isoforms (apparent molecular mass
from 22 to 26 kDa). Purified isoforms 1-1 to 6-6 presented
one single band whereas the heterodimer 3-6 displayed two
bands located at 24 and 23 kDa thus confirming the HPLC
results (Fig. 3). Moreover, isoforms 4-4, 5-5 and 6-6 also
gave a positive reaction in immunoblotting (data not
shown) when tested with a rabbit anti-(R. decussatus
GST) serum, produced in our laboratory [13].
Fig. 2. RP-HPLC of each affinity fraction (C1E1, C2E1, C1E2 and
C2E2). Protein samples (% 50 lg) were loaded on a Licrosorb RP 18
column. Elution conditions are given in Material and methods.

Fig. 3. RP-HPLC of each ionically purified
GST isoform. Protein samples (5 lg) were
loaded under the same conditions as given in
Fig. 2.
4362 P. Hoarau et al. (Eur. J. Biochem. 269) Ó FEBS 2002
GST isoforms 1-1, 2-2 and 4-4 were also recognized in
Western blotting analysis performed with rabbit anti-(alpha
class GST) serum (Fig. 4B), whereas the rabbit anti-(mu
class GST) serum only gave a positive reaction with
isoforms 4-4 and 5-5 (Fig. 4C).
N-Terminal amino acid sequence analysis
As subunit 4 is the only one which cross-reacts with the
three classes of mammalian GST antibodies, N-terminal
amino acid analysis was performed to determine to which
class this subunit belongs. Table 4 displays the results of the
amino acid sequence compared to published results. There
was 60% identity with GSH binding protein from the
mussel M. edulis and with a GST from the nematode
Clonorchis sinensis, and 53% with a GST from the
platyhelminth Fasciola hepatica.
N-Terminal sequence of R. decussatus presented 47, 26
and 20% of identity with the mu, alpha and pi class
consensus sequences from mammals, respectively. The
N-terminal sequence of subunit 4 of R. decussatus exhibits
a high similarity with the corresponding consensus mu GST
class from mammals.
DISCUSSION
In studies carried out on the blue mussel M. edulis [12],
Fitzpatrick and Sheehan (1993) have shown that four
different GST isoforms are induced in this animal in

response to pollutants. In previous work [13] we demon-
strated the same phenomenon in the clam R. decussatus
with chemical pollutants especially with 2,2-bis-(p-chloro-
phenyl)-1,1-dichloroethylene. Nevertheless, few studies have
concerned the purification and characterization of GST
isoforms in mollusks. The interest in using the clam
R. decussatus is a result of the fact that it is a very abundant
species commercialized for human consumption around the
Mediterranean.
In this paper, an attempt was made to purify the different
GST isoforms present in R. decussatus. For this purpose, the
use of two affinity columns (GSH–agarose and S-hexyl
GSH–agarose) demonstrated that R. decussatus GSTs have
different affinities for the chromatography matrices. These
results are in agreement with those published for nonverte-
brate GSTs [19]. Two elution concentrations were similarly
used by Kunze [20] for the purification of GSTs from porcine
liver. The originality of the method used in the present study
is the combination of two elution concentrations and the use
of two affinity columns mounted in series, which allowed the
GSTs to be separated into four distinct groups. Their analysis
by RP-HPLC showed that the first column, GSH–agarose,
binds seven isoforms, whereas the S-hexyl GSH column
allowed improvement of the purification yield only. In this
study, reduced GSH was used as affinity matrix competitor
for the two columns because all described GST isoforms have
good affinity for free GSH. Moreover, the GSH is nonin-
hibitory and may stabilize the enzyme [19], allowing further
purifications and assays. S-hexyl was used as a competitor
for the second column after elution with free GSH, no

CDNB activity was measurable in the recovered fractions.
Most of the GST affinity purifications reported [21,22] were
performed using only the GSH–agarose column and showed
arecoveryof% 50% of the initial CDNB activity. In the
present study, 75% of the total GST activity measured with
CDNB as a substrate was recovered in all the four affinity
fractions. When measuring the activity in the front fraction,
we observed that 25% of the GST activity was lost during the
charge step. Some GST isoforms may not be bound to the
two affinity matrices. Other types of substrate-linked mat-
rices are now investigated in the laboratory. Clark [19]
Table 2. Biochemical characteristics of R. decussatus GST isoforms. Results are expressed as means ± SD. Reverse phase HPLC retention time
(Rt) of subunits expressed in minutes; apparent molecular mass of each subunit expressed in kDa (see Fig. 4); GST specific activities (lmol substrate
per min per mg protein) of purified isoforms with CDNB and ETHA as substrates. ND, not detected.
GST isoforms 1-1 2-2 3-3 4-4 5-5 6-6 3-6
HPLC retention time (Rt) (min) 7.20 9.02 12.06 14.09 14.83 16.40 12.06/16.40
Subunit molecular mass (kDa) 26 24 24 25 25 23 24/23
Specific activity
(lmol substrateÆmin
)1
mg protein
)1
)
CDNB 38 ± 30 467 ± 41 290 ± 17 270 ± 35 86 ± 4 361 ± 21 91 ± 10.1
ETHA 59 ± 12 30 ± 3 15 ± 5 4.4 ± 2.4 ND 50 ± 10 0.8 ± 0.1
Table 3. Kinetic constants of purified GST isoforms (3-3; 5-5; 6-6; 3-6). V
max
and K
m
, k

cat
and ratio k
cat
/K
m
with GSH and CDNB as substrates.
Results are expressed as means ± SD.
Constant GST 3-3 GST 5-5 GST 6-6 GST 3-6
V
max (CDNB)
(lmolÆmin
)1
Æmg
)1
) 204.1 ± 4.2 242.1 ± 25.3 222.4 ± 7.9 200.2 ± 9.3
V
max (GSH)
(lmolÆmin
)1
Æmg
)1
) 181.6 ± 5.4 208.2 ± 16.3 199.6 ± 7.8 192.1 ± 10.4
K
m (CDNB)
(m
M
) 0.17 ± 0.02 1.65 ± 0.49 2.88 ± 0.37 0.86 ± 0.19
K
m (GSH)
(m

M
) 4.82 ± 0.33 0.37 ± 0.08 1.75 ± 0.43 2.20 ± 0.27
k
cat
(s
)1
) 214.3 ± 0.7 250.2 ± 6.4 234.4 ± 2.4 217.9 ± 4.8
k
cat
/K
m (CDNB)
(m
M
)1
Æs
)1
) 1244.97 151.72 81.34 252.69
k
cat
/K
m (GSH)
(m
M
)1
Æs
)1
) 44.417 672.95 133.82 99
Ó FEBS 2002 Purification of R. decussatus GST isoforms (Eur. J. Biochem. 269) 4363
reports that some GST isoforms are not linked to GSH
affinity matrices. The same phenomenon may occur in

R. decussatus. Moreover, some GSTs, particularly those
belonging to mammalian theta class, are not able to
metabolize CDNB and thus the activity of some GSTs
may be underestimated or even undetected when using this
assay.
The GST activities recorded for the four affinity fractions
varied according to the substrate used, the strongest activity
being obtained with CDNB and the lowest with ETHA.
ETHA activity, which was too low to be detected in the
original post-mitochondrial fraction, was detectable after
the separation of GST isoforms into several fractions.
The seven isoforms, separated by anion exchange chro-
matography, were partially characterized. K
m
, evaluated
using CDNB and GSH, gave significantly different results
for the four selected GST isoforms. K
m
using CDNB as a
substrate in nonvertebrate organisms, showed a range of
affinity between 0.05 and 1.8 m
M
[19]. This is in agreement
with K
m
values from R. decussatus. K
m
values for GSH
covered a wide range (0.3–2.9 m
M

) like those reported in the
literature for GST from nonvertebrate organisms (0.06–
2.7 m
M
). This allows one of the isoforms to catalyze the
conjugation in optimal conditions, whatever the concentra-
tion of endogenous GSH. This phenomenon is of great
interest as GSH is involved in many cellular processes,
especially in redox cycles [23,24]. The conjugation turnover
number, k
cat
, and the catalytic efficiency, k
cat/
K
m(substrate)
,
allowed us to distinguish the four GSTs tested. As few
studies gave such kinetics constants for GST and none for
GSTs from mollusc organisms, the comparison of the
kinetic parameters from R. decussatus GSTs remained
limited. Nevertheless, in the marsupial Antechinus stuartii,
Bolton and Ahokas [22] found k
cat
values for CDNB
rangingfrom40to290Æs
)1
. Prapanthadara et al.[25]
showed that the turnover of two GST isoforms called
GST-4 and GST-4c from Anopholes dirus were 149.85 and
18.69Æs

)1
for k
cat
, 172.24 and 62.30 m
M
)1
s
)1
for the ratio
k
cat/
K
m(GSH)
and 713.57 and 186.9 m
M
)1
s
)1
for the ratio
k
cat
/K
m(CDNB)
.Asregardsk
cat
, evaluated in R. decussatus,
our results are in good agreement with the data in the
literature; nevertheless the range of variation is narrower.
The same conclusion can be made for k
cat

/K
m(substrate)
.
Some of the commercial antibodies are very specific to
one class GST and are used for distinguishing GSTs from
a large range of organisms. In the present study, all
isoforms purified from R. decussatus were recognized by
rabbit antisera specific of mammalian pi-class GST.
Studies of aquatic species such as salmonids [26], Bufo
bufo [27] and M. edulis [28], showed that their GSTs
belong to the pi class. The GST isoforms 1-1 and 2-2
from R. decussatus were also recognized by rabbit antisera
Fig. 4. Immunoblotting of each anion exchange purified isoform (100 ng
per well). (A) Rabbit anti-pi class GST antisera (Chemicon). (B)
Rabbit anti-(alpha class GST) serum (Interchim). (C) Rabbit anti-(mu
class GST) serum (Interchim). MW, prestained low range SDS/PAGE
standards from Sigma (phosphorylase b, BSA, ovalbumin, carbonic
anhydrase, soybean trypsin inhibitor and lysozyme, respectively, 111,
73, 47.5, 33.9, 28.8 and 20.5 kDa) were not revealed by ECL but
visualized by superimposition of the X-ray film on the membrane.
Lane 1, purified 1-1 isoform; lane 2, purified 2-2 isoform; lane 3,
purified 3-3 isoform; lane 4, purified 4-4 isoform; lane 5, purified 5-5
isoform; lane 6, purified 6-6 isoform; lane 7, purified 3-6 isoform.
Table 4. Comparison between the N-terminal amino acid sequence of R. decussatus GST 4-4 isoform with other known GST sequences. Clonorchis
sinensis and F.hepaticaGST sequences are from the SWISS-PROT database; M. edulis binding protein and mu, alpha, pi GST classes consensus
sequences of mammals are from Fitzpatrick et al. [28].
Species N-terminal sequence Identity (%)
GST 44 Ruditapes decussatus
SELAYKKIRGLAQMN –
GSH binding protein Mytilus edulis

PTLGYWKIRGLAQPVR 60
GST Clonorchis sinensi
MAPVLGYWKIRGLAQPIR 60
GST Fasciola hepatica
MPAKLGY-KLRGLAQ 53
Consensus mu
PMTLGYWDIRGLAHAIR 47
Consensus alpha
AGKPVLHYFNARGRME 26
Consensus pi
PPYTVVYFPVRGGCAAMR 20
4364 P. Hoarau et al. (Eur. J. Biochem. 269) Ó FEBS 2002
specific for the mammalian alpha class GSTs. These two
GSTs, which are highly similar in their activities with
CDNB and ETHA, may be related to the mammalian
alpha class GST. The isoforms 3-3, 6-6, 3-6 and probably
also 5-5 may belong to the mammalian pi class according
to their immunological and biochemical properties.
However, the immunological and activity properties of
the 4-4 isoform are not sufficient to assign it to a GST
class; this isoform is recognized by the three rabbit
antisera (anti-alpha, anti-mu and anti-pi). It is now
accepted that mu, alpha and pi mammalian GST classes
have a common precursor the alpha/pi/mu class which
probably arose from theta gene duplication [29,30].
Some GST isoforms of the alpha/mu/pi precursor class
have been found in platyhelminths [29] and, more
recently, in the nematode Caenorhabditis elegans [30].
The primary amino acid sequence of subunit 4 from
R. decussatus showed 53% identity with GST from

F.hepatica (Fh 47) which is clearly an isoform of the
alpha/mu/pi precursor class. GST 4-4 shows similarity
with a conserved isoform of the common alpha/mu/pi
class; this hypothesis should be clarified by the determin-
ation of its mRNA sequence.
Immunological and biochemical properties are currently
used to support the classification of individual GST
isoforms [1]. In the case of the mollusk R. decussatus, direct
primary amino acid sequence analysis was also performed.
However, as in other invertebrates [28,31], the purified
isoforms of R. decussatus could not readily be assigned to
the present classification system which is based on mam-
malian GSTs.
ACKNOWLEDGEMENTS
P. Hoarau was supported by a joint fellowship from PACA Region and
SAFEGE-CETIIS, Aix-en-Provence. The authors thank Dr J. Trin-
chant, Laboratoire de Physiologie Ve
´
ge
´
tale, Universite
´
de Nice Sophia-
Antipolis for the use of an HPLC MERCK system.
REFERENCES
1. Mannervick, B. (1985) The isoenzyme of the glutathione
S-transferase. Adv. Enzymol. Relat. Areas. Mol. Biol. 57, 357–417.
2. Ketterer, B. (1988) Protective role of glutathione and glutathione
transferases in mutagenesis and carcinogenesis. Mutat. Res. 2,
343–361.

3. Daniel, V. (1993) Glutathione S-transferases: gene structure and
regulation of expression. CRCCrit.Rev.Biochem.Mol.Biol.28,
173–207.
4. Gallagher, E.P., Sheehy, K.M., Lame, M.W. & Segall, H.J. (2000)
In vitro kinetics of hepatic glutathione S-transferase conjugaison
in Largemouth Bass and Brown Bullheads. Environ. Toxicol.
Chem. 19, 319–326.
5. Gallagher, E.P. & Sheehy, K.M. (2000) Altered glutathione
S-transferase catalytic activities in female brown bullheads from a
contaminated central Florida lake. Mar. Environ. Res. 50, 399–
403.
6. Board, P.G., Baker, R.T., Chelvanayagam, G. & Jermin, L.S.
(1997) Zeta a novel class of glutathione transferases in a range of
species from plants to humans. Biochem. J. 328, 928–935.
7. Edwards, R., Dixon, D.P. & Walbot, V. (2000) Plant glutathione
S-transferases: enzymes with multiple functions in sickness and in
health. Trends. Plant. Sci. 5, 193–198.
8. Vuilleumier, S. (1997) Bacterial glutathione S-transferases. What
are they good for? J. Bacteriol. 179, 1431–1441.
9. Zhou, Z.H. & Syvanen, M. (1997) A complex glutathione
S-transferase gene family in the housefly Musca domestica. Mol.
Gen. Genet. 256, 187–194.
10. Satoh, K., Kitihara, A., Soma, Y., Inaba, Y., Hatayama, I. &
Sato, K. (1985) Purification, induction and distribution of pla-
cental glutathione transferase: a new marker enzyme for pre-
neoplastic cells in the rat chemical hepatocarcinogenesis. Proc.
Natl Acad. Sci. USA 82, 3964–3968.
11. Henson, K.L., Sheehy, K.M. & Gallagher, E.P. (2000) Con-
servation of a glutathione S-transferase in marine and freshwater
fish. Mar. Environ. Res. 50, 17–21.

12. Fitzpatrick, P.J. & Sheehan, D. (1993) Separation of multiple
forms of glutathione S-transferase from the blue mussel, Mytilus
edulis. Xenobiotica 23, 851–861.
13. Hoarau, P., Gnassia-Barelli, M., Rome
´
o, M. & Girard, J.P. (2001)
Differential induction of glutathione S-transferases in the clam
Ruditapes decussatus exposed to organic compounds. Environ.
Toxicol. Chem. 20, 523–529.
14. Habig, W.H., Pabst, M.J. & Jakobi, W.B. (1974) Glutathione
S-transferases: The first enzymatic step in mercapturic acid for-
mation. J. Biol. Chem. 249, 7130–7139.
15. Bradford, M. (1976) A rapid and sensitive method for the quan-
tification of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 72, 248–254.
16. Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage. Nature 227, 680–685.
17. Farrants, O., Meyer, D.J., Coles, B., Southan, C., Aitken, A.,
Johnson, P.J. & Ketterer, B. (1987) The separation of glutathione
transferase subunits by reverse-phase high-pressure liquide chro-
matography. Biochem. J. 245, 423–428.
18. Guinn, G. & Hendrix, D.L. (1985) Bracketing, a simple loading
technique that increases sample recovery, improves resolution, and
increases sensitivity in high performance liquid chromatography.
J. Chrom. 348, 123–129.
19. Clark, A.G. (1989) The comparative enzymology of the glutathi-
one S-transferases from non-vertebrate organisms. Comp. Bio-
chem. Physiol. 92, 419–446.
20. Kunze, T. (1997) Purification and characterisation of class alpha
andmuglutathioneS-transferases from porcine liver. Comp.

Biochem. Physiol. 116 B, 397–406.
21. Bolton, R.M. & Ahokas, J.T. (1997) Purification and character-
ization of hepatic glutathione transferase from an insectivorous
marsupial, the brown antechinus (Antechinus stuartii). Xenobiotica
27, 573–586.
22. Yuen, W.K. & Ho, J.W. (2001) Purification and characterization
of multiple glutathione S-transferase isozymes from Chironomidae
larvae. Comp. Biochem. Physiol. 129A, 631–640.
23. Canesi, L., Viarengo, A., Leonzio, C., Filippelli, M. & Gallo, G.
(1999) Heavy metals and glutathione metabolism in mussel tissues.
Aquat. Toxicol. 46, 67–76.
24. Lu, S.C. (2000) Regulation of glutathione synthesis. Curr. Top.
Cell Regul. 36, 95–116.
25. Prapanthadara, L., Promtet, N., Koottathep, S., Somboon, P. &
Ketterman, A.J. (2000) Isoenzymes of glutathione S-transferase
from the mosquito Anopheles dirus species B: the purification,
partial characterization and interaction with various insecticides.
Insect Biochem. Mol. Biol. 30, 395–403.
26. Dominey, R.J., Nimmo, I.A., Cronshaw, A.D. & Hayes, J.D.
(1991) The major glutathione S-transferase in Salmonid fish livers
is homologous to the mammalian pi class GST. Comp. Biochem.
Physiol. 100B, 93–98.
27. Di Ilio, C., Aceto, A., Bucciarelli, T., Dragani, B., Angelucci, S.,
Miranda, M., Poma, A., Amicarelli, F., Barra, D. & Frederici, G.
(1992) Glutathione transferase isoenzymes from Bufo bufo
embryos at an early development stage. Biochem. J. 283, 217–222.
28. Fitzpatrick, P.J., Krag, T.O.B., Hojrup, P. & Sheehan, D. (1995)
Characterization of glutathione S-transferase and a related
Ó FEBS 2002 Purification of R. decussatus GST isoforms (Eur. J. Biochem. 269) 4365
glutathione-binding protein from gill of the blue mussel Mytilus

edulis. Biochem. J. 305, 145–150.
29. Pemble, S.E. & Taylor, J.B. (1992) An evolutionary perspective on
glutathione S transferase inferred from class-theta glutathione
transferase cDNA sequences. Biochem. J. 287, 957–963.
30. Campbell, A.M., Teesdale-Spittle, P.H., Barrett, J., Liebau, E.,
Jefferies, J.R. & Brophy, P.M. (2001) A common class of
nematode glutathione S-transferase revealed by the theoretical
proteome of the model organism Caenorhabditis elegans. Comp.
Biochem. Physiol. 128B, 701–708.
31. Sheehan, D., Meade, G., Foley, V.M. & Dowd, C.A. (2001)
Structure, function and evolution of glutathione transferases:
implications for classification of non mammalian members of an
ancient enzyme superfamily. Biochem. J. 360, 1–16.
4366 P. Hoarau et al. (Eur. J. Biochem. 269) Ó FEBS 2002