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
purification was performed using anion exchange chroma-
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
Kinetic parameters were studied. Results showed that
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-
(3-3, 6-6 and 3-6) seem to be closely related to the pi-class
GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera
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 184.108.40.206) bind
lipophilic nonsubstrate ligands and compounds such as
heme and bilirubin , and are implicated in the biosynthe-
sis of prostaglandins . 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 .
The diversity of compounds metabolized by GSTs results
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 . 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 .
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
a delta class GST . 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 ) and in
fish , 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 
reported the purification of four GST isoforms in the
digestive gland and gills of the blue mussel Mytilus edulis.
In previous work , 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,
was performed by studying the kinetic parameters (Vmax,
Kmwith the commonly used substrate CDNB and GSH)
and by performing immunoblotting reactions. RP-HPLC
analysis was used to identify the different constitutive
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,
Abbreviations: GST, glutathione S-transferase; GSH, reduced gluta-
thione; CDNB, 1-chloro-2, 4-dinitrobenzene; ETHA, ethacrynic acid.
Enzyme: glutathione S-transferases (GST; EC 220.127.116.11).
(Received 14 May 2002, revised 11 July 2002, accepted 25 July 2002)
Eur. J. Biochem. 269, 4359–4366 (2002) ? FEBS 2002 doi:10.1046/j.1432-1033.2002.03141.x
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? FEBS 2002