Purification and partial characterization of seven glutathione S-transferase isoforms from the clam Ruditapes decussatus.
This paper deals with the purification and the partial characterization 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 chromatography. 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 Vmax 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, 6-6 and 3-6) seem to be closely related to the pi-class GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera 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/m micro /pi GST from Fasciola hepatica.
UMR 1112 INRA-UNSA, Laboratoire Réponse des Organismes aux Stress Environnementaux, Faculté des Sciences, Université de Nice-Sophia Antipolis, Nice, France. hoarau@unice.fr
Pascal
P
Hoarau
Ginette
G
Garello
Mauricette
M
Gnassia-Barelli
Michèle
M
Romeo
Jean-Pierre
JP
Girard
D000818:Animals; D049872:Bivalvia / Q000201:enzymology*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004137:Dinitrochlorobenzene / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017385:Sequence Homology; D013379:Substrate Specificity
D004137:Dinitrochlorobenzene; D007527:Isoenzymes; D010446:Peptide Fragments; D005982:Glutathione Transferase; D005978:Glutathione