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

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 fractionswere thus recovered. Further purification was performed using anion exchange chroma-tography. Seven fractions,whichpresent aGSTactivitywith 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, were collected and analyzed by RP-HPLC | Eur. J. Biochem. 269 4359-4366 2002 FEBS 2002 doi Purification and partial characterization of seven glutathione 5-transferase isoforms from the clam Ruditapes decussatus Pascal Hoarau Ginette Garello Mauricette Gnassia-Barelli Michele Romeo and Jean-Pierre Girard UMR 1112 INRA-UNSA Laboratoire Reponse des Organismes aux Stress Environnementaux Faculte des Sciences Universite de Nice-Sophia Antipolis Nice France 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 mp pi GST from Fasciola hepatica. Keywords clam glutathione S-transferase immunology kinetics N-terminal