Purification and characterisation of glutathione S-transferases from the freshwater clam Corbicula fluminea (Müller).
Glutathione S-transferases (GSTs) are involved in the phase II detoxification metabolism. To provide a molecular basis for their use as biomarkers of pollution, cytosolic GSTs from the freshwater clam Corbicula fluminea have been purified by glutathione-Sepharose affinity chromatography, anion-exchange chromatography (AEC) and reversed-phase (RP) HPLC. SDS-PAGE of visceral mass (VM) affinity-purified extracts revealed four subunits with apparent molecular masses (MW) of 30.2, 29.2, 28.5 and 27.2 kDa. Analysis by non-denaturing PAGE revealed three acidic dimeric proteins with apparent MW of 64, 55 and 45 kDa, named GSTc1, GSTc2 and GSTc3, respectively, based on their elution order by AEC. Only GSTc2 and GSTc3 exhibited GST activity towards 1-chloro-2,4-dinitrobenzene. A tissue-specific subunit pattern was obtained by RP-HPLC of affinity-purified extracts from VM and gills (GI): three major peaks were resolved, one of which was common to both tissues. MW of each VM subunit was determined by electrospray ionisation-mass spectrometry: 23602+/-1 Da for the major subunit and 23289+/-1 Da for the minor ones. Immunoblot analysis revealed all subunits from both tissues were related to the Pi-class GSTs. In addition, minor VM subunits were slightly related to the Mu-class ones. The interest of such molecular studies in biomonitoring programs is discussed.
Vidal
Mary-Laure
ML
Département de Toxicologie Biochimique, Laboratoire de Physico-Toxicochimie des Systèmes Naturels (LPTC), UPRES-A 5472 CNRS, Université Bordeaux 1, Avenue des Facultés, 33405, Talence, France. mlvidal@voila.fr
Rouimi
Patrick
P
Debrauwer
Laurent
L
Narbonne
Jean-François
JF
D000818:Animals; D049872:Bivalvia / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D005618:Fresh Water; D005982:Glutathione Transferase / Q000145:classification / Q000276:immunology / Q000302:isolation & purification*; D007526:Isoelectric Point; D008970:Molecular Weight; D021122:Protein Subunits
D021122:Protein Subunits; D005982:Glutathione Transferase