Biochemical characterization of glutathion-S-transferase and effects of lead acetate in Gammarus pulex

Abstract

Glutathione transferases (GST s; EC. 2.5.1.18) are a family of multifunctional proteins that catalyze the conjugation of GSH to the electrophilic centre of a large number of active molecules. A great number of drugs, halogenated hydrocarbons, epoxides and insectisides react with the sulphydryl group of glutathione. The reaction may be catalyzed by one or several glutathione trasferases, and it is therefore considered that a main function of these proteins is to aid detoxification of various xenobiotics. In the reactions catalyzed by these transferases, the sulfur atom of glutathione (GSH) provides electrons for nucleic attack on or reduction of the second, electrophilic substrate. A glutathione conjugate thus formed may be excreted as such or hydrolyzed to an S-(substituent)-cysteine derivative. The cysteine derivative can subsequently be N-acetylated to yield a mercapturic acid (mercaptate), which is a classical excretion product of xenobiotics. Alternatively, The cysteine moiety is cleaved at the C-S bond with elimination of pyruvate and NH3 and conversion of the S-substituent to a corresponding mercaptan. By this transformation, the sulfur of the new metabolite is the only remainder of the GSH molecule. The thiol group may be blocked by glucuronosylation or methylation and the resulting thioglucuronide or methylthio group, have also been identified as excretion products. These new sulfur containing end products of xenobiotics are important major metabolites arising from conjugation with glutathione. Thus, the importance of GSH transferase activity, is even Greater than realized in the past. In this study, optimum pH for glutathione-S-Transferase in Gammarus pulex, an invertebrata that is a member of the nourishment chain in aquatic environments and increasingly Gains importance as a test organism in environmental toxicology, is determined, and the activities for the same enzyme are assayed in different substrate concentrations. We analyzed them by Michealis-Menten and Lineweaver-Burk plots. Furthermore the effect of lead acetate on enzyme activity is investigated. GST was exposed to lead acetate EC50concentrations in order to get the changes of the GST activity after 4, 8, 16, 32 and 64 hours. As a result, in comparison with the control Group, an important decrease in GST activity, was observed. The inhibition of the GST activity in aquatic macro-invertebrates is in general comparable with that in rat liver.