BIOCHEMICAL-ANALYSIS OF RECOMBINANT GLUTATHIONE-S-TRANSFERASE OF FASCIOLA-HEPATICA

Four cDNAs encoding GST (rGST1, rGST7, rGST47 and rGST51) of Fasciola hepatica were expressed in Escherichia coli and the rGST proteins puri fied for biochemical analyses. The rGST proteins are 95% pure as indic ated by Coomassie staining of proteins separated by SDS-PAGE. Molecula r sieving by HPLC infers that, like the native protein, the rGST prote ins form homodimers under non-denaturing conditions. The rGST proteins are recognised by antisera raised to the native GST of F. hepatica. A ll four rGST proteins from F. hepatica actively conjugate glutathione to the universal substrate, 1-chloro-2,4-dinitrobenzene. The activity of the rGSTs was also measured for substrates which have been shown to have partial specificity for the Alpha, Mu or Pi classes of mammalian GSTs (trans-4-phenyl-3-buten-2-one, ethacrynic acid), for substrates known to be products of lipid peroxidation (trans-2-nonenal, trans,tra ns-2,4-decadienal) and for epoxy-3-(p-nitrophenoxy)-propane (EPNP), a known substrate for the theta class of GST. No rGST were active with E PNP. rGST47 and 51 showed activity with the other four substrates. rGS T7 was active with three substrates whereas rGST1 showed relatively lo w activity with all substrates except trans, trans-2,4-decadienal. The sensitivity of the rGST activity to inhibition by the GST inhibitors triphenyltin chloride and bromosulphophthalein also varied among the r GSTs with rGST1 showing a 800-fold difference in sensitivity between t he inhibitors. These results show that F. hepatica expresses a family of GST isoenzymes which exhibit unique substrate and inhibitor profile s.