Paragonimus westermani: A cytosolic glutathione S-transferase of a sigma-class in adult stage

Paragonimus westermani: A cytosolic glutathione S-transferase of a sigma-cl ass in adult stage. Experimental Parasitology 94, 180-189. We purified cyto solic glutathione S-transferase (GST) of adult Paragonimus westermani monit oring its activity with 1-chloro-2-4-dinitrobenzene (CDNB). The enzyme was purified 18.4-fold to electrophoretic homogeneity with 21 % recovery rate t hrough a three-step procedure. The purified enzyme (Pw28GST) has a subunit molecular weight of 28 kDa with an isoelectric point at 4.6. Monoclonal ant ibody (anti-Pw28GST) against Pw28GST did not cross-react with GSTs from oth er helminths. cDNA library was constructed in lambda ZAP II bacteriophage a nd screened with anti-Pw28GST. The corresponding gene containing a single o pen reading frame of 804 bp encoded 211 amino acids. The predicted amino ac id sequence exhibited a higher homology with catalytic domain near N-termin us of class sigma GSTs (58%) than with schistosome 28-kDa GSTs (45-41%) or with class sigma GSTs themselves (33-31%). The sequence contained both Tyr- 6 and Tyr-10 that are highly conserved in mammalian and helminth GSTs. The apparent K-m value of a recombinant enzyme was 0.78 mM. Both native and rec ombinant enzymes showed the highest activity against CDNB, relatively weak activity against ethacrynic acid and reactive carbonyls, and no activity ag ainst epoxy-3-(p-nitrophenoxy)-propane. The activities were inhibited by br omosulfophthalein, cibacron blue, and albendazole, but not by praziquantel. These findings indicate that adult P. westermani has a class sigma GST. (C ) 2000 Academic Press.