Purification and Characterization of Glutathione S-Transferases from the German Cockroach, Blattella germanica (L.)

Abstract

Glutathione S-transferases (GSTs) were purified and isolated from male adults of the German cockroach, Blattella germanica, by using a three-step procedure involving ammonium sulfate fractionation, glutathione-agarose affinity chromatography, and native polyacrylamide gel electrophoresis. Adult males possessed three GST isozymes, namely GST-1, GST-2, and GST-3, all of which were homodimers with a subunit molecular weight of 25,500. The affinity-purified GSTs had an apparent Km value of 0.16 mM and a Vmax of 78.4 μmol/min/mg protein (toward CDNB). The purified transferases were inhibited by dicumarol, ellagic acid, quercetin, ethacrynic acid, and triphenyltin chloride, showing I50 values (toward CDNB) of 1.6 μM, 7.3 μM, 2.0 μM, 0.35 μM, and 40 nM, respectively. These enzymes were particularly active toward CDNB compared with DCNB and p-nitrophenyl acetate, but had no activity toward 4-nitrobenzyl chloride, 1,2-epoxy-3-(p-nitrophenoxy) propane, trans-4-phenyl-3-buten-2-one, and ethacrynic acid. N-terminal amino acid sequence analysis of GST-1 indicated that the residues Tyr-4 and Leu-6 were highly conserved among 19 other GST sequences from various insect species. Immunological studies with enzyme-linked immunosorbent assay revealed that all three GST isozymes exhibited serum IgE antibody binding activity, indicating the allergenic nature of the enzymes.