D. melanogaster glutathione transferases E6 and E7 (DmGSTE6 and DmGSTE7) were successfully cloned, purified, and biochemically characterized. The recombinant proteins were readily purified using the combination of both anionic and BSP/GSH-agarose affinity chromatography. Although both GSTs have significant identity in their amino acid sequence, each enzyme displayed unique biochemical characteristics. Both recombinant proteins were only active toward 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and p-nitrobenzyl chloride (p-NBC) with significant difference in catalytic activities. The findings have shown that neither GSTE6 nor GSTE7 was able to counter oxidative stress. Comparatively, GSTE7 was a more efficient enzyme at turning over DCNB and p-NBC, based on its kcat/Km values which were of 0.183 and 2.25 min−1 mM−1, respectively. Thin-layer chromatography analysis showed that both isoforms were not able to conjugate several tested insecticides. The inhibition kinetics of natural products and dyes toward GSTs in vitro revealed that phenol red possessed inhibition effects only on GSTE6 while rose bengal and cardiogreen inhibit significantly on both GSTE6 and GSTE7. In contrast, methylene blue dye and trans-chalcone have been shown to stimulate GSTE7 activity toward CDNB.
Part of the book: Drosophila melanogaster