Studies on glutathione transferase of cowpea storage bruchid, Callosobrochus maculatus F

Insect glutathione transferases have been implicated in insecticides and herbicides metabolism. In this study, glutathione transferase was purified to apparent homogeneity from cowpea storage bruchid (Callosobruchusmaculatus) by anion exchange chromatography of DEAE-Sephacel and affinity chromatography of glutathione-Sepharose 4B. The purified enzyme is slightly acidic, pI 5.5, with a molecular weight of 48.5 ± 4 kDa and is composed of subunit molecular weight of 25 kDa. It exhibited an optimum pH and temperature of 8.0 and 40 °C, respectively. Kinetic data gathered from substrate specificity using 1-chloro-2,4-dintrobenzene,7-chloro-4-nitrobenzene-2-oxa-1,3-diazole, p-nitrophenyl acetate, ethacrynic acid, 1,2-dichloro-4-nitrobenzene and paranitrophenychloride and inhibition studies (cibacron blue, bromosulphophthalein, hematin, oxidized glutathione and S-hexylglutathione) of the enzyme showed that is of sigma class. The GST poorly conjugates 4-hyroxylnonenal, a product of lipid peroxidation, and cumene hydroperoxide and may not be involved in oxidative stress protection. Steady state kinetics and product inhibition studies were consistent with a random sequential detoxification mechanism. The interaction between the GST and the insecticides, Fenvalerate and Cypermethrin, was investigated by inhibition studies, circular dichroism and competitive inhibition spectroscopy. It demonstrated that enzyme was inhibited at a concentration that induced some minor changes in the secondary and tertiary structures of the enzyme consequently decrease the detoxification ability and enzyme stability but might not unfold it.

Fluorescence spectra of Callosobrochus maculatus GST on addition of different concentration 0–16 μM of (a) Fernevelate and (b) Cypermethrin. Fluorescence intensity was scanned from 300 to 400 nm with an excitation at 275 nm. Values are means of three independent replicates.Figure optionsDownload as PowerPoint slideHighlights
► Glutathione S-transferase was isolated from cowpea storage bruchid (Callosobruchusmaculatus).
► Kinetic properties and substrate specificity study of the enzyme showed that is of sigma class.
► The GST is not involved in oxidative stress protection.
► Pesticides alters the secondary and tertiary structures of the enzyme.