Purification and Biochemical Characterization of Glutathione S-Transferases inBactrocera minax(Diptera: Tephritidae)

Abstract

Glutathione S-transferases (GSTs) were purified from 3 developmental stages of Bactrocera minax through glutathione-agarose affinity chromatography, and characterized subsequently using the Michaelis-Menten kinetics toward the artificial substrates 1-chloro-2, 4-dinitrobenzene (CDNB) and reduced glutathione (GSH), respectively. Compared to the counterparts of third instar larva and adult, the highest specific activity of the purified GSTs towards CDNB was observed in the pupae. Although the specific activities of purified enzymes varied among 3 developmental stages, the purification yields were similar. SDS-PAGE revealed only one band at 23 kDa for all 3 stages. GSTs of the adults exhibited the highest Km value towards CDNB, while for GSH the pupae possessed the highest Km. The optimum temperature and pH for CDNB conjugation of the 3 stages were 37 °C and 7.5, respectively. Inhibition kinetics showed that ethacrynic acid, bromosulfalein, diethyl maleate, tetraethylthiuram disulfide and curcumin possessed excellent inhibitory effects on purified GSTs in B. minax. Moreover, the pupa showed the highest catalytic capability based on Vmax values for both the CDNB and GSH, which may suggest a potentially higher GSTs detoxification ability in the pupal stage than the other 2 developmental stages.