Purification and partial characterization of a glutathione S-transferase from the two-spotted spider mite, Tetranychus urticae

Abstract

Glutathione S -transferases (GSTs) are known to catalyze conjugations by facilitating the nucleophilic attack of the sulfhydryl group of endogenous reduced glutathione on electrophilic centers of a vast range of xenobiotic compounds, including insecticides and acaricides. Elevated levels of GSTs in the two-spotted spider mite, Tetranychus urticae Koch, have recently been associated with resistance to acaricides such as abamectin [Pestic. Biochem. Physiol. 72 (2002) 111]. GSTs from acaricide susceptible and resistant strains of T. urticae were purified by glutathione–agarose affinity chromatography and characterized by their Michaelis–Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene and monochlorobimane. The inhibitory potential of azocyclotin, dicumarol, and plumbagin was low (IC 50 values > 100 μM), whereas ethacrynic acid was much more effective, exhibiting an IC 50 value of 4.5 μM. GST activity is highest in 2–4-day-old female adults and dropped considerably with progressing age. Furthermore, molecular characteristics were determined for the first time of a GST from T. urticae , such as molecular weight (SDS–PAGE) and N-terminal amino acid sequencing (Edman degradation). Glutathione–agarose affinity purified GST from T. urticae strain WI has a molecular weight of 22.1 kDa. N-terminal amino acid sequencing revealed a homogeneity of ≈50% to insect GSTs closely related to insect class I GSTs (similar to mammalian Delta class GSTs).