Characterization of the major form of glutathione transferase in the mosquito Anopheles dirus A.

Abstract

Glutathione S-transferases (GSTs; EC 2.5.1.18) are a multigene family of dimeric multifunctional proteins that play a central role in detoxication of xenobiotic compounds including drugs, herbicides and insecticides (1 -3). The enzymes catalyse the nucleophilic attack of reduced glutathione (GSH) on the electrophilic centres of lipophilic compounds (e.g. halogenated aromatic and aliphatic). The GSTs in insects are of interest because of their potential role in insecticide resistance. Several insecticideresistant strains of housefly have been reported to have elevated GST activity in crude extracts (4-6). It has been shown that some GSTs from housefly possessed DDTdehydrochlorinase activity and thus that DDTdehydrochlorinase was a GST (7). The purification was performed by using sequential column chromatography on Q-sepharose, S-hexylglutathione agarose, hydroxylapatite and phenyl sepharose. An overloaded SDS-PAGE (9 pg of protein) showed only a single band present (not shown). When loading less protein only a single band was observed. From SDS-PAGE it was calculated the GST 4a had a subunit M, of 25.0 k 0.26 (S.E.; n=3). The activity with 1 mM CDNB and 10 mM GSH was measured at 340 nm in 0.1 M phosphate buffer, pH 6.5, at 22°C. This is the standard assay for GST activity during the purification procedure. The activity with DCNB was measured at 340 nm in the presence of 10 mM GSH and 0.95 mM DCNB. With all other substrates, the enzyme activity was measured as previously described (8). Glutathione peroxidase activity with cumene hydroperoxide as substrate was determined (9). Double-reciprocal plots of the initial-rate pattern for An. dirus A GST 4a with GSH and CDNB as substrates were linear and converging, indicating a sequential mechanism in which ternary complexes were formed (10). A k , for GSH and CDNB was calculated to be 150 set ' with a K,,, of 0.87 mM and a K,,, CDNs of 0.21 mM. In addition to CDNB, nine other possible GST substrates were examined for interaction with GST 4a. Of all the other compounds tested as substrates only cumene hydroperoxide and DCNB were utilised by the enzyme (TABLE 1). Therefore of ethacrynic acid, cumene hydroperoxide and p-nitrophenethyl bromide which are standard substrates for vertebrate Pi, Alpha and Theta class GSTs respectively, only the Alpha class substrate was utilised. However, several of the compounds interacted with GST 4a as observed by their inhibition of CDNB activity. The inhibition effect of these substrates on CDNB activity was studied using the same concentration of these compounds as was used for their substrate studies. Only 1,2epoxy-3-@-nitrophenoxy)propane and trans4-phenyl-3-buten2-one had no interaction detectable with GST 4a, the two being substrates for Theta and Mu class GSTs. The remaining compounds had different degrees of inhibition as shown in TABLE 1. IC, inhibition constants were determined for cibacron blue as 0.061 f 0.006 pM, for bromosulphophthalein as TABLE 1. Interaction of An. dims A GST 4a with possible GST substrates.