Isoenzymes of glutathione S-transferase from the mosquito Anopheles dirus species B: the purification, partial characterization and interaction with various insecticides

Previously we have purified and characterized a major glutathione S-transfe rase (GST) activity, GST-4a, from the Thai mosquito Anopheles dirus B, a mo del mosquito for study of anopheline malaria vectors [Prapanthadara, L. Koo ttathep, S., Promtet, N., Hemingway, J. and Ketterman, A.J. (1996) Insect B iochem. Mel. Biol. 26:3, 277-285]. In this report we have purified an isoen zyme, GST-4c, which has the greatest DDT-dehydrochlorinase activity. Three additional isoenzymes, GST-4b, GST-5 and GST-6, were also partially purifie d and characterized for comparison. All of the Anopheles GST isoenzymes pre ferred 1-chloro-2,4-dinitrobenzene (CDNB) as an electrophilic substrate. In kinetic studies with CDNB as an electrophilic substrate, the V-max of GST- 4c was 24.38 mu mole/min/mg which was seven-fold less than GST-4a. The two isoenzymes also possessed different K(m)s for CDNB and glutathione. Despite being only partially pure GST-4b had nearly a four-fold greater V-max for CDNB than GST-4c. In contrast, GST-4c possessed the greatest DDT-dehydrochl orinase specific activity among the purified insect GST isoenzymes and no a ctivity was detected for GST-5. Seven putative GST substrates used in this study were not utilized by An. dirus GSTs, although they were capable of in hibiting CDNB conjugating activity to different extents for the different i soenzymes. Bromosulfophthalein and ethacrynic acid were the most potent inh ibitors. The inhibition studies demonstrate different degrees of interactio n of the Art. dirus isoenzymes with various insecticides. The GSTs were inh ibited more readily by organochlorines and pyrethroids than by the phosphor othioates and carbamate. In a comparison between An. dirus and previous dat a from An. gambiae the two anopheline species possess a similar pattern of GST isoenzymes although the individual enzymes differ significantly at the functional level. The available data suggests there may be a minimum of thr ee GST classes in anopheline insects. (C) 2000 Elsevier Science Ltd. All ri ghts reserved.