Glutathione S-transferase 1 and 2 in susceptible and insecticide resistant Aedes aegypti

Abstract

Glutathione S-transferase enzymes (GST) were characterized in permethrin and in DDT resistant and susceptible strains of Aedes aegypti. The predominant GST, designated GST-1, was purified from susceptible and resistant larvae using S-hexyl-glutathione affinity chromatography and anion-exchange chromatography. Rabbit antiserum directed against the 26.8-kDa GST-1 subunit purified from the susceptible strain was specific for a 26.8-kDa subunit in the resistant strain. There was no significant difference in enzyme kinetic constants ( K m and V max) between GST-1 from susceptible and resistant strains. GST-1 enzyme activity varied during larval development in both resistant and susceptible strains, but was approximately 1.7 times greater in resistant larvae and pupae throughout development. The GST-1 amount (measured using a radioimmunoassay) also varied during development, and was generally higher in resistant larvae and pupae. GST-1 enzyme activity measured each day during the 6 days of larval development was significantly correlated with GST-1 protein amount on each of the previous days, suggesting that an inactive enzyme form is converted to the active form approximately 24 hr after it is synthesized. Another GST enzyme, GST-2, was also found in resistant and susceptible A. aegypti larvea. GST-2 was partially purified from resistant larvae using S-2,4-dinitrophenylglutathione affinity chromatography and anion-exchange chromatography. GST-2 has a molecular mass of 28 kDa, and an isoelectric point of less than 5.0. GST-2 was not cross-reactive with GST-1 antiserum. GST-2 enzyme activity varied during the development of both susceptible and resistant larvae, but the pattern was different than that for GST-1. Throughout development, GST-2 enzyme activity was substantially higher (approximately eight-fold) in resistant strains of A. aegypti compared to susceptible strains.