Evolution of glutathione S-transferase subunits in culicidae and related nematocera: Electrophoretic and immunological evidence for conserved enzyme structure and expression

The antigenic relatedness and molecular weights of glutathione S-transferase (GST) protein subunits expressed by representative members of the Dipteran suborder Nematocera were analyzed by immunoblotting and affinity chromatography. Ten species within the genus Aedes expressed two subunits which cross-reacted with an antiserum developed against the GST-1b isozyme purified from Aedes aegypti. One of these two subunits showed no discernible molecular weight variability within the ten Aedes species. Its molecular weight (25,900 Da) was identical to the GST-1a subunit found in A. aegypti. The molecular weight of the other subunits varied among the ten species, but in all cases was larger than 25,900 Da. In ten species within the genus Anopheles (considered to be among the more primitive genera in the Culicidae), only one immunologically related GST subunit was expressed. Its molecular weight was also identical to the GST-1a subunit found in the Aedes species. Members of genera considered phylogenetically intermediate between Anopheles and Aedes had either one or two cross-reacting subunits. Representative species of families closely related to the Culicidae (the Chaoboridae, Chironomidae, Simuliidae, and Cecidomyiidae) also expressed immunologically related GST subunits. GST isozymes were partially purified from Anopheles freeborni and A aegypti adults, larvae, and ovaries using S-hexylglutathione affinity chromatography. Adult and larval Anopheles freeborni expressed not only the immunologically related subunit, but an additional, immunologically unrelated subunit. In fully developed ovaries of both species, however, only subunits immunologically related to GST-1b were expressed. These results suggest that there is an immunologically conserved domain shared among specific GST isozymes within the Nemotocea. The conserved nature of these GST subunits, their correlation with oocyte development, and their localization within ovary tissue, suggests that they share a conserved in vivo function during oocyte maturation. In addition, the conserved antibody binding domain within these subunits apparently has been duplicated in several species of Culicidae.