Purification of Glutathione Dependent Enzymes in the Housefly

Abstract

The purification of DDT dehydrochlorinase and the glutathione S-transferases employing an affinity chromatography step and an isoelectric focussing step was carried out. A five step purification procedure was devised and found to be highly successful in obtaining purified fractions of the enzymes. DDT dehydrochlorinase was purified by 659 fold, the acidic glutathione S-transferase by 617 fold and the alkaline glutathione S-transferase by 602 fold relative to the initial specific activity of the homogenate. The acidic glutathione S-transferase was purified homogeneity while DDT dehydrochlorinase was purified to homogeneity. Attempts to separate DDT dehydroclrlorinase from the glutathione S-transferases were also carried out at the same time. It was not possible to separate these enzymes. The molecular weights studies of the subunits of the highly purified enzyme fractions were conducted. The subunits of the enzymes were similar in terms of molecular weights. The results obtained suggested that DDT dehydrochlorinase is a glutathione S-transferase and that these enzymes could have originated from a common precursor. Interstrain studies of DDT dehydrochlorinase and the glutathione S-transferases are in accordance with the previous findings of other workers. Different strains of houseflies contain different levels of these enzymes. The relationship between DDT dehydrochlorinase and one of the glutathione S-transferases was consistent throughout the strains investigated.