Glutathione-Dependent Enzymes of Houseflies

Abstract

1. DDT-dehydrochlorinase from DDT-susceptible houseflies has been partially purified by the following procedures: pH 5.0 fractionation,ammonium sulphate precipitation, DEAE-cellulose batchwise operation, hydroxylapatite column chromatography and Sephadex gel column chromatography. Glutathione added to enzyme preparations at the last three purification steps improves yield 2- to 3-fold. Gel electrophoresis of the final, highly active sample provides evidence of four major protein components. Sedimentation-velocity experiments show a single component of molecular weight about 40,000. 2. Comparative studies of DDT-dehydrochlorinase and GSH S-aryltransferase at all stages of the purification of DDT-dehydrochlorinase indicate some degree of similarity between these two enzymes. Evidence is also presented for the existence of at least three GSH S-transferases catalyzing the conjugation of GSH with CDNB. DDT-dehydrochlorinase is more closely associated with one of these transferases. 3. Isoelectric focusing of DDT-dehydrochlorinase gives between three to seven isoelectric forms of the enzyme but these are not reproducibly dependent on the duration of focusing. Isoelectric focusing of GSH S-aryltransferase from a crude enzyme preparation gives one major activity peak isoelectric at about pH 4.2. Focusing of a partially purified DDT-dehydrochlorinase preparation gives multiple GSH S-aryltransferase peaks. 4. Estimation of the molecular weight of DDT-dehydrochlorinase from different enzyme preparations by thin-layer gel filtration, Sephadex gel column chromatography, sedimentation-velocity experiments and sucrose gradient density centrifugation gives a value of about 40,000. Carrying out the experiments under the conditions favouring the association of DDT-dehydrochlorinase monomers as indicated by Dinamarca et al (1969, 1971) met with no success. 5. DDT-dehydrochlorinase is unstable to frothing but addition of GSH overcomes loss in activity GSH S-aryltransferase is stable.e to frothing.