A study of housefly esterases by means of a sensitive colorimetric method

Abstract

Esterases in organophosphate susceptible and resistant houseflies were studied by means of sensitive Gomori method. In susceptible flies the esterase activity to α-naphthylacetate was found to be mainly due to the cholinesterase and an ali-esterase identical with that responsible for most of the activity to methylbutyrate. The latter enzyme is much less active to β-naphthylacetate than to α-naphthylacetate.

The Km values of the cholinesterase for α- and β-naphthylacetate were 1·0 × 10−4 and 2·3 × 10−4 (M) respectively. The ali-esterase had a Km of approximately 10−4 (M) for α-naphthylacetate.

The activity to α-naphthylacetate of the cholinesterase was strongly, that of the ali-esterase only weakly pH-dependent in the range from pH 6 to 8. Both enzymes were more active at higher pH.

Eserine and diazoxon were used in inhibition experiments, acetylcholine and methylbutyrate in experiments on substrate competition. The Km value of the cholinesterase for acetylcholine was calculated as approximately 10−5 (M).

The addition of heat-inactivated homogenate strongly enhanced the ali-esterase activity to α-naphthylacetate. It did so much more at low than at high concentrations of the active homogenate and thus caused the disappearance of the disproportionality initially observed between enzyme concentration and activity. This activation phenomenon was, to a lesser extent, also observed with β-naphthylacetate. The ali-esterase activity to α-naphthylacetate in homogenates of organophosphate resistant strains was only about 15 per cent of that found in homogenates of organophosphate susceptible strains. No significant differences between the activities in susceptible and resistant strains were found if β-naphthylacetate and indophenylacetate were used as substrates.

Agar-gel electrophoresis of the supernatants obtained by high-speed centrifugation of homogenates proved the presence of about seven electrophoretically different esterases that occurred in more or less strain-specific patterns.