Purificação e Caracterização de uma esterase específica da fase larval em duas espécies de Drosophila do grupo repleta

Abstract

Esterases form a group of biochemically important enzymes in insects. Although they have been extensively studied, it is still unclear the physiological role of most of them. In this work, the purification and biochemical characterization of EST-4 (larval esterase) from two species of the Drosophila repleta group, Drosophila mulleri and Drosophila arizonae, was performed in order to establish comparative parameters between these enzymes in these species and to check for biochemical patterns that allow a functional differentiation of enzymes in these species. Both EST-4 were purified by molecular weight exclusion and ion exchange chromatographies and were biochemically analyzed, which revealed that, in D. mulleri, this enzyme has optimal activity at temperatures from 40 to 45°C an d pH 7.5, maintaining stability in alkaline pH in the range of pH 8.0 to 10. In this species it was classified as serine esterase, since its activity was inhibited in the presence of PMSF. The effect of metal ions revealed that no ion negatively modulated EST-4, and that iron had the most positive modulating effect exerted on this enzyme activity. For the EST-4 of D. arizonae, and enzyme activity showed the same optimum temperature (40°C) and pH (8.0), maintainin g stability in acidic pH in the pH range from 5.5 to 6.5 and was also classified as a serine esterase. The metal ions test demonstrated that Fe+2 had the opposite effect that was found in this enzyme of D. mulleri, serving as a negative modulator. The Al+3 inhibited the enzyme activity almost 100% and Na+ and Cu+2 had a positive modulation effect. Kinetic studies were performed investigating the influence of substrate Ï -nitrophenyl acetate in enzyme activity, and despite D. mulleri`s EST-4 had a higher affinity for this substrate, D. arizonae`s showed higher Vmax and catalytic efficiency in optimal conditions of reaction. Therefore, it can inferred, based on the characterization and kinetic data, that the EST-4 from D. mulleri and from D. arizonae presented biochemical and functional differentiation as they showed different properties compared to the substrate used, and slight differences with respect to the characterization tests.