Purification and properties of esterase-4 from Drosophila mojavensis

Abstract

One of the allozymes of esterase-4 from Drosophila mojavensis was purified to homogeneity. The purification was achieved by means of ammonium sulfate precipitation, anion-exchange chromatography, gel filtration, anion-exchange HPLC and gel-filtration HPLC. The resulting preparation was apparently homogeneous in SDS-polyacrylamide gel electrophoresis. The enzyme is most probably a dimer with a subunit molecular weight between 62 000 and 64 000. The purified enzyme showed maximal activity towards short-chain naphthyl esters, with a preference for α-maphthyl esters. Esterase-4 is tentatively classified as an acetyl esterase (EC 3.1.1.6) on the basis of inhibition experiments. The effects of four inhibitors were determined. Esterase-4 run on a non-denaturing gel was not inhibited by diisopropyl fluorophosphate, in contrast with other esterases present in a crude larval extract of D. mojavensis. In solution, the enzyme was only inhibited by a large excess of diisopropyl fluorophosphate. With antibodies raised against purified esterase-4, esterases immunologically related to esterase-4 were demonstrated by double-immunodiffusion in five closely related Drosophila species.