Studies of esterase 6 in Drosophila melanogaster. XIII. Purification and characterization of the two major isozymes.
Esterase-6 (EST 6; carboxylic-ester hydrolase; EC 3.1.1.1) from Drosophila melanogaster was purified to homogenity. Purified enzyme occurs as two closely moving isozymes, slow (EST 6S) and fast (EST 6F), on native polyacrylamide gel electrophoresis. Except for slight differences in their mobility, the two isozymes share similar molecular and catalytic properties. Both isozymes are glycoproteins and have an apparent molecular weight of 62,000 to 65,000 as judged by analytical gel filtration and sodium dodecyl sulfate (SDS) electrophoresis. They have identical mobility on SDS-polyacrylamide gels and an isoelectric point of 4.5. Each isozyme has a single active catalytic site as confirmed by titration with 0,0-diethyl-p-nitrophenyl phosphate (Paraoxon). We conclude that EST 6 is a monomeric enzyme. The amino acid composition of the two isozymes is very similar and both variants lack half-cystine residues. The low pI of the enzyme is due in part to a relatively high proportion of glutamic and aspartic amino acid residues. Characterization of the kinetic parameters of the isozymes using beta-naphthyl and p-nitrophenyl esters revealed no statistically significant differences in catalytic efficiency. There is, however, a suggestion that the two isozymes may differ in their substrate specificity.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight
D000596:Amino Acids; D029721:Drosophila Proteins; D007527:Isoenzymes; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; C468266:Est-6 protein, Drosophila