Partial purification and characterization of midgut leucyl aminopeptidase of Morimus funereus (Coleoptera : Cerambycidae) larvae

Abstract

Exopeptidases of Morimus funereus larvae were partially purified and characterized. Specific leucyl aminopeptidase (LAP) activity was increased eight-fold by gel filtration of the crude midgut extract. The partially purified LAP had a molecular mass greater than 100 kDa with pH optima from 7.0-9.0 and no strict substrate specificity. M. funereus LAP preferentially hydrolyzed p-nitroanilides with hydrophobic amino acids in the active site, with a K. for leucine-p-nitroanilide of 0.21 mM. Zymogram analysis of an electropherogram obtained by native polyacrylamide gel electrophoresis revealed four enzymatically active proteinases using leucine-p-nitroanilide and methionine-p-nitroanilide as substrates and two enzymatically active proteinases using lysine-p-nitroanilide as a substrate. Although the optimal temperature of LAP activity was 40 T, the enzyme was active over a broad temperature range from 2 to 60 T. Among a number of inhibitors tested, heavy metals and 1,10-phenanthroline completely inhibited the enzyme, while methanol, ethanol and EGTA stimulated somewhat LAP activity. (C) 2002 Elsevier Science Inc. All rights reserved.