CHARACTERIZATION AND PARTIAL-PURIFICATION OF A LEUCINE AMINOPEPTIDASEFROM FASCIOLA-HEPATICA

An aminopeptidase activity capable of hydrolyzing different aminomethy lcoumaring amino acids, but mainly leucine-7-amino-4-methylcoumarin (L eu-NHMc), was detected in deoxycholic soluble extracts from adult Fasc iola hepatica. The enzyme (EC 3.4.11.1) was partially purified by gel filtration and EAH-Sepharose affinity chromatography using bestatin as a ligand. Results obtained by gel filtration, direct fluorogenic subs trate analysis in polyacrylamide gel, and sodium dodecyl sulfate-polya crylamide gel electrophoresis suggest that in a native form the enzyme might be aggregated as a high molecular weight complex. By affinity c hromatography on concanavalin A-Sepharose, the enzyme did not bond to the column showing that it lacks mannose residues. The F. hepatica ami nopeptidase was characterized as a metalloproteinase based on its acti vation by Mn2+ and Mg2+, and its inhibition by EDTA, 1,10-phenanthroli ne, and bestatin. It has an optimal activity at a pH between 8.0 and 8 .5. Histochemical localization revealed strong leucine naphthylamidase activity at the cells lining the gut epithelium of the parasite.