Characterization and partial purification of a leucine aminopeptidase from Fasciola hepatica.
An aminopeptidase activity capable of hydrolyzing different aminomethylcoumarin amino acids, but mainly leucine-7-amino-4-methylcoumarin (Leu-NHMc), was detected in deoxycholic soluble extracts from adult Fasciola 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 substrate analysis in polyacrylamide gel, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that in a native form the enzyme might be aggregated as a high molecular weight complex. By affinity chromatography on concanavalin A Sepharose, the enzyme did not bond to the column showing that it lacks mannose residues. The F. hepatica aminopeptidase was characterized as a metalloproteinase based on its activation by Mn2+ and Mg2+, and its inhibition by EDTA, 1,10-phenanthroline, 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.
Acosta
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D
Unidad de Biología Parasitaria, Instituto de Higiene, Montevideo, Uruguay.
Goñi
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F
Carmona
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C
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003374:Coumarins / Q000378:metabolism; D003840:Deoxycholic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005210:Fasciola hepatica / Q000201:enzymology*; D006651:Histocytochemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents / Q000378:metabolism; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D003374:Coumarins; D007202:Indicators and Reagents; D008670:Metals; D011480:Protease Inhibitors; D003840:Deoxycholic Acid; D007931:Leucyl Aminopeptidase; C028743:7-amino-4-methylcoumarin