title: Leishmania mexicana: identification and characterization of an aspartyl proteinase activity.
abstract: An aspartyl proteinase activity was detected in the soluble fraction (SF) of Leishmania mexicana promastigotes by the use of the synthetic substrate benzoyl-Arg-Gly-Phe-Phe-Leu-4-methoxy-beta-naphthylamide selective for Cathepsin D like aspartyl-proteinases. This peptide was hydrolyzed with an apparent K(m) of 2.3+/-0.3 microM. The classic inhibitor of aspartyl-proteinases, diazo-acetyl-norleucinemethylester (DAN) inhibited the proteolytic activity with an IC(50) of 400 microM. The soluble fraction degraded (in absence of thiol groups) human fibrinogen with a specific activity of 533 U/mg protein. When tested for the ability to inhibit the "in vitro" proliferation of L. mexicana promastigotes, DAN showed concentration dependent anti-proliferative effects with a LD(50) of 466 microM at 48 h, with a significant fall in this value to 22 microM after 72 h. This is the first characterization of an aspartyl-proteinase activity in Leishmania, calling for further studies directed towards the physiologic role of these enzymes in the parasite. The anti-proliferative effect of its inhibition makes this enzyme a putative new target for the development of leishmanicidal drugs.
authors.affiliation: Laboratorio de Biología Celular de Parásitos, Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, Apartado Postal 47069, Caracas 1041-A, Venezuela. evaldivi@ciens.ucv.ve
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Valdivieso
authors.forename: Fracehuli
authors.initials: F
authors.lastname: Dagger
authors.forename: Ana
authors.initials: A
authors.lastname: Rascón
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007894:Leishmania mexicana / Q000201:enzymology*; D051379:Mice; D009646:Norleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: C000909:N-diazoacetylnorleucine methyl ester; D009646:Norleucine; D005340:Fibrinogen; D016282:Aspartic Acid Endopeptidases