title: Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease.
abstract: Pathogenic protozoan proteases play crucial roles in the host-parasite interaction, and its characterization contributes to the understanding of protozoan disease mechanisms. A Leishmania amazonensis promastigote protease was purified 36-fold, using aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography, yielding a total recovery of 49%. The molecular mass of active enzyme obtained from native gel filtration HPLC and SDS-PAGE under conditions of reduction and non-reduction was 68 kDa, suggesting that the enzyme may exist as a monomer. The protease isoelectric point (pI) was around 4.45 and, as demonstrated by deglycosylation assay, it did not have any carbohydrate content. The optimal pH and temperature of the enzyme were 8.0 and 28 degrees C, respectively, determined using alpha-N-rho-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 50% of the enzymatic activity was preserved after 4 min of pre-treatment at 42 degrees C and after 24 h of pre-treatment at 37 degrees C, both in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin, and both gelatin and peptide substrates containing arginine in ester bound were hydrolyzed by 68 kDa protease. The insulin beta-chain was also hydrolyzed by the protease, and four peptidic bonds (L11-V12, E13-A14, L15-Y16, and Y16-L17) were susceptible to the 68-kDa protease action. Inhibition studies suggested that the enzyme belonged to a serine protease class inhibited by calcium ions and activated by manganese ions. These findings demonstrate that the L. amazonensis 68-kDa serine protease differs from those of other protozoan parasites.
authors.affiliation: Laboratório de Bioquímica de Proteínas e Peptídeos, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21045-900, Rio de Janeiro, Brazil. rlopez@ioc.fiocruz.br
authors.forename: Raquel Elisa
authors.initials: RE
authors.lastname: da Silva-Lopez
authors.forename: Salvatore
authors.initials: S
authors.lastname: Giovanni-De-Simone
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry / Q000378:metabolism; D007526:Isoelectric Point; D007894:Leishmania mexicana / Q000201:enzymology*; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002413:Cations, Divalent; D007328:Insulin; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases