title: Partial characterization of proteolytic activity in Giardia duodenalis purified proteins.
abstract: This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.
authors.affiliation: Departamento de Parasitologia, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, SP, Brazil.
authors.forename: Erica Boarato
authors.initials: EB
authors.lastname: David
authors.forename: Semíramis
authors.initials: S
authors.lastname: Guimarães
authors.forename: Paulo Eduardo Martins
authors.initials: PE
authors.lastname: Ribolla
authors.forename: Silvana Torossian
authors.initials: ST
authors.lastname: Coradi
authors.forename: Diego Peres
authors.initials: DP
authors.lastname: Alonso
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005872:Giardia / Q000201:enzymology*; D006801:Humans; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D010447:Peptide Hydrolases