Partial characterization of proteolytic activity in Giardia duodenalis purified proteins Caracterização parcial da atividade proteolítica de frações de proteínas purificadas de trofozoítos de Giardia duodenalis

• Published in: Revista do Instituto de Medicina Tropical de São Paulo
• Main Authors: Erica Boarato David, Semíramis Guimarães, Paulo Eduardo Martins Ribolla, Silvana Torossian Coradi, Diego Peres Alonso
• Format: Article in Journal/Newspaper
• Language: English
• Published: Universidade de São Paulo (USP) 2007
• Subjects: Giardia duodenalis; Axenic strain; Protein fractionation; Protease activity; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doi.org/10.1590/S0036-46652007000600009; https://doaj.org/article/8d1a8c85c3424dfcbd2e38bc16ad38b2

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. O presente estudo consiste em uma caracterização preliminar da atividade proteolítica de frações de proteínas purificadas a partir de lisados de trofozoítos de cepa isolada e axenizada no Brasil. Frações obtidas por cromatografia ...