title: Characterization and purification of extracellular proteases of Trichomonas vaginalis.
abstract: Extracellular protease activity was detected in serum-free culture filtrates of Trichomonas vaginalis. The activity was demonstrated by hydrolysis of hide powder azure and possessed the characteristics of cysteine type proteases: inhibition by N-ethyl maleimide, Cu2+, antipain, N-tosyl-L-phenylalanine chloromethyl ketone, N-tosyl-L-lysine chloromethyl ketone, leupeptin, chymostatin, and iodoacetamide, and enhancement by cysteine, EDTA, and dithiothreitol. The activity was optimal at acid pH and the protease was also active on peptide nitroanilides with arginine derivatives. Purification of this activity by ethanol precipitation, ammonium sulfate fractionation, ion exchange chromatography, and gel filtration resulted in the isolation of two proteases estimated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to have molecular masses of 60 and 30 kilodaltons (kDa), respectively. The larger molecular mass protease broke down during purifications to two subunits of approximately 23 and 43 kDa, as determined by gel electrophoresis. Rabbit sera derived by immunization with the 23-kDa subunit cross-reacted by immunoblot with the 60- and 43-kDa subunits, but not with the 30-kDa protease. These soluble products of T. vaginalis growth could be important pathogenically in establishing T. vaginalis infection in the normally acid (pH less than or equal to 4.5) environment of the vagina.
authors.affiliation: Department of Medicine, University of Ottawa, Ont., Canada.
authors.forename: G E
authors.initials: GE
authors.lastname: Garber
authors.forename: L T
authors.initials: LT
authors.lastname: Lemchuk-Favel
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D014246:Trichomonas vaginalis / Q000201:enzymology*
chemical_list: D003546:Cysteine Endopeptidases