Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii.
Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.
Serrano-Luna
José de Jesús
Jde J
Departamento de Biología Celular, Cinvestav-IPN, México, D.F. México. jserrano@cell.cinvestav.mx
Cervantes-Sandoval
Isaac
I
Calderón
Jesús
J
Navarro-García
Fernando
F
Tsutsumi
Victor
V
Shibayama
Mineko
M
D000048:Acanthamoeba / Q000201:enzymology* / Q000378:metabolism; D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D010447:Peptide Hydrolases / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000187:drug effects / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013045:Species Specificity
D004791:Enzyme Inhibitors; D015842:Serine Proteinase Inhibitors; D007611:Aprotinin; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases