Immunocytochemical and biochemical detection of alpha-L-fucosidase in Trypanosoma cruzi.
The aim of the present study was to demonstrate the presence of alpha-L-fucosidase in Trypanosoma cruzi. Immunocytochemical and biochemical techniques were used to localize and characterize a membrane-associated, neutral-pH-optimum, alpha-L-fucosidase from the parasite. Light and electron microscopy localized the alpha-L-fucosidase specifically on the surface of the parasite and on membranes in the posterior region of the epimastigote stage. Although much less intense, labeling was also detected on the surface of trypomastigotes. At least 50% of the alpha-L-fucosidase activity was associated with epimastigote membrane solubilized with 1 M NaCl or 1% Triton X-100, suggesting that alpha-L-fucosidase is peripherally associated with membranes. The enzyme from epimastigotes had a neutral pH optimum (near 7) but displayed low specific activity when p-nitrophenyl-alpha-L-fucoside was employed as substrate (0.028 U/mg protein for epimastigotes and 0.015 U/mg protein for tissue culture-derived trypomastigotes). Polyacrylamide gel electrophoresis and Western blotting analysis both showed an expected 50-kDa polypeptide which was immunoreactive with anti-alpha-L-fucosidase antibodies.
Miletti
L C
LC
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brasil.
Almeida-de-Faria
M
M
Colli
W
W
Alves
M J M
MJ
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000254:growth & development / Q000648:ultrastructure; D005644:alpha-L-Fucosidase / Q000302:isolation & purification*
D005644:alpha-L-Fucosidase