Immunochemical studies on the FAD-dependent NADPH-cytochrome c reductase from Trypanosoma cruzi.
The cytosolic flavin enzyme from Trypanosoma cruzi was isolated by a modification of the previously reported method (T. Kuwahara, R. A. White, Jr., and M. Agosin (1985) Arch. Biochem. Biophys. 239, 18-28). In the present study, rabbits were inoculated with the purified enzyme and antibodies were purified from the sera. Ouchterlony double-diffusion analysis indicated that the antibodies reacted specifically with the flavoenzyme and not with other T. cruzi proteins. At the equivalence point, 1 ml of antibody neutralized about 4 nmol of enzyme. The IgG fraction had a small inhibitory effect on the catalytic activity of the enzyme as measured by cytochrome c reduction but only at IgG concentrations well above the equivalence point. Immunotitration of the enzyme in T. cruzi cultures showed that the enzyme corresponds to about 1% of the total protein during the logarithmic phase of growth, but this value decreases to about 0.6% during the stationary phase. Among various trypanosomatids tested, T. cruzi had the highest enzyme concentration; whereas, in other species it ranged from 0.25 to 2.4 micrograms/mg protein. These marked differences suggest that the antibody may be suitable for taxonomic purposes. The presence of the enzyme in amastigotes maintained in tissue culture cells was demonstrated by indirect immunofluorescence. The enzyme was found localized in the periphery of the cell, just beneath the subpellicular microtubules. However, distribution of the enzyme in epimastigotes was more diffuse. As immunofluorescence could be detected only in amastigotes and not in the tissue culture cells, it is suggested that the antibody may be suitable for histopathological diagnosis of Chagas' disease.
D000818:Animals; D000913:Antibodies, Protozoan / Q000096:biosynthesis / Q000502:physiology; D001666:Binding Sites, Antibody; D002522:Chlorocebus aethiops; D005260:Female; D005455:Fluorescent Antibody Technique; D007106:Immune Sera; D005779:Immunodiffusion; D007150:Immunohistochemistry; D007424:Intracellular Fluid / Q000032:analysis / Q000469:parasitology; D009251:NADPH-Ferrihemoprotein Reductase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000254:growth & development / Q000276:immunology
D000913:Antibodies, Protozoan; D007106:Immune Sera; D009251:NADPH-Ferrihemoprotein Reductase