Immunolocalization of a cysteine protease within the lysosomal system of Trypanosoma congolense.
A cysteine protease has been purified from bloodstream forms of Trypanosoma congolense by affinity chromatography on cystatin-Sepharose. A polyclonal antibody was raised against the purified enzyme and used for immunocytochemical localization of the enzyme by electron microscopy. Antibody labeling of the cysteine protease, using colloidal gold-labeled protein A (PrA-Au), was observed over amorphous material within subcellular organelles which have the appearance of lysosome-like bodies. This intracellular labeling colocalized in organelles containing bovine serum albumin-gold (BSA-Au) that had been endocytosed by the living parasites. The PrA-Au/antibody also labeled the flagellar pocket and parasite cell surface, albeit less consistently. Volume density analysis showed that the organelles containing endocytosed BSA-Au, after 30 min incubation at 37 degrees C in BSA-Au, comprised approximately 22% of the total parasite cell volume. Under similar conditions, but employing horseradish peroxidase (HRP) as a fluid-phase marker of the lysosomal system, only 5.7% of the cell contained HRP. This value dropped to 3.6% after 60 min incubation. Volume density analysis showed that the amorphous material which was labeled by the antibody to the cysteine protease occupied 6.9% of the cell volume. This amorphous material was contained within a membrane-bound lysosome-like organelle that occupied 11.5% of the cell. Thus, the cysteine protease appears to be present in half, or less, of the lysosomal system of T. congolense.
Mbawa
Z R
ZR
International Laboratory for Research on Animal Diseases, Nairobi/Kenya.
Webster
P
P
Lonsdale-Eccles
J D
JD
D000818:Animals; D000918:Antibody Specificity; D003546:Cysteine Endopeptidases / Q000032:analysis* / Q000302:isolation & purification; D004705:Endocytosis / Q000502:physiology*; D006046:Gold / Q000378:metabolism; D007150:Immunohistochemistry; D008247:Lysosomes / Q000201:enzymology* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014348:Trypanosoma congolense / Q000201:enzymology* / Q000648:ultrastructure
D012710:Serum Albumin, Bovine; D006046:Gold; D003546:Cysteine Endopeptidases