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 o...

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Bibliographic Details
Published inEuropean journal of cell biology Vol. 56; no. 2; p. 243
Main Authors Mbawa, Z R, Webster, P, Lonsdale-Eccles, J D
Format Journal Article
LanguageEnglish
Published Germany 01.12.1991
Subjects
Animals
Antibody Specificity
Cysteine Endopeptidases - analysis
Cysteine Endopeptidases - isolation & purification
Endocytosis - physiology
Gold - metabolism
Immunohistochemistry
Lysosomes - enzymology
Lysosomes - ultrastructure
Microscopy, Immunoelectron
Serum Albumin, Bovine - metabolism
Trypanosoma congolense - enzymology
Trypanosoma congolense - ultrastructure
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Summary: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.
ISSN:0171-9335