Isolation and characterization of the Golgi complex of the protozoan Trypanosoma cruzi.
In this study the Golgi complex of the epimastigote forms of Trypanosoma cruzi were isolated and characterized. Using well-controlled sonication to rupture the cells and centrifugation on a discontinuous sucrose density gradient, a highly enriched Golgi fraction was obtained. The Golgi fraction contained most of the beta-galactosyltransferase (beta-Gal transferase) and UDP-N-acetyl-glucosamine: polypeptide-alpha-N-acetyl-glucosaminyltransferase (O-alpha-GlcNAc transferase) activities with minimal contamination of other organelles, as observed by enzymatic assays and electron microscopy analysis. To characterize the Golgi from T. cruzi cells further, it was incubated with a monoclonal antibody against a 58 kDa protein involved in the association of the Golgi complex with microtubules in mammalian cells. Immunofluorescence microscopy showed that the 58 kDa protein is localized in the T. cruzi Golgi region, a result confirmed by high resolution scanning electron microscopy immunocytochemistry. Thus, our results show, for the first time, that the beta-Gal transferase, the O-alpha-GlcNAc transferase and the 58 kDa protein are present in the Golgi complex of T. cruzi and are novel biochemical markers which can be used in the characterization of this organelle in T. cruzi.
Morgado-Díaz
J A
JA
Divisão de Biologia Celular, Coordenação de Pesquisa, Instituto Nacional de Câncer, Rio de Janeiro, Brasil.
Nakamura
C V
CV
Agrellos
O A
OA
Dias
W B
WB
Previato
J O
JO
Mendonça-Previato
L
L
De Souza
W
W
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000201:enzymology / Q000502:physiology* / Q000648:ultrastructure; D006593:Hexokinase / Q000378:metabolism; D007150:Immunohistochemistry; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000502:physiology / Q000648:ultrastructure*; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000378:metabolism
D015800:Protozoan Proteins; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase; D006593:Hexokinase; D000135:Acid Phosphatase