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Isolation and characterization of the Golgi complex of the protozoan Trypanosoma cruzi

Published online by Cambridge University Press:  23 July 2001

J. A. MORGADO-DÍAZ ,
C. V. NAKAMURA ,
O. A. AGRELLOS ,
W. B. DIAS ,
J. O. PREVIATO ,
L. MENDONÇA-PREVIATO  and
W. DE SOUZA
J. A. MORGADO-DÍAZ
Affiliation:
Divisão de Biologia Celular, Coordenação de Pesquisa, Instituto Nacional de Câncer, Praça Cruz Vermelha, 23, 6° Andar, 20 230 130, Rio de Janeiro, RJ, Brasil
C. V. NAKAMURA
Affiliation:
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, CCS-Bloco G, UFRJ, 21 949 900 Cidade Universitária, Rio de Janeiro, RJ, Brasil Permanent address: Laboratório de Microbiologia, Departamento de Análises Clínicas, Universidade Estadual de Maringá, 87 020 900 Maringá, PR, Brasil.
O. A. AGRELLOS
Affiliation:
Instituto de Microbiologia, CCS-Bloco I, Universidade Federal do Rio de Janeiro, 21 949 900 Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, Brasil
W. B. DIAS
Affiliation:
Instituto de Microbiologia, CCS-Bloco I, Universidade Federal do Rio de Janeiro, 21 949 900 Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, Brasil
J. O. PREVIATO
Affiliation:
Instituto de Microbiologia, CCS-Bloco I, Universidade Federal do Rio de Janeiro, 21 949 900 Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, Brasil
L. MENDONÇA-PREVIATO
Affiliation:
Instituto de Microbiologia, CCS-Bloco I, Universidade Federal do Rio de Janeiro, 21 949 900 Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, Brasil
W. DE SOUZA
Affiliation:
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, CCS-Bloco G, UFRJ, 21 949 900 Cidade Universitária, Rio de Janeiro, RJ, Brasil
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Abstract

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 β-galactosyltransferase (β-Gal transferase) and UDP-N-acetyl-glucosamine: polypeptide-α-N-acetyl-glucosaminyltransferase (O-α-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 β-Gal transferase, the O-α-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.

Keywords

Golgi complexsubcellular fractionationglycosyltransferasesTrypanosoma cruzi
Type
Research Article
Information
Parasitology , Volume 123 , Issue 1 , July 2001 , pp. 33 - 43
Copyright
© 2001 Cambridge University Press

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