Purification and biochemical characterization of the hydrogenosomes of the flagellate protozoan Tritrichomonas foetus.
A highly purified hydrogenosomal fraction was obtained from Tritrichomonas foetus by differential and Percoll gradient centrifugations. Transmission electron microscopy and assay of the malic enzyme activity were used to evaluate the isolation method and the integrity of the organelle. The isolated hydrogenosomes showed the same morphology as observed in intact cells, including the presence of a peripheral vesicle with an electron-dense content. SDS-PAGE revealed the presence of several protein bands, with those of 120, 66, 60, 59, 48, 45, and 35 kDa as the major ones. The hydrogenosome membrane was solubilized with Triton X-100 leaving a fraction containing its matrix attached to the peripheral vesicle. Further treatment with proteinase K solubilized the matrix components, leaving a pure peripheral vesicle fraction. Enzymatic assay during all procedures suggested that malate dehydrogenase was localized in the hydrogenosomal membrane. SDS-PAGE showed that proteins of 66, 45 and 32 kDa were localized in the peripheral vesicle. Western blot analysis of all fractions using alkaline phosphatase-conjugated wheat germ agglutinin revealed the presence of glycoproteins, with a major one of 45 kDa, in the peripheral vesicle of the hydrogenosome.
Díaz
J A
JA
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil.
De Souza
W
W
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D006859:Hydrogen; D007425:Intracellular Membranes / Q000201:enzymology; D037102:Lectins; D008291:Malate Dehydrogenase / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis / Q000737:chemistry; D008970:Molecular Weight; D015388:Organelles / Q000737:chemistry* / Q000648:ultrastructure; D015800:Protozoan Proteins / Q000032:analysis / Q000737:chemistry; D016835:Tritrichomonas foetus / Q000166:cytology* / Q000201:enzymology
D037102:Lectins; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D006859:Hydrogen; D008291:Malate Dehydrogenase