The pyruvate:ferredoxin oxidoreductase enzyme is located in the plasma membrane and in a cytoplasmic structure in Entamoeba

This work investigated the cellular location of the pyruvate:ferredoxin oxidoreductase (PFO) enzyme in Entamoeba. A 1.9 kb fragment located at the 3' end of the Ehpfo gene was cloned in the pRSETB vector and expressed. The recombinant peptide was purified and inoculated in rabbits. By Western blot assays the antibodies detected a single 130 kDa band in all E. histolytica strains tested and in E. moshkovskii. By immunofluorescence, the antibodies showed the presence of PFO in the plasma membrane and in a cytoplasmic structure that appeared as a ring or as a compact small body in E. histolytica strains. In E. invadens and E. moshkovskii (strains FIC and Laredo) PFO was located in the plasma membrane showing different fluorescence patterns. Immunofluorescence on E. histolytica synchronized cultures showed that the cytoplasmic structure appeared in 85, 60, 20 and 10% of the trophozoites in mitosis, G1, S and G2 phases, respectively. By in situ hibridization the Ehpfo gene was found in the nuclei and the trophozoites of the clone A, strain HMI:IMSS, differed in the Ehpfo gene content. (C) 1998 Academic Press.