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:ferredoxi n 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 ex pressed. The recombinant peptide was purified and inoculated in rabbit s. By Western blot assays the antibodies detected a single 130 kDa ban d in all E. histolytica strains tested and in E. moshkovskii. By immun ofluorescence, 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 me mbrane showing different fluorescence patterns. Immunofluorescence on E. histolytica synchronized cultures showed that the cytoplasmic struc ture appeared in 85, 60, 20 and 10% of the trophozoites in mitosis, G1 , S and G2 phases, respectively. By in situ hibridization the Ehpfo ge ne 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 .