title: Entamoeba histolytica, heterologous expression and in situ immunolocalization of ornithine decarboxylase (EhODC).
abstract: Previous studies from this laboratory have dealt with the purification and biochemical characterization of ornithine decarboxylase (ODC) from Entamoeba histolytica. Enzyme compartmentalization has been described as a major mechanism in the regulation of polyamine metabolism. However, the subcellular location of ODC in the human parasite has remained unresolved. To examine this issue, we cloned the full-length gene (Ehodc) encoding for the parasite enzyme, whose open reading frame encodes for a peptide of 412 amino acids with an estimated molecular mass of 46kDa that exhibits similarity to other ODCs. Heterologous overexpression of the gene allowed us to purify the recombinant protein (rEhODC) by metal affinity chromatography. The purified polypeptide was used to raise heteroclonal antibodies that were utilized to localize the enzyme in situ by immunofluorescence and confocal microscopy. EhODC was observed to be associated with the plasma membrane, in vesicles close to the plasma membrane and in the EhkOs organelle.
authors.affiliation: Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal No. 187, Guanajuato Gto. 36000, México.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Terán-Figueroa
authors.forename: Pablo
authors.initials: P
authors.lastname: Arteaga-Nieto
authors.forename: America
authors.initials: A
authors.lastname: Bethancourt-Rodríguez
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Labra-Barrios
authors.forename: Juan Pedro
authors.initials: JP
authors.lastname: Luna-Arias
authors.forename: Arturo
authors.initials: A
authors.lastname: Flores-Carreón
authors.forename: Carmen
authors.initials: C
authors.lastname: Cano-Canchola
authors.forename: Everardo
authors.initials: E
authors.lastname: López-Romero
authors.forename: Carlos
authors.initials: C
authors.lastname: Calvo-Méndez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009955:Ornithine Decarboxylase / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D009955:Ornithine Decarboxylase