Entamoeba histolytica, heterologous expression and in situ immunolocalization of ornithine decarboxylase (EhODC).
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.
Terán-Figueroa
Yolanda
Y
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal No. 187, Guanajuato Gto. 36000, México.
Arteaga-Nieto
Pablo
P
Bethancourt-Rodríguez
America
A
Labra-Barrios
Maria Luisa
ML
Luna-Arias
Juan Pedro
JP
Flores-Carreón
Arturo
A
Cano-Canchola
Carmen
C
López-Romero
Everardo
E
Calvo-Méndez
Carlos
C
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
D009955:Ornithine Decarboxylase