Entamoeba histolytica: purification and characterization of ornithine decarboxylase.
Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis in eukaryotes, was stabilized and purified from trophozoites of the parasite protozoan E. histolytica. Analytical electrophoresis revealed the presence in the purified preparations of a major polypeptide of 45 kDa and barely detectable amounts of two other proteins of 70 and 120 kDa. Both the 45 and 70 kDa polypeptides were recognized by a mouse anti-ODC monoclonal antibody. The major polypeptide exhibited amino terminal sequence homology in the range of 40-73% with ODCs from other organisms. The immunoreactive polypeptide of 70 kDa was not identified. The molecular masses of 216 and 45 kDa determined for the native enzyme by gel filtration and for the major polypeptide by SDS-PAGE, respectively, suggest that the amoeba ODC is a homopentamer. Dialysis against hydroxylamine rendered the enzyme activity fully dependent on pyridoxal 5'-phosphate (PLP). As expected for an oligomeric enzyme, ODC activity exhibited sigmoidal kinetics when it was measured as a function of increasing concentrations of L-ornithine and PLP yielding S(0.5) values of 0.45 and 0.18 mM, respectively. Purified ODC was inhibited by 1,3-diaminopropane and 2,4-diamino-2-butanone but was largely insensitive to inhibition by alpha-difluoromethylornithine (DFMO), indicating that the enzyme may not be a suitable target for this anti-parasitic drug. Other features of the amoeba ODC were common with the enzyme from prokaryotes and eucaryotes.
Arteaga-Nieto
Pablo
P
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Mexico.
López-Romero
Everardo
E
Terán-Figueroa
Yolanda
Y
Cano-Canchola
Carmen
C
Luna Arias
Juan P
JP
Flores-Carreón
Arturo
A
Calvo-Méndez
Carlos
C
D000595:Amino Acid Sequence; D000818:Animals; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009952:Ornithine / Q000494:pharmacology; D009955:Ornithine Decarboxylase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D065108:Ornithine Decarboxylase Inhibitors; D011732:Pyridoxal Phosphate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D065108:Ornithine Decarboxylase Inhibitors; D011732:Pyridoxal Phosphate; D009952:Ornithine; D009955:Ornithine Decarboxylase