title: Alternative 2-keto acid oxidoreductases in Trichomonas vaginalis: artifact of histochemical staining.
abstract: Trichomonas vaginalis has been reported to possess alternative 2-keto acid oxidoreductases (KORs). These enzymes preferentially used indolepyruvate in a reaction that resembled that of pyruvate:ferredoxin oxidoreductase (PFO). However, the KORs did not reduce ferredoxin and remained active in metronidazole-resistant trichomonads lacking PFO. Therefore, it was proposed that the KORs may help trichomonads to survive in the presence of metronidazole. The KORs were identified using activity staining on native gels (Brown DM, Upcroft JA, Dodd HN, et al. Alternative 2-keto acid oxidoreductase activities in T. vaginalis. Mol Biochem Parasitol 1999;98:203-14). In the current study, we showed that the apparent KOR activity was caused by the non-enzymatic reduction of the indicator dye, nitroblue tetrazolium, by indolepyruvate, which is facilitated by Triton X-100 used to prepare the membrane fractions. We could not confirm the presence of KORs in metronidazole-resistant T. vaginalis. The low level indolepyruvate-dependent activity that is present in T. vaginalis strains sensitive to metronidazole is catalyzed by PFO, which was verified using the pure enzyme. Therefore, our results suggest that alternative 2-keto acid oxidoreductases do not exist in T. vaginalis.
authors.affiliation: Department of Parasitology, Charles University in Prague, Viničná 7, 128 44 Prague 2, Czech Republic.
authors.forename: Věra
authors.initials: V
authors.lastname: Zedníková
authors.forename: Neritza Campo
authors.initials: NC
authors.lastname: Beltrán
authors.forename: Jan
authors.initials: J
authors.lastname: Tachezy
authors.forename: Ivan
authors.initials: I
authors.lastname: Hrdý
mesh_terms: D016477:Artifacts; D006651:Histocytochemistry / Q000379:methods*; D007211:Indoles / Q000378:metabolism; D007651:Keto Acids / Q000378:metabolism; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D017830:Octoxynol / Q000378:metabolism; D010088:Oxidoreductases / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods*; D014246:Trichomonas vaginalis / Q000201:enzymology*
chemical_list: D007211:Indoles; D007651:Keto Acids; D009580:Nitroblue Tetrazolium; D017830:Octoxynol; D010088:Oxidoreductases