PFOR

C—The pfor H2P04 is nearest to the pH value needed. Thus, the simplest buffer would involve this ion. The phosphoric acid in E would lower the pH too much. 

Giardia intestinalis contains PFOR and has been shown to produce hydrogen (Lloyd et al. 2002) but has not been found to contain hydrogeno-somes. Its iron-sulfur composition appears to be quite distinct from those of the trichomonads. EPR showed both soluble and membrane-bound iron-sulfur proteins of the [4Fe-4S] type (ElUs et al. 1993). 

Fig. 2 SDS-polyacrylamide gel electrophoresis of T. vaginalis hydrogenosomes purified by isopycnic centrifugation on a Percoll gradient. 1 PFOR 2 malic enzyme, 64-kDa hy-drogenase, and Cpn 60 3 succinyl CoA synthetase fi subunit 4 Hmp35 5 succinyl CoA synthetase a subunit 6 Hmp31 (ATP/ADP carrier) 7 adenylate kinase 8 thiol peroxidase. Well-resolved but unmarked bands mostly belong to malic enzyme fragments or unknown proteins. Proteins were identified by mass spectroscopy. The 12% gel is stained with Coomassie Brilliant Blue R 250 (authors original)...

A pharmacological implication of the presence of PFOR in T vaginalis should be briefly mentioned. PFOR is the key enzyme in the activation of... 

Fig. 2 Scheme of metronidazole activation in the hydrogenosome the pyruvate pathway. In the presence of metronidazole, electrons generated by pyruvate ferredoxin oxidore-ductase (PFOR) are transferred by ferredoxin (Fd) to the prodrug form of metronidazole (MTZ0X) and not to their natural acceptor hydrogenase (HY). The cytotoxic nitro free radicals R-N02- are generated as intermediate products of the metronidazole reduction (MTZred)... 

Fig. 3 Signal of metronidazole anion radicals generated by hydrogenosomes of T. vaginalis in the presence of 45 mM metronidazole, pyruvate, CoA, and ferredoxin (activity of PFOR). Recorded by EPR spectroscopy at 25 °C with 20 mW of microwave power, a frequency of 9.64 GHz, and a modulation amplitude of 0.19 mT. The hyperfine coupling constants were n0no2 = 1 565 mT, Hfl4 = 0.542 mT, and - 0.229 mT...

An alternative hydrogenosomal pathway of metronidazole activation, which is independent of PFOR activity, was discovered in T. vaginalis (Hrdy et al. 

T. vaginalis NDH transfers electrons to ferredoxin, which serves as a terminal electron carrier donating the electrons to the drug (Fig. 4). Attention to this pathway was drawn by earlier metabolic studies on the biochemical background of drug resistance development in T. vaginalis (Rasoloson et al. 2002), in particular by a need to explain the partial susceptibility of PFOR-deficient T. vaginalis lines to metronidazole (see below). Hrdy et al. (2005) provided direct evidence that the pathway is functional in both wild-... 

Development of anaerobic resistance in both species was accompanied by a progressive decrease and eventual loss of PFOR and hydrogenase activities and ferredoxin levels. For development of anaerobic resistance in T. foetus, the decrease in PFOR activity was of crucial importance (Cerkasovova et al. 

Fig. 8 SDS-PAGE (a) and Western blot (b) analysis of the purified hydrogenosomal fractions isolated from the metronidazole-susceptible T. vaginalis strain TV 10-02 (P) and its metronidazole-resistant derivatives MR-3, MR-5, MR-30, MR-50, and MR-100 displaying the aerobic (3), early anaerobic (5), advanced anaerobic (30, 50), and fully developed anaerobic resistance (100) to metronidazole. Numbers in the designation of MR strains indicate the concentrations of metronidazole in ixg/ml at which the organisms multiply in culture. About 10 pg protein was loaded per line. PFOR pyruvate ferredoxin oxidoreduc-tase, a-STK a subunit of succinate thiokinase (hydrogenosomal enzyme not involved in metronidazole resistance used as control), Fdx ferredoxin. From Rasoloson et al. (2002) by courtesy of the Society of General Microbiology...

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