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Inactivation of mitochondrial adenosine triphosphatase from Trypanosoma cruzi by oxygen radicals
Abstract:
Incubation of Trypanosoma cruzi mitochondrial ATPase (Fo-F1) with the xanthine oxidase system (XO), Fenton's reagent (Fe2+ + H2O2) and the ascorbate-Cu system, caused gradual loss of enzyme activity, which increased as a function of incubation time and rate of oxygen radical generation. The essential role of OH. radicals for ATPase inactivation was supported by a) the enzyme protection afforded by superoxide dismutase, catalase and mannitol, when using the XO system; b) the similar effect of mannitol and benzoate with Fenton's reagent; c) the similar effect of catalase, EDTA and histidine with the ascorbate-Cu system; d) the increased rate of ATPase inactivation by 1) the XO system supplemented with chelated iron, and 2) the ascorbate-Cu system supplemented with H2O2. Comparison of oxygen radical generators for their action on membrane-bound (Fo-F1) and soluble F1 revealed that ascorbate-Cu was the most effective one, possibly because of its capability of producing OH. radicals that react preferentially with the enzyme at their formation site.
Insights
Trypanosoma cruzi mitochondrial ATPase (Fo-F1) activity is lost due to oxygen radicals generated by various systems. Hydroxyl radicals (OH.) are key to this inactivation, with the ascorbate-Cu system being most effective.
Area of Science:
- Biochemistry
- Molecular Biology
- Parasitology
Background:
- Mitochondrial ATPase (Fo-F1) is crucial for energy production in Trypanosoma cruzi.
- Oxygen radicals can damage essential cellular components like enzymes.
- Understanding enzyme inactivation mechanisms is vital for antiparasitic drug development.
Purpose of the Study:
- To investigate the inactivation of Trypanosoma cruzi mitochondrial ATPase (Fo-F1) by different oxygen radical generating systems.
- To identify the specific reactive oxygen species responsible for ATPase inactivation.
- To compare the efficacy of various radical generating systems in damaging the enzyme.
Main Methods:
- Incubation of purified Trypanosoma cruzi mitochondrial ATPase (Fo-F1) with xanthine oxidase (XO), Fenton's reagent, and ascorbate-Cu systems.
- Assay of residual enzyme activity after incubation.
- Use of radical scavengers (superoxide dismutase, catalase, mannitol, benzoate, EDTA, histidine) to identify reactive species.
- Supplementation of radical generating systems with iron or hydrogen peroxide to enhance radical production.
Main Results:
- All tested oxygen radical systems (XO, Fenton's reagent, ascorbate-Cu) caused a gradual loss of ATPase activity.
- Hydroxyl radicals (OH.) were identified as the primary species responsible for enzyme inactivation, evidenced by protection from scavengers.
- The ascorbate-Cu system was the most potent in inactivating both membrane-bound and soluble F1 ATPase, likely due to localized OH. radical production.
- Chelated iron and H2O2 addition enhanced ATPase inactivation rates in respective systems.
Conclusions:
- Oxygen radicals, particularly hydroxyl radicals, significantly inactivate Trypanosoma cruzi mitochondrial ATPase (Fo-F1).
- The ascorbate-Cu system is highly effective in generating radicals that damage the enzyme, suggesting potential as a research tool.
- These findings contribute to understanding parasite biochemistry and potential therapeutic targets.