Journal
PARASITOLOGY
Volume 140, Issue 3, Pages 328-337Publisher
CAMBRIDGE UNIV PRESS
DOI: 10.1017/S003118201200162X
Keywords
Trypanosoma; mitochondrion; dehydrogenase; respiration; NDH2
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Funding
- Grant Agency of the Czech Republic [204/09/1667]
- Czech Ministry of Education [LC07032, 6007665801]
- Praemium Academiae award
- Scientific Grant Agency of the Slovak Ministry of Education
- Academy of Sciences [1/0393/09]
- The National Scholarship Programme of the Slovak Republic
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The respiratory chain of the procyclic stage of Trypanosoma brucei contains the standard complexes I through IV, as well as several alternative enzymes contributing to electron flow. In this work, we studied the function of an alternative NADH: ubiquinone oxidoreductase (NDH2). Depletion of target mRNA was achieved using RNA interference (RNAi). In the non-induced and RNAi-induced cell growth, membrane potential change, alteration in production of reactive oxygen species, overall respiration, enzymatic activities of complexes I, III and/or IV and distribution of NADH: ubiquinone oxidoreductase activities in glycerol gradient fractions were measured. Finally, respiration using different substrates was tested on digitonin-permeabilized cells. The induced RNAi cell line exhibited slower growth, decreased mitochondrial membrane potential and lower sensitivity of respiration to inhibitors. Mitochondrial glycerol-3-phosphate dehydrogenase was the only enzymatic activity that has significantly changed in the interfered cells. This elevation as well as a decrease of respiration using NADH was confirmed on digitonin-permeabilized cells. The data presented here together with previously published findings on complex I led us to propose that NDH2 is the major NADH: ubiquinone oxidoreductase responsible for cytosolic and not for mitochondrial NAD(+) regeneration in the mitochondrion of procyclic T. brucei.
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