Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples.

The Journal of Biological Chemistry
Fariha AnsariAlexander Galkin

Abstract

Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 104 min-1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mous...Continue Reading

References

Oct 26, 2010·Journal of Visualized Experiments : JoVE·Irina Gostimskaya, Alexander Galkin
Sep 10, 2020·Transplantation Direct·Lu WangJohn H Dark

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