Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples
| Autor: | Edward Owusu-Ansah, Christian Garcia, Sergey Sosunov, Zoya Niatsetskaya, Anna Stepanova, Vadim Ten, Alexander Galkin, Belem Yoval-Sánchez, Fariha Ansari, Ilka Wittig |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Enzyme complex
Flavin mononucleotide HEK293 human embryonic kidney 293 cells mitochondrial respiratory chain complex I Mitochondrion HAR hexaammineruthenium chloride (II) Biochemistry chemistry.chemical_compound Mice ROS reactive oxygen species Animals Humans Mitochondrial respiratory chain complex I KGDHC α-ketoglutarate dehydrogenase complex Ketoglutarate Dehydrogenase Complex Molecular Biology Flavin adenine dinucleotide chemistry.chemical_classification enzyme turnover Dihydrolipoamide dehydrogenase DLD dihydrolipoyl dehydrogenase Electron Transport Complex I Methods and Resources flavin adenine dinucleotide Brain DDM n-dodecyl-β-d-maltoside SMP submitochondrial particle Cell Biology FAD flavin adenine dinucleotide flavin mononucleotide stoichiometry Mitochondria Enzyme HEK293 Cells chemistry MSE mannitol/sucrose/EGTA RET reverse electron transfer Ubiquinone reductase BSA bovine serum albumin Electrophoresis Polyacrylamide Gel fluorescence |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X 0021-9258 |
| Popis: | 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 mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies. |
| Databáze: | OpenAIRE |
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