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Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples
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 p...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8503622/ https://www.ncbi.nlm.nih.gov/pubmed/34543622 http://dx.doi.org/10.1016/j.jbc.2021.101204 |
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author | Ansari, Fariha Yoval-Sánchez, Belem Niatsetskaya, Zoya Sosunov, Sergey Stepanova, Anna Garcia, Christian Owusu-Ansah, Edward Ten, Vadim Wittig, Ilka Galkin, Alexander |
author_facet | Ansari, Fariha Yoval-Sánchez, Belem Niatsetskaya, Zoya Sosunov, Sergey Stepanova, Anna Garcia, Christian Owusu-Ansah, Edward Ten, Vadim Wittig, Ilka Galkin, Alexander |
author_sort | Ansari, Fariha |
collection | PubMed |
description | 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 10(4) 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 × 10(3) 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. |
format | Online Article Text |
id | pubmed-8503622 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-85036222021-10-18 Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples Ansari, Fariha Yoval-Sánchez, Belem Niatsetskaya, Zoya Sosunov, Sergey Stepanova, Anna Garcia, Christian Owusu-Ansah, Edward Ten, Vadim Wittig, Ilka Galkin, Alexander J Biol Chem Methods and Resources 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 10(4) 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 × 10(3) 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. American Society for Biochemistry and Molecular Biology 2021-09-17 /pmc/articles/PMC8503622/ /pubmed/34543622 http://dx.doi.org/10.1016/j.jbc.2021.101204 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Methods and Resources Ansari, Fariha Yoval-Sánchez, Belem Niatsetskaya, Zoya Sosunov, Sergey Stepanova, Anna Garcia, Christian Owusu-Ansah, Edward Ten, Vadim Wittig, Ilka Galkin, Alexander Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples |
title | Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples |
title_full | Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples |
title_fullStr | Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples |
title_full_unstemmed | Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples |
title_short | Quantification of NADH:ubiquinone oxidoreductase (complex I) content in biological samples |
title_sort | quantification of nadh:ubiquinone oxidoreductase (complex i) content in biological samples |
topic | Methods and Resources |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8503622/ https://www.ncbi.nlm.nih.gov/pubmed/34543622 http://dx.doi.org/10.1016/j.jbc.2021.101204 |
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