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

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 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.

Redox-dependent loss of flavin by mitochondria complex I is different in brain and heart

“…It strongly suggested that the mechanism of inactivation of the brain enzyme is in fact redox-dependent dissociation of tightly bound complex I flavin. In order to directly identify the mechanism of complex I inactivation, we developed a method to accurately determine only complex I-associated FMN in mitochondrial preparations [ 39 ]. A typical Coomassie-stained hrCN PAGE gel of DDM-solubilized intact mitochondria from the brain and heart is shown in Fig.…”

“…After denaturation of the native protein by SDS [ 39 ], we observed two major flavin-containing bands corresponding to mitochondrial α-ketoglutarate dehydrogenase (αKGDHC) and complex I ( Fig. 3 A, lanes 4–5, ∼1200 kDa and 850 kDa bands, respectively).…”

“…A dramatic difference in FMN dissociation kinetics between the brain and heart enzyme in RET-like conditions was observed using the H 2 O 2 release as an indirect real-time measure for complex I activity as well as assessing complex I activity directly. Using our novel approach for quantification of complex I FMN [ 39 ], we unambiguously demonstrated that, unlike in mitochondria from the heart, incubation in RET-like conditions decreases complex I FMN content in the brain enzyme. These observations suggest a difference in the vicinity of the complex I FMN-binding site between the brain and heart enzyme.…”

“…Sample solubilization with DDM and running conditions for high resolution clear native (hrCN) electrophoresis of mitochondrial membranes and tissues was essentially done as described [ 38 , 39 ]. Gels were analyzed either for complex I in-gel activity, stained with Coomassie or scanned for flavin fluorescence in a Typhoon 9000 gel scanner (GE) using a 473 nm laser and BPB1 filter (530 nm maximum, 20 nm bandpass).…”

“…For the Western Blot, we used an antibody against the S8 subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I) (Rabbit monoclonal NDUFS8 - C-terminal; Abcam Cat# ab180183: dilution 1:2000; 2% milk; 2h at room temperature). Blot images were obtained using Fluorchem M Western Imaging System (Protein Simple, San Jose, CA) [ 39 ].…”