Abstract
ABSTRACTThe in-gel activity assay (IGA) is a powerful technique that uses enzymatic activity and compares intensities of detected bands in mitochondrial respiratory chain supercomplexes, and it is applicable to eukaryotic organisms. However, no IGA has been established for complex III because of the difficulty of access by ubiquinol, a substrate for complex III. Herein, we demonstrate that cytochrome c (Cyt c) showed peroxidase activity on IGA as a component of complexes III and IV. We used pre-incubation with sodium dodecyl sulfate (SDS) before IGA to loosen complexes in the gel after high-resolution clear native polyacrylamide gel electrophoresis (hrCN-PAGE), a refinement of blue native PAGE. The signals of IGA based on peroxidase activity were obtained using enhanced chemiluminescence solution. Then, the gel was directly used in western blotting or hrCN/SDS two-dimensional PAGE. Our findings indicate that IGA for Cyt c reflected the indirect activity of complexes III and IV.
Highlights
The in-gel activity assay (IGA) has been used to detect and simultaneously compare associations of mitochondrial respiratory chain complexes by semi-quantification of proportional fluctuations in the electron transfer chain (ETC) activity of supercomplexes (Diéguez-Casal et al, 2014; Van Coster et al, 2001; Wittig et al, 2007)
Triton X-100 is a nonionic detergent that solubilizes the mitochondrial membrane for extraction of supercomplexes (Schägger and Pfeiffer, 2000), and it was fortunate that enhancement of IGA only at positions corresponding to the molecular mass of CIII was advantageous in analyzing the indirect activity of CIII
Using an in-gel peroxidase activity assay, dubbed IGA-cytochrome c (Cyt c), we demonstrated that Cyt c was visualized in gel as a flexible component of CIII and CIV
Summary
The in-gel activity assay (IGA) has been used to detect and simultaneously compare associations of mitochondrial respiratory chain complexes (called ‘supercomplexes’) by semi-quantification of proportional fluctuations in the electron transfer chain (ETC) activity of supercomplexes (Diéguez-Casal et al, 2014; Van Coster et al, 2001; Wittig et al, 2007). It was reported that supercomplex formation was enhanced in genetically adipogenic differentiated human mesenchymal stem cells (Hofmann et al, 2012). It was summarized in a review that deterioration of supercomplex formation modulates cristae morphology and leads to mitochondrial dysfunction (Baker et al, 2019). It has been gradually revealed that supercomplex formation and mitochondrial dynamics are tightly related. Since IGA does not require specific antibodies, it can be a powerful tool for the investigation of supercomplexes in Department of Biology, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
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