Musiol 2018 MiPschool Tromso C4
Event: MiPschool Tromso-Bergen 2018
In mitochondrial respiration measurements, digitonin is a widely used substance for the permeabilization of the plasma membrane. Digitonin is a mixture of several different saponins, naturally varying in their composition. Therefore, the effective concentration of each digitonin batch must be determined individually. We recently identified the secondary plant metabolite α-tomatine to exhibit the same permeabilizing properties as digitonin. Furthermore, it can not only permeabilize the plasma membrane but also the outer mitochondrial membrane.
In this study, we used α-tomatine to selectively permeabilize the cellular and mitochondrial membrane of HEK-293 cells in respirometric measurements. The permeabilization of the outer mitochondrial membrane was combined with an excess of reduced cytochrome C to directly modulate the activity of complex IV. In standardized substrate-uncoupler-inhibitor-titration protocols, complex IV activity is usually influenced by changes in substrate availability, the substrate oxidation capacity of different enzymes and the size of the ubiquinone and cytochrome C pool.
We demonstrate that our assay bypasses these limitations. Thus, the maximal activity of complex IV within the inner mitochondrial membrane can be assessed more specifically. In conclusion, the use of α-tomatine allows a more precise determination of factors responsible for changes in state 3 respiration and to increase our basic understanding of the role of complex IV in the respiratory chain.
Musiol E(1,2), Weber D(1,2), Dawid C(3), Hofmann T(3), Fromme T(1,2)
- Chair Molecular Nutritional Medicine, Technische Univ München, Else Kröner-Fresenius Zentrum Ernährungsmedizin. - Eva.Musiol@tum.de
- ZIEL – Inst Food Health, Technische Univ München
- Chair Food Chemistry Molecular Sensory Science, Technische Univ München
Labels: MiParea: Respiration, Pharmacology;toxicology
Organism: Human Tissue;cell: HEK
Enzyme: Complex IV;cytochrome c oxidase Regulation: Cyt c
HRR: Oxygraph-2k Event: C4, Oral