Baglivo 2023 ESCI Prague
Event: ESCI 2023 Prague CZ
Cytochrome c is the redox link between Complexes CIII and CIV of the mitochondrial electron transfer system. Cytochrome c release across the mitochondrial outer membrane (mtOM) is associated with apoptosis. Stimulation of respiration by added cytochrome c indicates damage of the mtOM. This respirometric test for mtOM integrity provides a quality control to exclude potential injuries caused experimentally during mitochondrial preparation by tissue homogenization or plasma membrane permeabilization.
Selecting the step of cytochrome c addition in substrate-uncoupler-inhibitor-titration (SUIT) protocols is of primary importance for diagnostic analysis. This study presents a quality control of SUIT protocols using the cytochrome c test.
We applied SUIT protocols RP1 and RP2 to address the cytochrome c effect in different respiratory states. The first steps in RP1 use pyruvate & malate to analyze coupling control in the NADH-pathway. RP2 focuses on fatty acid oxidation (FAO), considering malate-linked anaplerosis. We tested these protocols in HEK 293T permeabilized cells and beef liver and heart homogenate.
Cytochrome c control efficiency  was correlated when cytochrome c was added in the ADP-activated OXPHOS state of the NADH-pathway and in FAO. Following 0.1 mM malate in RP2, addition of cytochrome c before or after titration of fatty acid did not affect the O2 flux in FAO. Optimization of digitonin concentrations for plasma membrane permeabilization of cultured cells was essential to avoid damage of the mtOM. Conclusions: Underestimating respiratory capacities in mitochondrial preparations is avoided by adding cytochrome c as an early step of SUIT protocols. However, cytochrome c added in the LEAK state causes an unexplained activation of respiration.
- Oroboros Instruments GmbH, Oroboros Ecosystem, Innsbruck, Austria.
- Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. https://doi.org/10.26124/bec:2020-0002
List of abbreviations, terms and definitions - MitoPedia
Labels: MiParea: Respiration, Instruments;methods, mt-Membrane
Organism: Human, Bovines Tissue;cell: Heart, Liver, HEK Preparation: Permeabilized cells, Homogenate
Regulation: Cyt c