Abstract: Hand SC, Gnaiger E (2014) Flux control ratios in isolatd mitochondria. OXPHOS capacity and respiratory control in isolated mitochondria. Mitochondr Physiol Network 12.11(06):1-5.
Methodological and conceptual features of high-resolution respirometry are illustrated in an experiment with isolated mitochondria in the Oroboros O2k. The experiment demonstrates manual titrations applied to study respiratory control ratios and respiratory capacity in isolated mitochondria. Application of the DatLab software is shown for real-time data analysis. The following guideline describes the experimental protocol and includes a short discussion of results. The experiments were carried out by participants of an O2k-Workshop on HRR in December 2006 (IOC36; Schröcken, Austria).
The coupling control protocol (Fig. 1) was developed for respiratory studies of isolated mitochondria, and has general implications on critically assessing experiments with isolated mitochondria or in a variation of the protocol for permeabilized cells and tissues. As a substrate, succinate+rotenone or succinate without rotenone was chosen in the present example (MiPNet11.09). The experiment was aimed at assessing (1) respiratory OXPHOS and LEAK capacity, in the coupled ADP activated state and LEAK state after phosphorylation of ADP to ATP (States P and L; check for possible limitations by non-saturating ADP-concentrations or loss of cytochrome c), (2) capacity of the Electron transfer-pathway (ET-pathway) in the noncoupled state (State E; uncoupler titration, avoiding inhibition by high uncoupler concentrations), and (3) coupling control of OXPHOS (underestimated by the conventional RCR if the phosphorylation system is limiting).
Labels: MiParea: Respiration, Instruments;methods
Organism: Artemia, Crustaceans
Preparation: Isolated mitochondria
Regulation: Coupling efficiency;uncoupling Coupling state: LEAK, OXPHOS, ET Pathway: S HRR: Oxygraph-2k, O2k-Protocol