MiPNet12.11 MitoRespiration
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» Open Access-MiPNet12.11.pdf; Versions
OROBOROS (2014-02) Mitochondr Physiol Network
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 Oxygraph-2k (O2k). The experiment demonstrates manual titrations applied to study respiratory control ratios and respiratory capacity in isolated mitochondria. Application of the DatLab 4.2 software is shown for on-line 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 protocol (Fig. 1) was developed for respiratory studies of isolated mitochondria, and has general implications on critically assessing experiments with isolated mitochondria or 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 transport system (ETS) in the uncoupled 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).
- >> Product: OROBOROS Oxygraph-2k, O2k-Catalogue
• O2k-Network Lab: AT_Innsbruck_OROBOROS, US LA Baton Rouge Hand SC
Labels: MiParea: Respiration, Instruments;methods
Organism: Artemia
Preparation: Isolated Mitochondria"Isolated Mitochondria" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property.
Regulation: Coupling efficiency;uncoupling Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property.
HRR: Oxygraph-2k, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.
O2k-Demo
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