- high-resolution terminology - matching measurements at high-resolution
Uncoupler titration
Description
In uncoupler titrations various uncouplers, such as FCCP or DNP [MiPNet03.02] are applied to uncouple mitochondrial electron transport through Complexes I to IV from phosphorylation (Complex V or ATP synthase, ANT and phosphate transport), particularly with the aim to obtain the non-coupled state E at maximum oxygen flux [MiPNet12.05].
Uncouplers may be used not only in isolated mitochondria or permeabilized tissue preparations, but also in intact cells. Uncouplers are permeable through the cell membrane, and intact cells contain energy substrates for mitochondrial respiration. The non-coupled (uncoupler-activated) state may be compared with ROUTINE respiration of the intact cells, in terms of the R/E or ROUTINE control ratio (compare: uncoupler control ratio, UCR). Or the non-coupled state may be the basis for evaluating LEAK respiration in the mitochondrial resting state induced by the addition of oligomycin (inhibitor of ATP synthase) or atractyloside (inhibitor of ANT), obtaing the L/E or LEAK control ratio (compare respiratory control ratio, RCR).
Optimum Uncoupler Concentrations
Uncouplers must be titrated carefully up to an optimum concentration for maximum stimulation of flux, since excess concentrations of uncoupler exert a strongly inhibitory effect.
The optimum FCCP (or DNP) concentration for the non-coupled state varies over a large concentration range, depending on the medium ('binding' of FCCP), type and concentration of sample. This is also true for other uncouplers, such as DNP (ref. 1). To evaluate the optimum concentration, a FCCP titration has to be performed initially (refs. 1-2). For subsequent routine applications, we recommend a few titrations starting close to optimum concentration (ref. 3). Optimum FCCP concentrations range over an order of magnitude, from <0.5 to >4.0 µM.
Uncoupling Control Ratio, UCR
There are strong mathematical arguments to replace the conventional UCR and RCR by the inverse ratios [MiPNet08.09].
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Labels:
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., Permeabilized Cells and Tissues; Homogenates"Permeabilized Cells and Tissues; Homogenates" 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: OXPHOS and ETS Capacity"OXPHOS and ETS Capacity" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Coupling; Membrane Potential"Coupling; Membrane Potential" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.
HRR: Chemicals and Media"Chemicals and Media" 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., Method"Method" 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.
