MiPNet12.23 FiberRespiration: Difference between revisions
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results. The experiments was carried out by participants of an O2k-Course on HRR in December 2007 [http://www.oroboros.at/index.php?id=ioc44 (IOC44; Schroecken, Austria).] | results. The experiments was carried out by participants of an O2k-Course on HRR in December 2007 [http://www.oroboros.at/index.php?id=ioc44 (IOC44; Schroecken, Austria).] | ||
[[Image:MiPNet12.23.jpg|center|100px|frame]] | |||
Figure 1. Oxygen concentration ([ฮผM] blue line) and oxygen flux per mg wet weight of muscle ([pmolโs-1โmg-1] red lines) in O2k chamber B, in permeabilized fibres from horse skeletal muscle with the standard titration protocol. | |||
A multiple substrate-uncoupler-inhibitor titration protocol (Fig. 1) was developed for respiratory studies of permeabilized muscle fibres. The experiment was aimed at an assessment of a sequence of titrations, inducing stepwise defined respiratory states. | A multiple substrate-uncoupler-inhibitor titration protocol (Fig. 1) was developed for respiratory studies of permeabilized muscle fibres. The experiment was aimed at an assessment of a sequence of titrations, inducing stepwise defined respiratory states. | ||
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# LEAK state, L: Non-phosphorylating resting state with substrates for Complex I (glutamate+malate; GMN; without adenalytes, N). | #<span style="color:red"> '''LEAK state, L:'''</span> Non-phosphorylating resting state with substrates for Complex I (glutamate+malate; GMN; without adenalytes, N). | ||
# OXPHOS capacity with Complex I substrates, P: Respiratory capacity in the active coupled state (with ADP, GMD). | #<span style="color:green">'''OXPHOS capacity with Complex I substrates, P:'''</span> Respiratory capacity in the active coupled state (with ADP, GMD). | ||
#Cytochrome c test for quality control: Further addition of cytochrome c yields a test for integrity of the outer mitochondrial membrane (loss of cytochrome c would be indicated by a stimulation of respiration). | #<span style="color:black">'''Cytochrome c test for quality control:'''</span> Further addition of cytochrome c yields a test for integrity of the outer mitochondrial membrane (loss of cytochrome c would be indicated by a stimulation of respiration). | ||
#OXPHOS capacity with Complex I+II substrates, P: Respiratory stimulation by convergent electron flow from Complexes I+II at the Q-junction, in the coupled state after further addition of succinate, as an estimate of OXPHOS capacity (Gnaiger 2009). | #<span style="color:green">'''OXPHOS capacity with Complex I+II substrates, P:'''</span> Respiratory stimulation by convergent electron flow from Complexes I+II at the Q-junction, in the coupled state after further addition of succinate, as an estimate of OXPHOS capacity (Gnaiger 2009). | ||
#Electron transfer system (ETS) capacity with CI+II substrates, E: Uncoupling by FCCP titration (avoiding inhibition by high FCCP concentrations), as a test for limitation of OXPHOS relative to ETS capacity by the phosphorylation system. | #<span style="color:blue">'''Electron transfer system (ETS) capacity with CI+II substrates, E:'''</span> Uncoupling by FCCP titration (avoiding inhibition by high FCCP concentrations), as a test for limitation of OXPHOS relative to ETS capacity by the phosphorylation system. | ||
#ETS capacity with CII substrate, E: ETS capacity with succinate, after blocking Complex I with rotenone. | #<span style="color:blue">'''ETS capacity with CII substrate, E:'''</span> ETS capacity with succinate, after blocking Complex I with rotenone. | ||
#Residual oxygen consumption (ROX) due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (Inhibitor of Complex III). | #'''Residual oxygen consumption (ROX)''' due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (Inhibitor of Complex III). | ||
Revision as of 12:11, 29 November 2013
Lemieux H, Votion DM, Gnaiger E (2007) Mitochondrial respiration in permeabilized fibres: Needle biopsies from horse skeletal muscle. Mitochondr Physiol Network 12.23. |
ยป Open Access-MiPNet12.23.pdf; Versions
OROBOROS (2007) Mitochondr Physiol Network
Abstract: O2k-Protocol for Mitochondrial Respiration in Permeabilized Fibres: Needle Biopsies from Horse Skeletal Muscle
Methodological and conceptual features of highresolution respirometry are illustrated in an experiment with permeabilized fibres in the OROBOROS Oxygraph- 2k (O2k). The experiment demonstrates manual titrations applied to study respiratory capacity in permeabilized fibres. Application of the DatLab 4 software is shown for on-line data analysis MiPNet12.09 The following guideline describes the experimental protocol and includes a short discussion of results. The experiments was carried out by participants of an O2k-Course on HRR in December 2007 (IOC44; Schroecken, Austria).
Figure 1. Oxygen concentration ([ฮผM] blue line) and oxygen flux per mg wet weight of muscle ([pmolโs-1โmg-1] red lines) in O2k chamber B, in permeabilized fibres from horse skeletal muscle with the standard titration protocol.
A multiple substrate-uncoupler-inhibitor titration protocol (Fig. 1) was developed for respiratory studies of permeabilized muscle fibres. The experiment was aimed at an assessment of a sequence of titrations, inducing stepwise defined respiratory states.
- LEAK state, L: Non-phosphorylating resting state with substrates for Complex I (glutamate+malate; GMN; without adenalytes, N).
- OXPHOS capacity with Complex I substrates, P: Respiratory capacity in the active coupled state (with ADP, GMD).
- Cytochrome c test for quality control: Further addition of cytochrome c yields a test for integrity of the outer mitochondrial membrane (loss of cytochrome c would be indicated by a stimulation of respiration).
- OXPHOS capacity with Complex I+II substrates, P: Respiratory stimulation by convergent electron flow from Complexes I+II at the Q-junction, in the coupled state after further addition of succinate, as an estimate of OXPHOS capacity (Gnaiger 2009).
- Electron transfer system (ETS) capacity with CI+II substrates, E: Uncoupling by FCCP titration (avoiding inhibition by high FCCP concentrations), as a test for limitation of OXPHOS relative to ETS capacity by the phosphorylation system.
- ETS capacity with CII substrate, E: ETS capacity with succinate, after blocking Complex I with rotenone.
- Residual oxygen consumption (ROX) due to oxidative side reactions in the permeabilized fibres, estimated after addition of Antimycin A (Inhibitor of Complex III).
- >> O2k-Protocols:Overall contents
- >> Product: OROBOROS Oxygraph-2k, O2k-Catalogue
โข O2k-Network Lab: AT_Innsbruck_OROBOROS, BE_Liege_Votion DM
Labels: MiParea: Respiration, Instruments;methods
Organism: Horse
Tissue;cell: Skeletal muscle
Preparation: Permeabilized tissue
Regulation: Coupling efficiency;uncoupling, Cyt c 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
Further information
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- >> MiPNet04.07 is integrated in MiPNet12.23.
- Version 1: 2007
- Version 6: 2011-12-11