MiPNet12.01 Suppl T-issue: Difference between revisions
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'''OROBOROS (2014) MitoPathways at the Q-junction. Mitochondr Physiol Network 12.01(02): Supplementary T-issue.''' | '''OROBOROS (2014) MitoPathways at the Q-junction. Mitochondr Physiol Network 12.01(02): Supplementary T-issue.''' | ||
High-resolution respirometry presented as supplementary '''''T-issue''''' (OROBOROS T-shirt). OXPHOS analysis in O2k-experiment 2007-04-14 A-03 (IOC39, April 2007, SchrΓΆcken, Vorarlberg, Austria). | High-resolution respirometry presented as supplementary '''''T-issue''''' (OROBOROS T-shirt). OXPHOS analysis in O2k-experiment 2007-04-14 A-03 (IOC39, April 2007, SchrΓΆcken, Vorarlberg, Austria). Pyruvate+glutamate+malate (PGM) were used in combination to induce C<sub>I</sub>-linked [[LEAK respiration]] in permeabilized mouse skeletal muscle. Saturating ADP (D) stimulated respiration to the level of [[OXPHOS capacity]] (''P'' state), with a small effect of 10 Β΅M cytochrome c (''c''), expressed as the [[Cytochrome c control factor |cytochrome ''c'' control factor]] (''FCF<sub>c</sub>''<0.5; indicating integrity of the outer mt-membrane). Addition of succinate (S) stimulated respiration by convergent e-input through the [[Q-junction]]. C<sub>I+II</sub> OXPHOS capacity was not stimulated further by uncoupler titration (U). Therefore, the capacity of the phosphorylation system matched the [[ETS capacity]] (''E'' state). At ''E=P'' the [[E-P coupling control factor |''E-P'' coupling control factor]] is zero, indicating that there is no ETS excess capacity over ''P''. Inhibition of C<sub>I</sub> by rotenone (Rot) inhibited respiration to the level of C<sub>II</sub>-linked ETS capacity, which was higher than C<sub>I</sub>-linked respiratory capacity (''E=P''). C<sub>I+II</sub>-linked respiratory capacity was higher than respiration with any single e-input substrate state, indicating an additive effect at the Q-junction. However, since C<sub>I+II</sub> < C<sub>I</sub> + C<sub>II</sub>, the additive effect was incomplete, which indicates that any electron channelling through supercomplexes to C<sub>IV</sub> was incomplete. Addition of azide (Azd) inhibited respiration to the level of [[residual oxygen consumption]] (ROX). Β | ||
:>> Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]] | :>> Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]] | ||
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|additional=MitoPathways, O2k-Demo, O2k-Core | |additional=MitoPathways, O2k-Demo, O2k-Core | ||
}} | }} | ||
== Limitations of the protocol == | |||
=== Maximum OXPHOS and ETS capacity === | |||
Evaluation of maximum respiratory capacities requires titration of further substrates activating additional respiratory complexes at the Q-junction ([[Electron-transferring flavoprotein complex |C<sub>ETF</sub>]] and [[glycerophosphate dehydrogenase complex |C<sub>GpDH</sub>]]). | |||
=== ROX correction === | |||
The fact that ROX was higher in the C<sub>I+II</sub> substrate state compared to C<sub>I</sub>-linked LEAK respiration indicates that ROX is partially controlled by the substrate state, and a single measurement of ROX can thus not be applied for correction of total oxygen consumption. Total respiration, therefore, represents apparent coupling states ''L''Β΄, ''P''Β΄ and ''E''Β΄. | |||
=== Malate concentration === | |||
The [[malate]] concentration was 2 mM, to saturate CI-linked respiration. However, at 2 mM malate, the fumarate concentration is increased to a level which inhibits succinate dehydrogenase. Then CI+II- and CII-linked respiratory capacities are underestimated. A malate concentration of 0.5 mM, which saturates CI-linked respiration and inhibits CII-linked respiration to a lesser extent, represents and improved standard. | |||
>> [[Talk:Malate |Optimum malate concentration in SUIT protocols]] |
Revision as of 19:28, 19 July 2014
MitoPathways at the Q-junction. |
OROBOROS (2014-07) Mitochondr Physiol Network
Abstract:
OROBOROS (2014) MitoPathways at the Q-junction. Mitochondr Physiol Network 12.01(02): Supplementary T-issue.
High-resolution respirometry presented as supplementary T-issue (OROBOROS T-shirt). OXPHOS analysis in O2k-experiment 2007-04-14 A-03 (IOC39, April 2007, SchrΓΆcken, Vorarlberg, Austria). Pyruvate+glutamate+malate (PGM) were used in combination to induce CI-linked LEAK respiration in permeabilized mouse skeletal muscle. Saturating ADP (D) stimulated respiration to the level of OXPHOS capacity (P state), with a small effect of 10 Β΅M cytochrome c (c), expressed as the cytochrome c control factor (FCFc<0.5; indicating integrity of the outer mt-membrane). Addition of succinate (S) stimulated respiration by convergent e-input through the Q-junction. CI+II OXPHOS capacity was not stimulated further by uncoupler titration (U). Therefore, the capacity of the phosphorylation system matched the ETS capacity (E state). At E=P the E-P coupling control factor is zero, indicating that there is no ETS excess capacity over P. Inhibition of CI by rotenone (Rot) inhibited respiration to the level of CII-linked ETS capacity, which was higher than CI-linked respiratory capacity (E=P). CI+II-linked respiratory capacity was higher than respiration with any single e-input substrate state, indicating an additive effect at the Q-junction. However, since CI+II < CI + CII, the additive effect was incomplete, which indicates that any electron channelling through supercomplexes to CIV was incomplete. Addition of azide (Azd) inhibited respiration to the level of residual oxygen consumption (ROX).
- >> Product: OROBOROS Oxygraph-2k, O2k-Catalogue
β’ O2k-Network Lab: AT_Innsbruck_OROBOROS
Labels: MiParea: Respiration
Organism: Mouse
Tissue;cell: Skeletal muscle
Preparation: Permeabilized tissue
Coupling state: LEAK, ROUTINE, 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.
MitoPathways, O2k-Demo, O2k-Core
Limitations of the protocol
Maximum OXPHOS and ETS capacity
Evaluation of maximum respiratory capacities requires titration of further substrates activating additional respiratory complexes at the Q-junction (CETF and CGpDH).
ROX correction
The fact that ROX was higher in the CI+II substrate state compared to CI-linked LEAK respiration indicates that ROX is partially controlled by the substrate state, and a single measurement of ROX can thus not be applied for correction of total oxygen consumption. Total respiration, therefore, represents apparent coupling states LΒ΄, PΒ΄ and EΒ΄.
Malate concentration
The malate concentration was 2 mM, to saturate CI-linked respiration. However, at 2 mM malate, the fumarate concentration is increased to a level which inhibits succinate dehydrogenase. Then CI+II- and CII-linked respiratory capacities are underestimated. A malate concentration of 0.5 mM, which saturates CI-linked respiration and inhibits CII-linked respiration to a lesser extent, represents and improved standard. >> Optimum malate concentration in SUIT protocols