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| | {{OROBOROS header page name}} |
| {{Publication | | {{Publication |
| |title=[[Image:O2k-Protocols.jpg|right|80px|link=http://wiki.oroboros.at/index.php/O2k-Protocols|O2k-Protocols contents]] MitoPathways at the Q-junction. | | |
| |info=[[Gnaiger 2014 MitoPathways]] | | |title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols contents]] MitoPathways at the Q-junction: mouse skeletal muscle fibers. |
| |authors=OROBOROS | | |info=[[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/2/2d/MiPNet12.01_Suppl_T-issue.pdf |Bioblast pdf]] »[http://www.bioblast.at/index.php/File:MiPNet12.01_Suppl_T-issue.pdf Versions] |
| |year=2014-07 | | |authors=Oroboros |
| | |year=2018-11-18 |
| |journal=Mitochondr Physiol Network | | |journal=Mitochondr Physiol Network |
| |abstract=[[File:MiPNet12.01 SupplementaryT-issue.jpg|left|500px|Supplementary T-issue: MitoPathways at the Q-junction]] | | |abstract= Oroboros (2018) MitoPathways at the Q-junction: mouse skeletal muscle fibers. Mitochondr Physiol Network 12.01(03): Suppl T-issue. |
| '''OROBOROS (2014) MitoPathways at the Q-junction. Mitochondr Physiol Network 12.01(02): Supplementary T-issue.'''
| | {{MiPNet pdf page linking to MitoPedia}} |
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| [[High-resolution respirometry]] and OXPHOS analysis is presented as supplementary '''''T-issue''''' ([[OROBOROS]] T-shirt).
| | |mipnetlab=AT_Innsbruck_Oroboros |
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| [[Pyruvate]]+[[glutamate]]+[[malate]] (PGM) were used in combination to induce C<sub>I</sub>-linked [[LEAK respiration]] in permeabilized mouse skeletal muscle (O2k-experiment 2007-04-14 A-03; IOC39, April 2007, Schröcken, Vorarlberg, Austria). Saturating [[ADP]] (D) stimulated respiration to the level of [[OXPHOS capacity]] (''P'' state), with a small effect of 10 µM [[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).
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| |mipnetlab=AT_Innsbruck_OROBOROS
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| {{Labeling | | {{Labeling |
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| |tissues=Skeletal muscle | | |tissues=Skeletal muscle |
| |preparations=Permeabilized tissue | | |preparations=Permeabilized tissue |
| |couplingstates=LEAK, ROUTINE, ETS | | |couplingstates=LEAK, ROUTINE, ET |
| |substratestates=CI, CII, CI+II, ROX | | |pathways=N, S, NS, ROX |
| |instruments=Oxygraph-2k, Protocol | | |instruments=Oxygraph-2k, O2k-Protocol |
| |additional=MitoPathways, O2k-Demo, O2k-Core | | |additional=MitoPathways, O2k-Demo, O2k-Core |
| }} | | }} |
| == Limitations of the protocol ==
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| === Maximum OXPHOS and ETS capacity ===
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| 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>]]).
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| === ROX correction ===
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| 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. Therefore, a single measurement of ROX cannot be applied for correction of total oxygen consumption in the different substrate states. Total respiration, therefore, represents apparent coupling states ''L''´, ''P''´ and ''E''´ (Fig. 1). ROX correction is possible in the present experiment only for C<sub>I+II</sub>- and C<sub>II</sub>-linked respiration. [[Azide]] inhibits not only C<sub>IV</sub> but other heme-based oxidases and peroxidases, and therefore may interfere with ROX beyond blocking respiratory electron transfer. Based on this argument, a combination of C<sub>II</sub>- and C<sub>III</sub>-inhibitors (malonic acid, antimycin A, myxothiazol) may yield more consistent results, although any ROS scavenged by cytochrome ''c'' may in the absence of a C<sub>IV</sub>-inhibitor result in respiratory oxygen consumption through C<sub>IV</sub>.
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| === Malate concentration ===
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| The [[malate]] concentration was 2 mM, to saturate C<sub>I</sub>-linked respiration. However, at 2 mM malate, the fumarate concentration is increased to a level which inhibits succinate dehydrogenase. Then C<sub>I+II</sub>- and C<sub>II</sub>-linked respiratory capacities are underestimated. A malate concentration of 0.5 mM, which saturates C<sub>I</sub>-linked respiration and inhibits C<sub>II</sub>-linked respiration to a lesser extent, represents and improved standard.
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| » [[Talk:Malate |Optimum malate concentration in SUIT protocols]]
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| :» Product: [http://www.oroboros.at/?oxygraph OROBOROS Oxygraph-2k], [[O2k-Catalogue_OROBOROS| O2k-Catalogue]]
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MiPNet12.01 Suppl T-issue
| MitoPathways at the Q-junction: mouse skeletal muscle fibers.
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» 
»Versions
Oroboros (2018-11-18) Mitochondr Physiol Network
Abstract: Oroboros (2018) MitoPathways at the Q-junction: mouse skeletal muscle fibers. Mitochondr Physiol Network 12.01(03): Suppl T-issue.
- Open the pdf document above.
• O2k-Network Lab: AT_Innsbruck_Oroboros
Labels: MiParea: Respiration
Organism: Mouse
Tissue;cell: Skeletal muscle
Preparation: Permeabilized tissue
Coupling state: LEAK, ROUTINE, ET
Pathway: N, S, NS, ROX
HRR: Oxygraph-2k, O2k-Protocol
MitoPathways, O2k-Demo, O2k-Core
