Difference between revisions of "MiPNet12.01 Suppl T-issue"
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[[High-resolution respirometry]] with a [[SUIT protocol]] for [[OXPHOS]]<sup>1</sup> analysis is presented as supplementary '''''T-issue''''' ([[OROBOROS]] T-shirt). | [[High-resolution respirometry]] with a [[SUIT protocol]] for [[OXPHOS]]<sup>1</sup> analysis is presented as supplementary '''''T-issue''''' ([[OROBOROS]] T-shirt). | ||
[[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) (Fig. | [[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) (Fig. O2).<sup>2</sup> Saturating [[ADP]] (D; 2.5 mM final concentration) 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'', in striking contrast to human skeletal muscle mitochondria.<sup>3</sup> 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 [[Respiratory complexes |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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=== Maximum OXPHOS and ETS capacity === | === 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>]]). | 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>]]). | ||
=== Malate concentration === | === Malate concentration === | ||
Revision as of 13:02, 20 July 2014
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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 with a SUIT protocol for OXPHOS1 analysis is presented as supplementary T-issue (OROBOROS T-shirt).
Pyruvate+glutamate+malate (PGM) were used in combination to induce CI-linked LEAK respiration in permeabilized mouse skeletal muscle (O2k-experiment 2007-04-14 A-03; IOC39, April 2007, Schröcken, Vorarlberg, Austria) (Fig. O2).2 Saturating ADP (D; 2.5 mM final concentration) 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 (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, in striking contrast to human skeletal muscle mitochondria.3 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).
• 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 SUIT 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).
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
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. 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 CI+II- and CII-linked respiration. Azide inhibits not only CIV 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 CII- and CIII-inhibitors (malonic acid, antimycin A, myxothiazol) may yield more consistent results, although any ROS scavenged by cytochrome c may in the absence of a CIV-inhibitor result in respiratory oxygen consumption through CIV.
References
- ↑ Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 3rd ed. Mitochondr Physiol Network 19.12. OROBOROS MiPNet Publications, Innsbruck: 64 pp. »Open Access
- ↑ OROBOROS (2014) Oxygraph-2k manual titrations: SUIT protocols with mitochondrial preparations. Mitochondr Physiol Network 09.12(11): 1. »Open Access
- ↑ Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837-1845. »PMID: 19467914
- » Product: OROBOROS Oxygraph-2k, O2k-Catalogue
