Cytochrome c control efficiency: Difference between revisions
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|abbr=''FCF''<sub>c</sub> | |abbr=''FCF''<sub>c</sub> | ||
|description=The '''cytochrome ''c'' control factor''' expresses the control of respiration by externally added [[cytochrome c | cytochrome ''c'']], c, as a fractional change of flux from substrate state CHO to CHOc. In this [[flux control factor]] (''FCF''<sub>c</sub>), CHOc is the [[reference state]] with stimulated flux; CHO is the [[background state]] with CHO substrates, upon which c is added, ''FCF''<sub>c</sub> = (''J''<sub>CHOc</sub>-''J''<sub>CHO</sub>)/''J''<sub>CHOc</sub>. | |description=The '''cytochrome ''c'' control factor''' expresses the control of respiration by externally added [[cytochrome c | cytochrome ''c'']], c, as a fractional change of flux from substrate state CHO to CHOc. In this [[flux control factor]] (''FCF''<sub>c</sub>), CHOc is the [[reference state]] with stimulated flux; CHO is the [[background state]] with CHO substrates, upon which c is added, ''FCF''<sub>c</sub> = (''J''<sub>CHOc</sub>-''J''<sub>CHO</sub>)/''J''<sub>CHOc</sub>. | ||
[[Cytochrome_c_control_factor#Cytochrome_c_control:_a_test_for_outer_mt-membrane_integrity |» '''MiPNet article''']] | |||
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= Cytochrome ''c'' control: a test for outer mt-membrane integrity = | |||
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The outer mitochondrial membrane integrity can be evaluated by stimulation of respiration by exogenously added cytochrome ''c'' and expressing the normalized c-effect as the [[flux control factor]]<ref> Gnaiger E. Flux control factor: normalization of mitochondrial respiration. Mitochondr Physiol Network. »[[Flux control factor]]</ref>, ''FCF''<sub>c</sub>. | The outer mitochondrial membrane integrity can be evaluated by stimulation of respiration by exogenously added cytochrome ''c'' and expressing the normalized c-effect as the [[flux control factor]]<ref> Gnaiger E. Flux control factor: normalization of mitochondrial respiration. Mitochondr Physiol Network. »[[Flux control factor]]</ref>, ''FCF''<sub>c</sub>. | ||
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Treatment-triggered cytochrome ''c'' release, e.g. cell death induction, has to be distinguished from preparation induced damage. | Treatment-triggered cytochrome ''c'' release, e.g. cell death induction, has to be distinguished from preparation induced damage. | ||
If cytochrome ''c'' is released as a result of apoptosis induction, this is a biological phenomenon and a relevant result. | If cytochrome ''c'' is released as a result of apoptosis induction, this is a biological phenomenon and a relevant result. | ||
=== Interpretation of the cytochrome ''c'' control factor === | |||
Cytochrome ''c'' release throughout the mitochondrial population in a tissue ''in vivo'' may induce a pathophysiological limitation of respiratory capacity, since the released cytochrome c accumulates in the cytosol at a low (non-saturating) concentration. If all mitochondria in a particular cell release their cytochrome ''c'', then the cytosolic concentration increases to a level nearly saturating respiration (Brown and Buritaite #Ref). If the pathology involves a change of mt-membrane properties which render the mitochondria more vulnerable to experimental damage during tissue preparation, then the cytochrome ''c'' effect is to be interpreted differently. Such a pathophysiological modification is specifically revealed by the respirometric approach to OXPHOS analysis with SUIT protocols. | |||
== References == | == References == | ||
<references/> | <references/> | ||
Revision as of 07:09, 29 June 2014
- high-resolution terminology - matching measurements at high-resolution
Cytochrome c control efficiency
Description
[[Description::The cytochrome c control factor expresses the control of respiration by externally added cytochrome c, c, as a fractional change of flux from substrate state CHO to CHOc. In this flux control factor (FCFc), CHOc is the reference state with stimulated flux; CHO is the background state with CHO substrates, upon which c is added, FCFc = (JCHOc-JCHO)/JCHOc. » MiPNet article]]
Abbreviation: FCFc
Reference: [[Info::Gnaiger 2013 Abstract MiP2013]]
MitoPedia methods:
Respirometry
MitoPedia topics:
Respiratory control ratio,
Substrate and metabolite
Cytochrome c control: a test for outer mt-membrane integrity
| Cytochrome c control: a test for outer mt-membrane integrity. |
Abstract: [[has abstract::Gnaiger E (2014) Cytochrome c control: a test for outer mt-membrane integrity. Mitochondr Physiol Network 2014-04-21.
The cytochrome c control factor, FCFc, provides an index for the outer mt-membrane integrity.]]
• O2k-Network Lab: AT Innsbruck Gnaiger E
Labels:
HRR: Theory, Protocol
O2k-SOP
The outer mitochondrial membrane integrity can be evaluated by stimulation of respiration by exogenously added cytochrome c and expressing the normalized c-effect as the flux control factor[1], FCFc.
Determination of cytochrome c loss
Addition of cytochrome c (10 µM final concentration)[2] may stimulate OXPHOS capacity or ETS capacity and thus provides evidence for the occurrence of cytochrome c loss.
Cytochrome c test
When using cytochrome c as a quality control for permeabilised muscles from various species, which cytochrome c should we use (a wide range of types of cytochrome c is available from Sigma-Aldrich) and at which concentration?
We apply routinely cytochrome c from Sigma C 7752.[3] It would be interesting to compare the consequence of application of different sources of cytochrome c.
In a detailed discussion on the dependence of respiration in isolated mitochondria and permeabilized cardiac fibers, we showed for the first time showing that cytochrome c kinetics is different when studying a segment of the ETS (CII-linked: succinate+rotenone) versus the isolated step of cytochrome c oxidase (CIV; ascorbate+TMPD+antimycin A). For CII-linked respiration, an exernal cytochrome c concentration of 10 µM yields kinetic saturation (monophasic hyperbolic), but kinetics is biphasic for CIV and 10 µM is not saturating. [4],[5]
Importantly, cytochrome c increases the chemical background oxygen flux in the presence of ascorbate and TMPD, dependent on oxygen concentration and cytochrome c concentration, and appropriate chemical background corrections are required. [6],[7] Without ascorbate and TMPD, added cytochrome c is stable.
Evaluation of the cytochrome c effect, when respiration is slightly unstable: Mark respiration just before cytochrome c addition and after. Take these to values to calculate the increase of respiration due to cytochrome c addition.
At which step of the protocol should cytochrome c be added?
If a stimulatory effect of cytochrome c is observed, respiratory capacities measured before cytochrome c addition might be cytochrome c limited and therefore underestimated. This provides an argument to add cytochrome c at an early state of the protocol.
It is important not to add cytochrome c in a LEAK state: There is always an unexplained activation of respiration, unrelated to the injury of the outer mt-membrane. Add cytochrome c only after activation by ADP.
Cytochrome c release
Cytochrome c release induced by sample preparation
A preparation induced damage of the outer mitochondrial membrane and as a result subsequent loss of cytochrome c can be detected by a stimulation of respiration after the addition of cytochrome c. The preparation induced damage can also affect the respiratory complexes. Therefore, experimental runs showing a preparation induced cytochrome c release should be excluded from the final data set. In perfectly prepared muscle fibers cytochrome c should have no stimulatory effect on maximum respiratory activity, in liver biopsies a small effect is observed, even in carefully prepared samples.
Cytochrome c release induced by treatment
Treatment-triggered cytochrome c release, e.g. cell death induction, has to be distinguished from preparation induced damage. If cytochrome c is released as a result of apoptosis induction, this is a biological phenomenon and a relevant result.
Interpretation of the cytochrome c control factor
Cytochrome c release throughout the mitochondrial population in a tissue in vivo may induce a pathophysiological limitation of respiratory capacity, since the released cytochrome c accumulates in the cytosol at a low (non-saturating) concentration. If all mitochondria in a particular cell release their cytochrome c, then the cytosolic concentration increases to a level nearly saturating respiration (Brown and Buritaite #Ref). If the pathology involves a change of mt-membrane properties which render the mitochondria more vulnerable to experimental damage during tissue preparation, then the cytochrome c effect is to be interpreted differently. Such a pathophysiological modification is specifically revealed by the respirometric approach to OXPHOS analysis with SUIT protocols.
References
- ↑ Gnaiger E. Flux control factor: normalization of mitochondrial respiration. Mitochondr Physiol Network. »Flux control factor
- ↑ Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am J Physiol Heart Circ Physiol 286: H1633–H1641. »Open Access
- ↑ Fontana-Ayoub M, Fasching M, Gnaiger E (2014) Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr Physiol Network 03.02(17): 1-9. »Open Access
- ↑ Gnaiger E, Kuznetsov AV (2002) Mitochondrial respiration at low levels of oxygen and cytochrome c. Biochem Soc Trans 30: 242-248. »PMID: 12023860
- ↑ Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am J Physiol Heart Circ Physiol 286: H1633–H1641. »Open Access
- ↑ Renner K, Amberger A, Konwalinka G, Kofler R, Gnaiger E (2003) Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells. Biochim Biophys Acta 1642: 115-123. »PMID: 12972300
- ↑ Kuznetsov AV, Gnaiger E (2010) Oxygraph assay of cytochrome c oxidase activity: chemical background correction. Mitochondr Physiol Network 06.06(07): 1-4. »Open Access
