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.
MitoPedia concepts: Respiratory control ratio
MitoPedia methods: Respirometry
MitoPedia topics: Substrate and metabolite
Cytochrome c control: a test for outer mt-membrane integrity
|Gnaiger E (2014) Cytochrome c control: a test for outer mt-membrane integrity. Mitochondr Physiol Network 2014-04-21.|
Abstract: 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, O2k-Protocol Additional: O2k-SOP
Determination 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. 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 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. ,
- 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. , 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.
- ↑ 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. »Bioblast link«
- ↑ Gnaiger E, Kuznetsov AV (2002) Mitochondrial respiration at low levels of oxygen and cytochrome c. Biochem Soc Trans 30: 242-248. »Bioblast link«
- ↑ 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. »Bioblast link«
- ↑ 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. »Bioblast link«
- ↑ Laner V, Boushel RC, Hamilton KL, Miller BF, Williamson KK, Davis MS, Gnaiger E (2014) Cytochrome c flux control factor as a quality criterion in respiratory OXPHOS analysis in canine permeabilized fibres. Mitochondr Physiol Network 19.13. »Bioblast link«