SUIT-022 O2 ce D051

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SUIT-022 O2 ce D051

Description

Ce1;ce2KCN;ce3SHAM.v2.png Ce1;ce2KCN;ce3SHAM.png

Abbreviation: AOX-ce CN+SHAM

Reference: A Determination of the respiration due to the alternative oxidase pathway -SUIT-022

SUIT number: D051_ce1;ce2KCN;ce3SHAM

O2k-Application: O2

SUIT protocol pattern: ce1;ce2KCN;ce3SHAM

The SUIT-022 O2 ce D051 provides an estimation of mitochondrial respiration due to the Q-alternative oxidase pathway (Q-AOX pathway), which is cyanide-and antimycin A-resistant, for the specific case of microalgal cells. This protocol must be complemented with the study in parallel of mitochondrial respiration through the CIII- cytochrome c oxidase pathway (CIII-CIV; see SUIT-023).

Communicated by Huete-Ortega M and Gnaiger E (last update 2019-06-15)
MitoPedia: SUIT

Steps and respiratory states

Ce1;ce2KCN;ce3SHAM.v2.png Ce1;ce2KCN;ce3SHAM.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
ce1 ROUTINE ce1
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
ce2KCN ROUTINE or ROX ce1;ce2KCN
  • Cyanide (usually added as KCN) is a competitive inhibitor of cytochrome c oxidase (CIV). In the presence of an active alternative oxidase (AOX) pathway, after the inhibition of CIV, cells remain in the physiological coupling state ROUTINE. In those occasions where the AOX is not active, after the inhibition of CIV, the (ROX state) is normally achieved, where Rox is the residual oxygen consumption due to oxidative side reactions estimated either after inhibition of CIII, CIV or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration. Cyanide inhibition is reversed by pyruvate and high oxygen levels.
ce3SHAM ROX ce1;ce2KCN;ce3SHAM


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Strengths and limitations

  • SUIT-022 O2 ce D051 provides an estimation of the cyanide-and antimycin A-resistant mitochondrial respiration due to the activity of the alternative oxidase in microalgal cells.
  • The concentrations of inhibitors used in this protocol have been optimised for the specific case of the microalgal model species Chlamydomonas reinhardtii
+ Short duration of experiment
- No estimation of coupling control
- The use of cyanide requires a careful cleaning of the chambers after use and careful handling of the chemical to guarantee health safety.


Compare SUIT protocols

  • SUIT-023 O2 ce D053 has to be used in parallel to obtain complete information of the contribution of both Q-AOX and CIII-CIV pathways to mitochondrial respiration.


References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry