SUIT-023 O2 ce D053

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SUIT-023 O2 ce D053

Description

Ce1;ce2SHAM;ce3KCN.png

Abbreviation: AOX-ce SHAM+CN

Reference: A Determination of the respiration due to the cytochrome c oxidase pathway -SUIT-023

SUIT number: D053_ce1;ce2SHAM;ce3KCN

O2k-Application: O2

SUIT protocol pattern: ce1;ce2SHAM;ce3KCN

The SUIT-023 O2 ce D053 provides an estimation of mitochondrial respiration in microalgal living cells through the CIII-cytochrome c oxidase pathway (CIII-CIV), which is inhibited by antimycin A and cyanide. This protocol must be complemented with the study in parallel of the mitochondrial respiration through the Q-alternative oxidase pathway (see SUIT-022).

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

Steps and respiratory states

Ce1;ce2SHAM;ce3KCN.png Ce1;ce2SHAM;ce3KCN.v2.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.
ce2SHAM ROUTINE OR ROX ce1;ce2SHAM
  • Salicylhydroxamic acid (SHAM) is an inhibitor of the alternative oxidase (AOX). When AOX is blocked by SHAM, electrons are forced through the cytochrome c oxidase (CIV) pathway.
  • After inhibition with SHAM, the ROUTINE state is normally achieved, defined as the respiration in the physiological coupling state where the mitochondrial respiration is through the cytochrome c oxidase (CIV) pathway (CIV). Occassionally, the cytochrome c oxidase pathway may not be active and then we have the ROX state, where Rox is the residual oxygen consumption due to oxidative side reactions estimated either after inhibition of CIII or 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.
ce3KCN ROX ce1;ce2SHAM;ce3KCN


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Strengths and limitations

  • SUIT-023 O2 ce D053 provides an estimation of the cyanide-and antimycin A-non-resistant mitochondrial respiration in microalgal living 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 careful cleaning of the chambers after use and careful handling of the chemical to guarantee health safety.


Compare SUIT protocols

  • SUIT-022 O2 ce D051 has to be used in parallel to obtain complete information of the contribution of the Q-AOX and CIII-CIV pathways to mitochondrial respiration.


References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry