SUIT-022 O2 ce D051

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SUIT-022 O2 ce D051

Description

Abbreviation: ce

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

SUIT number: D051_ce1;ce2KCN;ce3SHAM

O2k-Application: O2

MitoPedia: SUIT

SUIT-category: ce

SUIT protocol pattern: linear ce1;ce2KCN;ce3SHAM

The SUIT-022 O2 ce D051 has been designed to provide an estimation of the mitochondrial respiration due to the alternative oxidase 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 the mitochondrial respiration due to the cytochrome c oxidase (CIV) pathway (see SUIT-023).

Communicated by Huete-Ortega M and Gnaiger E (last update 2019-04-09)

Steps and respiratory states

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), after the inhibition of CIV, cells respiration is branched through the alternative oxidase (AOX) pathway, achieving the B(CN)) respiration. 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 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. Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) 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.

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Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

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

- Estimation of the linear coupling control is not done

- The use of cyanide requires a careful and in deep 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 alternative oxidase and cytochrome c oxidase (CIV) pathways to mitochondrial respiration.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry