SUIT-023

high-resolution terminology - matching measurements at high-resolution

SUIT-023

Description

Abbreviation: ce

Reference: A: Determination of the respiration due to the cytochrome c oxidase pathway »Versions

SUIT-category: ce

SUIT protocol pattern: linear ce1;ce2SHAM;ce3KCN

The SUIT-023 is designed to provide an estimation of the mitochondrial respiration in cells due to the cytochrome c oxidase (CIV) pathway, which is non-resistant to cyanide-and antimycin A inhibition. This protocol must be complemented with the study in parallel of the mitochondrial respiration due to the alternative oxidase pathway (see SUIT-022).

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

Specific SUIT protocols

SUIT-023 O2 ce D053

SUIT-023 O2 ce D053 for intact microalgal cells (ce)

Steps and respiratory states

Step	State	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 cytochrome c oxidase (CIV) branched respiration, B(SHAM), 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 Cyanide (usually added as KCN) is a competitive inhibitor of cytochrome c oxidase (CIV). 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.

Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

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-023 provides an estimation of the cyanide-and antimycin A-non-resistant mitochondrial respiration due to the activity of the cytochrome c oxidase (CIV) pathway in cells. To complement the study with the estimation of the mitochondrial respiration due to the alternative oxidase pathway, it must be carried out in parallel to SUIT-022.

+ This protocol can be used with many type of cells, although optimal concentrations of inhibitors have to be optimised.

+ 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-022 O2 ce D051 for microalgal intact cells

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry