SUIT-006

high-resolution terminology - matching measurements at high-resolution

SUIT-006

Description

Abbreviation: N, S, F, Gp

Reference: A: Coupling control protocol, mtprep »Versions

SUIT-category: N, S, F, Gp

SUIT protocol pattern: linear coupling control protocol 1X;2D;3Omy;4U-

The SUIT-006 protocol is a coupling control protocol for mitochondrial preparations. Depending on the aims of the researcher, different combinations of substrates can be used to assess coupling control at a defined electron transfer-pathway state. The use of oligomycin is optional, however, it provides important information when endogenous adenylates are present, a situation that may lead to overestimated LEAK respiration measured before ADP addition.

Communicated by Cardoso LH, Gnaiger E (last update 2019-01-21)

DLP applications

SUIT-006 O2 pce D029 for N-pathway with permeabilized cells. Omy may be added in order to not overestimate LEAK respiration, that might be overestimated in 1PM if there are endogenous adenylates.

SUIT-006 O2 mt D022 for S-pathway with isolated mitochondria.

References

	Year	Reference	Organism	Tissue;cell
MiPNet26.10 MgG data analysis	2021-09-16	Measurement of mitochondrial ATP production using Magnesium Green: DL-Protocols and data analysis with DatLab 7.4
Cardoso 2021 BEC MgG	2021	Cardoso LHD, Doerrier C, Gnaiger E (2021) Magnesium Green for fluorometric measurement of ATP production does not interfere with mitochondrial respiration. Bioenerg Commun 2021.1. https://doi.org/10.26124/bec:2021-0001	Mouse	Heart
Komlodi 2021 BEC AmR-O2	2021	Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004	Saccharomyces cerevisiae	Other cell lines
Krumschnabel 2014 Methods Enzymol	2014	Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. https://doi.org/10.1016/B978-0-12-416618-9.00009-1	Mouse	Nervous system

Steps and respiratory states

Step	State	Comment - Events (E) and Marks (M)
1X	X_L(n)	1X X: combination of substrates and inhibitors chosen according to the aims. Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	X_P	1X;2D OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	Xc_P	1X;2D;2c OXPHOS capacity P (with saturating [ADP]), active OXPHOS state. Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (OXPHOS capacity P with saturating [ADP]).
3Omy	X_LOmy	1X;2D;2c;3Omy Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin. The addition of oligomycin is optional depending on the experimental question to resolve. If it is known that L_n has the same values for L_Omy for a kind of sample and substrates, this step may be skipped. If the carrier of oligomycin (EtOH) is used instead, it is possible to evaluate whether it affects OXPHOS. In these cases, LEAK state is not reached again.
4U	X_E	1X;2D;2c;3Omy;4U Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency.
5Ama	ROX	1X;2D;2c;3Omy;4U;5Ama Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).

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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

This protocol is indicated when the aim is to analyse coupling control in mtprep (see MitoPedia: Sample preparations) at a defined electron transfer-pathway state.
Different combinations of substrates and inhibitors (X) can be used depending on the specific aims, e.g.: PGM, PM, GM, MnaPM or other combinations for N-pathway control state; S or RotS for S-pathway control state.
CIV activity can be measured as an extension.

+ The combinations of substrates for one ET-pathway state with inhibitors for other pathways provide the best coupling control protocols specific for one ET-pathway state, e.g.: MnaPM for N-pathway control state, RotS for S-pathway control state.

+ Short duration of the experiment.

- This protocol does not include internal ET-pathway control steps.

- Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors, if applied (e.g., rotenone & succinate, SRot-pathway control state).

- LEAK respiration in the absence of Omy may be overestimated due to ATP turnover stimulated by endogenous adenylates. This can be evaluated by SUIT 1X;2D;3Omy;4U;-. In mouse brain homogenate with succinate & rotenone, L_n and L_Omy are identical (Krumschnabel et al 2014).

Compare SUIT protocols

SUIT-001: Extended protocol with SUIT-006 as the initial coupling control module.
SUIT-004: Shortened version of SUIT-001, also with SUIT-006 as the initial coupling control module.
SUIT-003: Coupling control protocol for non-permeabilized cells (ce), counterpart to SUIT-006 for mt-preparations (mtprep).

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry, Fluorometry