SUIT-006

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SUIT-006

Description

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Abbreviation: N or S

Reference: A »Versions

SUIT-category: N or S

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

DLP applications

SUIT-006 O2

References

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 mt preparations.
Different combinations of substrates and inhibitors can be used depending on the specific aims, e.g.: PM, GM, MnaPM or other combinations for N-pathway control state; S or RotS for S-pathway control state.

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

+ Short duration of the experiment.

- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

- CIV activity is not measured, to save experimental time.

Compare SUIT protocols

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry