SUIT-006 O2 mt D047

high-resolution terminology - matching measurements at high-resolution

SUIT-006 O2 mt D047

Description

Abbreviation: N(PM)

Reference: A SUIT-006 is to analyse the coupling control in NADH-pathway on mitochondrial prepartions (isolated mitochondria and tissue homogenate).

SUIT number: D047_1PM;2D;2c;3Omy;4U;5Ama

O2k-Application: O2

SUIT-category: N(PM)

SUIT protocol pattern: Bidirectional linear coupling control protocol 1PM;2D;2c;3Omy;4U;5Ama

Communicated by Cardoso LH and Komlodi T (last update 2019-02-20) 

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1PM	PML(n)	N	CI	1PM NADH-linked substrates (type N-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	PMP	N	CI	1PM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	PMcP	N	CI	1PM;2D;2c NADH-linked substrates (type N-pathway to Q). 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	PML(Omy)	N	CI	1PM;2D;2c;3Omy NADH-linked substrates (type N-pathway to Q). 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 respiration. In these cases, LEAK state is not reached again.
4U	PME	N	CI	1PM;2D;2c;3Omy;4U NADH-linked substrates (type N-pathway to Q). 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			1PM;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 isolated mitochondria or tissue homogenates in NADH-pathway using PM as substrates.

+ Reasonable duration of the experiment.

+ This protocol can be extended with the Complex IV module.

- This protocol analyses the coupling control in NADH-pathway (with PM), however, it is less specific than a similar protocol containing the complex II inhibitor malonic acid, that would allow to analyse the NADH-pathway without any contribution of the S-pathway.

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

Compare SUIT protocols

• SUIT-001 O2 mt D001: Extended protocol with SUIT-006 without oligomycin as the initial coupling control module, for mitochondrial preparations.
• SUIT-003: Coupling control protocol for non-permeabilized cells (ce), counterpart to SUIT-006 for mt-preparations (mtprep).

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry