SUIT-006 O2 mt D047

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SUIT-006 O2 mt D047

Description

1PM;2D;2c;3Omy;4U;5Ama.png

Abbreviation: CCP mt PM

Reference: A SUIT-006 - is a coupling control protocol in the N-pathway for isolated mitochondria, tissue homogenate and cells permeabilized before addition to the O2k chamber.

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

O2k-Application: O2

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

SUIT-006 O2 mt D047 is designed to study different coupling control states (L(n) - P - L(Omy) - E) at N(PM) pathway control state. The use of oligomycin is optional, however, it provides important information when endogenous adenylates are present and ATPases are active in the sample. This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately.

Communicated by Cardoso LHD, Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-06-28) 

Representative traces

SUIT-006 O2 mt D047.png

MitoPedia: SUIT

Steps and respiratory states

1PM;2D;2c;3Omy;4U;5Ama.png


Step State Pathway Q-junction Comment - Events (E) and Marks (M)


1PM PML(n) N CI 1PM
2D PMP N CI 1PM;2D
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. 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 (P, OXPHOS-capacity 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
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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Strengths and limitations

  • This protocol is indicated when the aim is to analyse coupling control in mitochondrial preparations 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. A DLPU can be created, with the addition of an event for malonic acid right before the addition of the N-linked substrates.
- Oligomycin may lead to underestimation of ET capacity. This may be prevented by the addition of low concentrations of oligomycin, which should be previously tested.
- 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