SUIT-004 O2 pfi D010

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SUIT-004 O2 pfi D010

Description

1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd.png

Abbreviation: NS(PM)

Reference: A: Short protocol linked to SUIT-001 O2 pfi D002 - SUIT RP1 (human skeletal muscle)-SUIT-004

SUIT number: D010_1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd

O2k-Application: O2

MitoPedia: SUIT
SUIT-category: NS(PM)
SUIT protocol pattern: orthogonal 1PM;2D;2c;3U;4S;5Rot

The SUIT-004 O2 pfi D010 protocol provides information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS and S) can be evaluated by using this SUIT protocol. The SUIT-004 O2 pfi D010 can be extended with the CIV assay module.


Communicated by Iglesias-Gonzalez J and Gnaiger E (last update 2019-01-21)
MitoPedia: SUIT

Steps and respiratory states

1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd.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).
  • 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]).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
3U PME N CI 1PM;2D;3U
4S PMSE NS CI&II 1PM;2D;3U;4S
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • Noncoupled electron transfer state, ET-state, with ET-capacity, E.
5Rot SE S CII 1PM;2D;3U;4S;5Rot
6Ama ROX 1PM;2D;3U;4S;5Rot;6Ama
  • 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).
Step Respiratory state Pathway control ET-Complex entry into Q-junction Comment
## Tm TmE CIV CIV
## Azd ROX


Questions.jpg


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Strengths and limitations

  • A succinate concentration of >10 mM may be required for saturating SE capacity.
  • CIV assessment is skipped if a prolonged protocol is not practical due to time limitation.
  1. RP1-6Ama may be skipped since correction of the initial states by ROX measured in RP1-6 Ama may represent an over-correction, and inhibition takes a very long time in several cases (Pesta 2012 Methods Mol Biol).
+ Linear coupling control (L- P- E) with NADPH-linked substrate substrates (PM).
+ Pathway control in ET state (N, NS, S).
+ This protocol can be extended with the Complex IV module.
+ Short experimental time.
- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-004 in favor of reducing the time consumed during the experiment.

Compare SUIT protocols

  • SUIT-001 O2 pfi D002: A comparable but more comprehensive protocol comprising additional substrate states.
Specific cases
  • imt, ROX: NS(PM)01_imt,1PM,2D(c),3U,4S,5Rot,6Ama
  • pfi, high O2, no ROX: NS(PM)01_pfiO2,1PM,2D(c),3U,4S,5Rot

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry