SUIT-004

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SUIT-004

Description

1PM;2D;3U;4S;5Rot.png

Abbreviation: NS(PM)

Reference: A: short RP1 Bioblast pdf »Versions

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

The SUIT-004 protocols are designed to provide a quick assessment of the linear coupling control (L- P- E) with NADH-linked substrates (PM) and the contribution of the S-pathway to the ET state (N, NS, S). SUIT-004 can be extended with the CIV assay module.

Communicated by Doerrier C, Iglesias-Gonzalez J, Krumschnabel G and Gnaiger E (last update 2019-01-21)

Specific SUIT protocols

1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd.png SUIT 004.png

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.
+ Linear coupling control (L- P- E) with N 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 assayed in SUIT-004 in favour of reducing the experimental time.

Compare SUIT protocols

  • SUIT-001 (RP1): SUIT-004 is a shortened version of RP1; without G, F and Gp.

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry