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: RP1-short pfi

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

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

O2k-Application: O2

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. Permeabilized muscle fibers are sensitive to oxygen supply due to limited diffusion of oxygen to the fiber bundle core. To counteract this limitation, hyperoxic conditions (400-250 µM O2) must be employed. To set the optimal oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.

Communicated by Doerrier C, Gnaiger E (last update 2019-06-05)

Representative traces

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
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
## AsTm AsTmE CIV CIV
## Azd ROX


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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

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

Chemicals and syringes

Step Chemical(s) and link(s) Comments
1PM Pyruvate (P) and Malate (M)
2D ADP (D)
2c Cytochrome c (c)
3U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
4S Succinate (S)
5Rot Rotenone (Rot)
6Ama Antimycin A (Ama)
Step Chemical(s) and link(s) Comments
## AsTm Ascorbate (As) and TMPD (Tm)
## Azd Azide (Azd)
Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry