SUIT-021 O2 mt D035

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SUIT-021 O2 mt D035

Description

1GM;2D;2c;3S;4Rot;5Omy;6U;7Ama.png

Abbreviation: NS(GM)

Reference: A:short protocol for simultaneous determination of O2 flux and mitochondrial membrane potential in mitochondrial preparations (isolated mitochondria, permabilized cells, and tissue homogenate)-SUIT-021

SUIT number: D035_1GM;2D;2c;3S;4Rot;5Omy;6U;7Ama

O2k-Application: O2

MitoPedia: SUIT
SUIT-category: NS(GM)
SUIT protocol pattern: orthogonal 1GM;2D;2c;3S;4Rot;5Omy;6U-

This is a protocol to assess the additivity between N-pathway and S- pathway in the Q-junction and to investigate the N- and NS-pathway control state in mitochondrial preparations . Oligomycin (Omy) is used to induce a LEAK state of respiration through inhibition of the ATP synthase. Higher concentration of Omy can decreases the ET state initiated by an uncoupler, therefore the required concentration of Omy has to be determined. Uncoupler increases the respiration and induces the ET state. Antimycin A (Ama), which is an inhibitor of Complex III), blocks the respiration. Originially this is a protocol for simultaneous determination of O2 flux and mitochondrial membrane potential on isolated mitochondria and tissue homogenate. It can be used as a control experiment for SUIT-021 Fluo mt D036 without using the fluorescence dye in order to evaluate its effect on the O2 flux as a control.

Communicated by Huete-Ortega M, Komlodi T and Gnaiger E  (last update 2019-04-03)
MitoPedia: SUIT

Steps and respiratory states

1GM;2D;2c;3S;4Rot;5Omy;6U;7Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1GM GML(n) N CI 1GM
2D GMP N CI 1GM;2D
2c GMcP N CI 1GM;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.
3S GMSP NS CI&II 1GM;2D;2c;3S
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
4Rot SP S CII 1GM;2D;2c;3S;4Rot
5Omy SL(Omy) 1GM;2D;2c;3S;4Rot;5Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
6U* SE S CII 1GM;2D;2c;3S;4Rot;5Omy;6U
7Ama ROX 1GM;2D;2c;3S;4Rot;5Omy;6U;7Ama
  • 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


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Strengths and limitations

  • NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
  • Comparison of GM- with PM-capacity yields important information on N-pathway respiratory control upstream of CI.
  • A succinate concentration of >10 mM may be required for saturating SE capacity.
+ You obtain information in a single protocol about the NS-, S and N- pathway control state.
+ CIV activity determination and Cytochrome c test can be performed.
- Oligomycin concentration has to be determined. Higher concentrations of oligomycin may inhibit the ET state.
- Careful washing is required after the experiment to avoid carry-over of the inhibitors and uncoupler.

Compare SUIT protocols

  • SUIT-001;SUIT-001 O2 mt D001for mitochondrial preparations: a complex protocol to get information not only about the NS(PGM) in the OXPHOS state, but also in ET state and about the FGp-pathways.
  • SUIT-004;SUIT-004 O2 pfi D010: provides information about the NS(PM) pathway in the ET state without the contribution of G.
  • SUIT-008;SUIT-008 O2 pfi D014 and SUIT-008 O2 ce-pce D025: provide information about the NS(PGM) pathway in the OXPHOS and ET-states, but without the addition of Omy.
  • SUIT-011: provides information about the NS(GM) pathway in the OXPHOS and ET-states, but without the addition of Omy.
  • SUIT-012: provides information about the N(PGM) pathway in the OXPHOS and ET-states without the contribution of S-pathway and without the addition of Omy.
  • SUIT-014: a protocol similar to SUIT-021 O2 mt D035, but with the addition of P in the OXPHOS state before S. Omy is not added in the protocol.


References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry