SUIT-020 O2 mt D032

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SUIT-020 O2 mt D032

Description

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

Abbreviation: Q-junction additivity and respiratory control for membrane potential

Reference: A: short protocol for determination of O2 flux in mitochondrial preparations (isolated mitochondria, permeabilized cells, and tissue homogenate)-SUIT-020

SUIT number: D032_1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama

O2k-Application: O2

MitoPedia: SUIT
SUIT protocol pattern: orthogonal 1PM;2D;2c;3G;4S;5Rot;6Omy;7U-

This is a protocol to assess the additivity between N-pathway and S- pathway in the Q-junction as well as investigate the N- and NS-pathway control state in mitochondrial preparations. It can serve as a diagnostic tool for the activity of glutamate dehydrogenase and its linked pathway. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Oligomycin has to be titrated in small amounts to avoid overtitration and inhibition of the flux in the ET-state. The Complex III inhibitor Antimycin A (Ama) 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 is can be used as a control experiment for SUIT-020 Fluo mt D033 without using the fluorescence dye to evaluate its effect on the O2 flux as a control.

Communicated by Huete-Ortega M, Komlodi T and Gnaiger E  (last update 2019-06-06)  
MitoPedia: SUIT

Steps and respiratory states

1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama.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.
3G PGMP N CI 1PM;2D;2c;3G
4S PGMSP NS CI&II 1PM;2D;2c;3G;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.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
5Rot SP S CII 1PM;2D;2c;3G;4S;5Rot
6Omy SL(Omy) 1PM;2D;2c;3G;4S;5Rot;6Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
7U* SE S CII 1PM;2D;2c;3G;4S;5Rot;6Omy;7S
8Ama ROX 1PM;2D;2c;3G;4S;5Rot;6Omy;7S;8Ama
  • 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.
  • The fluorescence dye can be added after cytochrome c to test its effect on the respiration.
+ You obtain information in a single protocol about the NS-, S and N- pathway control state.
+ This protocol can be used as a control for SUIT-020 Fluo mt D033 to test the effect of the fluorescence dyes on the respiration.
+ 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 on 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 on 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