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-

SUIT-020 O2 mt D032 is design 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. 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. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the required concentration of Omy has to be assessed by the Omy titration test. 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
## AsTm AsTmE 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