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
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 2019-04-03)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
|Step||Respiratory state||Pathway control||ET-Complex entry into Q-junction||Comment|
- » Selected keywords in SUIT protocols
- Pathway control
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-020 O2 mt D035, but with the addition of P in the OXPHOS state before S. Omy is not added in the protocol.
MitoPedia methods: Respirometry