SUIT-020 O2 mt D032: Difference between revisions
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ย Communicated by [[Huete-Ortega M]], | ย Communicated by [[Huete-Ortega M]], [[Komlodi T]] and [[Gnaiger E]]ย (last update 2019-04-03) | ||
ย ย ย | ย ย ย | ||
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:::* The fluorescence dye can be added after cytochrome c to test its effect on the respiration. | :::* 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-pathway control state| NS-]], [[Succinate pathway control state| S]] and [[NADH Electron transfer-pathway state| N-]] pathway control state. | :::+ You obtain information in a single protocol about the [[NS-pathway control state| NS-]], [[Succinate pathway control state| S]] and [[NADH Electron transfer-pathway state| N-]] pathway control state. | ||
:::+ This protocol can be used as a control for [[SUIT-020 Fluo mt D033]] to test the effect of | :::+ 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. ย | :::+ CIV activity determination and Cytochrome c test can be performed. ย | ||
:::- [[Oligomycin| Oligomycin]] concentration has to be determined. Higher concentrations of Oligomycin may inhibit the [[Electron transfer-pathway| ET state]]. | :::- [[Oligomycin| Oligomycin]] concentration has to be determined. Higher concentrations of Oligomycin may inhibit the [[Electron transfer-pathway| ET state]]. | ||
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:::* [[SUIT-001]];[[SUIT-001 O2 mt D001]]for 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-001]];[[SUIT-001 O2 mt D001]]for 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-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 | :::* [[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 | :::* [[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-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. | :::* [[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. |
Revision as of 10:25, 3 April 2019
Description
Abbreviation: NS(PGM)
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-category: NS(PGM)
- SUIT protocol pattern: orthogonal 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) |
---|---|---|---|---|
1PM | PML(n) | N | CI | 1PM
|
2D | PMP | N | CI | 1PM;2D
|
2c | PMcP | N | CI | 1PM;2D;2c
|
3G | PGMP | N | CI | 1PM;2D;2c;3G
|
4S | PGMSP | NS | CI&II | 1PM;2D;2c;3G;4S
|
5Rot | SP | S | CII | 1PM;2D;2c;3G;4S;5Rot
|
6Omy | SL(Omy) | 1PM;2D;2c;3G;4S;5Rot;6Omy
| ||
7U* | SE | S | CII | 1PM;2D;2c;3G;4S;5Rot;6Omy;7S
|
8Ama | ROX | 1PM;2D;2c;3G;4S;5Rot;6Omy;7S;8Ama
|
Step | Respiratory state | Pathway control | ET-Complex | Comment |
---|---|---|---|---|
## AsTm | AsTmE | CIV | CIV | |
## Azd | CHB |
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- ยป Electron transfer pathway
- ยป Fatty acid oxidation pathway control state, F
- ยป NADH electron transfer-pathway state, N
- ยป Succinate pathway control state, S
- ยป NS-pathway control state, NS
- ยป Glycerophosphate pathway control state, Gp
- ยป Complex IV single step, CIV
- ยป Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- ยป Glutamate, G
- ยป Glycerophosphate, Gp
- ยป Malate, M
- ยป Octanoylcarnitine, Oct
- ยป Pyruvate, P
- ยป Succinate, S
- Main fuel substrates
- Glossary
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.
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry