SUIT-020 O2 mt D032: Difference between revisions
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::: '''[[SUIT protocol pattern]]:''' orthogonal 1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama | ::: '''[[SUIT protocol pattern]]:''' orthogonal 1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama | ||
This is a protocol to investigate the [[NADH Electron transfer-pathway state| N-]] and [[NS-pathway control state| NS-pathway]] control state on isolated mitochondria and tissue homogenate.ย Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. [[Oligomycin| Oligomycin]] has to be titrated in small amount in order to avoid overtitration and therefore, inhibition of the flux in the ET-state. Antimycin (Ama), which is an inhibitor of Complex III) blocks the respiration.ย ย | This is a protocol to investigate the [[NADH Electron transfer-pathway state| N-]] and [[NS-pathway control state| NS-pathway]] control state on isolated mitochondria and tissue homogenate.ย Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. [[Oligomycin| Oligomycin]] has to be titrated in small amount in order to avoid overtitration and therefore, inhibition of the flux in the ET-state. Antimycin (Ama), which is an inhibitor of Complex III) blocks the respiration.ย ย | ||
Originially this is a | Originially this is a protocol for simultaneous determination of O<sub>2</sub> 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 in order to detect the effect of it on the O<sub>2</sub> flux as a control. ย | ||
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Revision as of 11:46, 20 February 2019
Description
Abbreviation: NS(PGM)
Reference: A: SUIT-020 short protocol for determination of O2 flux in mitochondrial preparations (isolated mitochondria and tissue homogenate).
SUIT number: D032_1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama
O2k-Application: O2
- SUIT-category: NS(PGM)
- SUIT protocol pattern: orthogonal 1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama
This is a protocol to investigate the N- and NS-pathway control state on isolated mitochondria and tissue homogenate. 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 amount in order to avoid overtitration and therefore, inhibition of the flux in the ET-state. Antimycin (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 is can be used as a control experiment for SUIT-020 Fluo mt D033 without using the fluorescence dye in order to detect the effect of it on the O2 flux as a control.
Communicated by Huete-Ortega M, Komlodi T and Gnaiger E (last update 2019-02-20)
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 |
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- 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
- + You can get information in one protocol about the NS-, S and N- pathway control state.
- + This protocol can be used as a control for SUIT-020 Fluo mt D033.
- + CIV activity and Cytochrome c test can be performed.
- - Oligomycin concentration has to be determined. Higher concentration of Oligomycin than required can inhibit the ET state.
Compare SUIT protocols
- SUIT-001;SUIT-001 O2 mt D001for isolated mitochondria and tissue homogenate: 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 additionally.
- SUIT-004;SUIT-004 O2 pfi D010: it provides an information about the NS(PM) pathway in the ET state withou the contribution of G.
- SUIT-008;SUIT-008 O2 pfi D014 and SUIT-008 O2 pce D025: it provides information about the NS(PGM) pathway in the OXPHOS and ET-states, but without the addtion of Omy.
- SUIT-011:it provides information about the NS(GM) pathway in the OXPHOS and ET-states, but without the addtion of Omy.
- SUIT-012:it provides information about the N(PGM) pathway in the OXPHOS and ET-states wihtout the contribution of S-pathway and without the addtion of Omy.
- SUIT-014: it is similar protocol 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