SUIT-031 O2 mt D075: Difference between revisions
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|info='''A'''ย to analyse O<sub>2</sub> fluxย in the NS- pathway control state on isolated mitochondria- '''[[SUIT-031]]''' | |info='''A'''ย to analyse O<sub>2</sub> fluxย in the NS- pathway control state on isolated mitochondria- '''[[SUIT-031]]''' | ||
|application=Q | |application=Q | ||
|SUIT number=D0745_1mt;1PM;2D;3S;4Rot;5U; | |SUIT number=D0745_1mt;1PM;2D;3S;4Rot;5U;6Ama | ||
}} | }} | ||
{{Template:SUIT text D075}} | {{Template:SUIT text D075}} |
Revision as of 10:55, 2 December 2021
Description
Abbreviation: PM+S+Rot
Reference: A to analyse O2 flux in the NS- pathway control state on isolated mitochondria- SUIT-031
SUIT number: D0745_1mt;1PM;2D;3S;4Rot;5U;6Ama
O2k-Application: Q
This is a protocol to investigate oxygen flux N- and NS-pathway and S-pathway control state in the OXPHOS state and S-pathway control state in the ET state in isolated mitochondria, tissue homogenate and cells permeabilized before addition in the O2k-chamber. SUIT-031 Q mt D072 and SUIT-031 Q ce-pce D074 are designed to measure simultaneously O2 flux and the Q-redox state in isolated mitochondria and permeabilized cells.
Communicated by Komlodi T (last update 2021-12-02)
Representative traces
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
|
3S | PMSP | NS | CI&II | 1PM;2D;2c;3S
|
4Rot | SP | S | CII | 1PM;2D;2c;3S;4Rot
|
5U | SE | N | CII | 1PM;2D;2c;3S;4Rot;5U
|
6Ama | ROX | 1PM;2D;2c;3S;4Rot;5U;6Ama
|
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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
- SUIT-031 Q ce-pce D074 in combination with SUIT-006 Q ce-pce D073 provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:
- 1. to obtain reference values.
- 2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.
- 3. to screen specific defects.
- Cytochrome c test can be performed in the following protocol: [[ ]].
- This protocol can be extended with the Complex IV module in the following protocol: [[ ]].
- To study the effect of CoQ2 on mitochondrial respiration, the following protocol can be used in a parallel experiment: [[ ]].
- + Reasonable duration of the experiment.
- - The fully oxidized CoQ2 cannot be detected with rotenone in this protocol. This protocol can be run simultaneously with SUIT-006 Q ce-pce D073 to determine the fully oxidized CoQ2. If it is not applicable, the effect of rotenone can be tested on the Q-signal in a separate experiment using the same sample under the same experimental conditions (same respiration medium, same sample concentration). If rotenone does not have a further effect on the Q signal in the presence of sample and coenzyme Q2, this test can be omitted.
- - Omy concentration has to be determined if used. Higher concentrations of Omy may inhibit the ET state.
- - Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors.
- - The concentration of the oxidized and reduced Q fraction cannot be determined.
- - CIV activity cannot be determined and cytochrome c test cannot be performed together with the Q-Sensor.
Compare SUIT protocols
- SUIT-006 O2 mt D047 is a coupling-control protocol in the N-pathway for isolated mitochondria, tissue homogenate and cells permeabilized before addition to the O2k- chamber.
- SUIT-004 is designed to provide a quick assessment of the linear coupling control (L- P- E) with NADH-linked substrates (PM) and the contribution of the S-pathway to the ET state (N, NS, S).
- SUIT-008 is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PM | Pyruvate (P) and Malate (M) | |
2D | ADP (D) | |
3S | Succinate (S) | |
4Rot | Rotenone (Rot) | |
5U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler. |
6Anox | The O2 concentration in the O2k-chamber can be decreased by N2 or H2 injection to reach faster anoxia, see: Setting the oxygen concentration. | |
7Ama | Antimycin A (Ama) | This step can be omitted. |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet24.12 NextGen-O2k: Q-Module | 2021-10-29 | NextGen-O2k: Q-Module manual | ||
Komlodi 2021 BEC Q | 2021 | Komlรณdi T, Cardoso LHD, Doerrier C, Moore AL, Rich PR, Gnaiger E (2021) Coupling and pathway control of coenzyme Q redox state and respiration in isolated mitochondria. Bioenerg Commun 2021.3. https://doi.org/10.26124/bec:2021-0003 | Mouse | Heart Nervous system |
MitoPedia concepts:
SUIT protocol,
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MitoPedia methods:
Respirometry