Description
Abbreviation: PM+S+Rot
Reference: A to analyse O2 flux and the redox state of the Q-pool in the N-,NS- and S- pathway control state on isolated mitochondria- SUIT-031
SUIT number: D072_1PM;2D;3S;4Rot;5U;5Anoxia;6Ama
O2k-Application: Q
SUIT-031 Q mt D072 is a protocol to investigate simultaneously oxygen flux and the Q-redox state in the N- and S-pathway control state in OXPHOS and in the S-pathway control state in the ET state in isolated mitochondria. After the addition of mitochondria in the absence of fuel substrates and ADP, Rox with endogenous substrates (Ren) is detected due to oxidation of endogenous substrates remaining after mitochondrial isolation.
Directly after the sample addition, coenzyme Q2 mimetic was titrated since the naturally occurring CoQ is trapped in the mitochondrial inner membrane. CoQ2 reacts both with the mitochondrial complexes at the Q-binding site (CI, CII, and CIII) and with the detecting electrode of the Q-Sensor. Application of the lowest possible CoQ2 concentration is recommended to avoid any side reactions on the ETS caused by the mimetics. Our study shows that 1 µM CoQ2 was sufficient to detect the Q-redox change without an influence on respiration.
The addition of PM leads to a partial reduction of CoQ2 which is reflected in the increase of the Q-signal. ADP oxidizes CoQ2, reflected in the decrease of the Q-signa. Addition of S initiates NS-OXPHOS capacity and results in further reduction of CoQ2 in the OXPHOS state. Rotenone via CI inhibition reduces O2 flux to S-linked OXPHOS capacity and causes oxidation of CoQ2. The uncoupler CCCP oxidizes CoQ2, however, using mouse cardiac mitochondria (see figures) U did not influence either O2 flux or the Q redox state which means that the respiration is not limited by the phosphorylation system in this sample type.
Anoxia was reached when the mitochondria consumed the oxygen in the O2k-chambers. In the absence of O2, the ETS upstream of CIV is reduced and thus leads to the full reduction of CoQ2. This step is used as a reference step when calculating the Q redox fraction. At the end of the protocol, the CIII inhibitor antimycin A can be added to check its effect on the fully reduced CoQ2 under anoxia.
in the DatLab software, SUIT-031 DLP files are currently provided for different applications. For O2 application with ce-pce, choose [[]], for mt, choose SUIT-031. For Q redox state detection with ce-pce, choose SUIT-031 Q ce-pce D074.
How to measure the Q redox state, see: MiPNet24.12 NextGen-O2k: Q-Module in preparation
Communicated by Komlodi T (last update 2021-02-08)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1Q2 | 1Q2
| |||
1PM | PML(n) | N | CI | 1Q2;1PM
|
2D | PMP | N | CI | 1Q2;1PM;2D
|
3S | PMSP | NS | CI&II | 1Q2;1PM;2D;3S
|
4Rot | SP | S | CII | 1Q2;1PM;2D;3S;4Rot
|
5U | SE | N | CII | 1Q2;1PM;2D;3S;4Rot;5U
|
6Anox | 1Q2;1PM;2D;3S;4Rot;5U;6Anox
| |||
7Ama | ROX | 1Q2;1PM;2D;3S;4Rot;5U;6Anox;7Ama
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- To study the effect of coenzyme Q2 on mitochondrial respiration, the following protocol can be used in a parallel experiment after permeabilization of the plasma membrane with digitonin: [[ ]].
- Addition of Ama can be omitted if a sufficient sample amount is available to detect the fully reduced Q state.
- + Reasonable duration of the experiment.
- - The fully oxidized Q state cannot be detected with rotenone in this protocol. This protocol can be run simultaneously with SUIT-006 Q mt D071 to determine the fully oxidized Q-pool. 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.
- - CIV activity cannot be determined and cytochrome c test cannot be performed together with the Q-Sensor.
- + Cytochrome c test can be performed in the following protocol: [[ ]].
- + This protocol can be extended with the Complex IV module in the following protocol: [[ ]].
- - 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.
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 |
---|---|---|
1Q2 | Coenzyme Q2 (Q) |
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 | Komlódi 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:
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