Description
Abbreviation: RET
Reference: A - SUIT-026
SUIT number: D064_1S;2Rot;3D;4Ama
O2k-Application: AmR
- MitoPedia: SUIT - Protocol for Evaluation of ROS production by RET in isolated mitochondria
- SUIT protocol pattern: 1S;2Rot;3D;4Ama
SUIT-026 AmR mt D064 has been designed to study the RET-initiated ROS production in isolated mitochondria. The protocol is focused on LEAK state for the S-pathway to provide the conditions of high membrane potential and redox power in the Q-junction. Under these conditions, in several samples has been described that a reverse flow of the electrons into the membrane arm and the quinone binding site of the Complex I occurs, promoting the production of ROS. The addition of rotenone provides excellent control to this mechanism because this compound blocks the point on which the electrons leak from the Complex I leading to decreased ROS production. If rotenone would have been added before S titration, it had inhibited the RET. The titration of ADP at saturating concentrations to reach OXPHOS allows us to harmonize this protocol with SUIT-006 O2 mt D022 for quality control and a full assessment of the coupling control. Addition of Ama leads to increased ROS production via inhibition of Complex III at the Qi site.
Communicated by Komlodi T, Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-26)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
0DTPA |
| |||
0SOD |
| |||
0HRP |
| |||
0AmR |
|
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1S | SL(n) | S | CII | 1S
|
2Rot | SL(n) | S | CII | 1S;2Rot
|
3D | SP | S | CII | 1S;2Rot;3D
|
4Ama | ROX | 1S;2Rot;3D;4Ama
|
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- Coupling control
- Pathway control
- Β» Electron transfer pathway
- Β» Fatty acid oxidation pathway control state, F
- Β» NADH electron transfer-pathway state, N
- Β» Succinate pathway control state, S
- Β» NS-pathway control state, NS
- Β» Glycerophosphate pathway control state, Gp
- Β» Complex IV single step, CIV
- Β» Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- Β» Glutamate, G
- Β» Glycerophosphate, Gp
- Β» Malate, M
- Β» Octanoylcarnitine, Oct
- Β» Pyruvate, P
- Β» Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- The conditions on which RET is observable in isolated mitochondria are not physiological but it has been suggested that corresponds to some pathological states.
- + This protocol must be run parallel with SUIT-026 O2 mt D063 to have a respiratory control.
- + Short protocol.
- + Rotenone provides an excellent control step for RET.
- - The protocol does not allow to obtain all the coupling states of the sample. However, doing a DLPU to add an uncoupler will resolve this limitation.
Compare SUIT protocols
- SUIT-006 O2 mt D022: CCP mtprep for S-pathway.
- SUIT-026 O2 mt D063: Respiratory control protocol..
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry