Description
Abbreviation: RET (Respiratory control)
Reference: A - SUIT-026
SUIT number: D064_1S;2Rot;3D;4Ama
O2k-Application: AmR
- MitoPedia: SUIT - Protocol for the respiratory control of SUIT-026 AmR mt D064
- SUIT protocol pattern: 1S;2Rot;3D;4Ama
SUIT-026 O2 mt D063 has been designed to serve as a respiratory control of the SUIT-026 AmR mt D064 study the RET production of ROS in isolated mitochondria. The protocol is focused on LEAK state for the S-pathway to provide the conditions of high membrane potential, redox power in the Q-junction and no presence of reduced NADH. 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. The last step, the titration of ADP at saturating concentrations to reach OXPHOS, allow us to harmonize this protocol with SUIT-006 O2 mt D022 for quality control and a full assessment of the coupling control.
Communicated by 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) |
---|---|---|---|---|
1S | SL(n) | S | CII | 1S
|
2Rot | SL(n) | S | CII | 1S;2Rot
|
3D | SP | S | CII | 1S;2Rot;3D
|
3c | SP | S | CII | 1S;2Rot;3D;3c
|
4Ama | ROX | 1S;2Rot;3D;3c;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.
- - The protocol does not allow to obtain all the coupling states of the sample.
- + Short protocol.
- + Rotenone provides an excellent control step for RET.
Compare SUIT protocols
- SUIT-006 O2 mt D022: CCP mtprep for S-pathway.
- SUIT-026 AmR mt D064: Protocol to evaluate the RET production of ROS.
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Fluorometry