SUIT-026

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SUIT-026

Description

1S;2Rot;3D;4Ama.png

Abbreviation: RET

Reference: A Short protocol to study RET-related H2O2 production

SUIT protocol pattern: 1S;2Rot;3D;4Ama

SUIT-026 AmR mt D064 has been designed to study the reverse electron transport (RET)-initiated ROS production, while SUIT-026 O2 mt D063 serves as a respiratory control protocol for SUIT-026 AmR mt D064 in mitochondrial preparations: isolated mitochondria and tissue homogenate and permeabilized cells (which are already permeabilized when they are added to the chamber). 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. 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.


Communicated by Komlodi T and Gnaiger E (last update 2019-06-26)

Specific SUIT protocols

SUIT-026 AmR mt D064

1S;2Rot;3D;4Ama.png SUIT-026 AmR mt D064 O2 trace.png SUIT-026 AmR mt D064 AmR trace.png

SUIT-026 AmR mt D064 to study RET-initiated H2O2 production using isolated mtiochondria, tissue homogenate or permeabilized cells.

SUIT-026 O2 mt D063

1S;2Rot;3D;3c;4Ama.png SUIT-026 O2 mt D063 O2 trace.png

SUIT-026 O2 mt D063 serves as a control for SUIT-026 AmR mt D064 in order to study the effect of the AmR assay on the respiration.


MitoPedia: SUIT

Steps and respiratory states

1S;2Rot;3D;4Ama.png


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
  • Addition of cytochrome c yields a test for integrity of the mtOM. Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (P, OXPHOS-capacity with saturating [ADP]).
  • Succinate, S ( type S-pathway to Q).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
4Ama ROX 1S;2Rot;3D;3c;4Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of Antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).



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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.
  • Many fluorescence dyes can inhibit components of the ET system, thus affecting the respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye. Choose SUIT-026 O2 mt D063 as a control for SUIT-026 AmR mt D064.
+ Rotenone provides an excellent control step for RET owing to its inhibitory effect on RET leading to decreased ROS formation.
+ Short duration of the experiment.
- CIV activity and cytochrome c test cannot be performed together with the fluorescence. SUIT-026 O2 mt D063 can be used in parallel, with cytochrome c addition to assess the mt outer membrane integrity as a quality control of the sample.
- This protocol does not provide information about all the coupling control states (LEAK, OXPHOS and ET). However, it is possible to create a DLPU by adding an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.
- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.


Compare SUIT protocols

  • SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potential on the N-control state in isolated mitochondria and tissue homogenate.
  • SUIT-006 O2 mt D022: CCP mtprep for S-pathway.


References

MitoPedia concepts: SUIT protocol, SUIT protocol, Recommended 


MitoPedia methods: Fluorometry