SUIT-026 AmR mt D064

high-resolution terminology - matching measurements at high-resolution

SUIT-026 AmR mt D064

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. 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				DTPA is an iron chelator, which decreases the chemical fluorescence background created by the Amplex UltraRed assay. Administration of DTPA into the O2k-chamber is recommended before all other chemicals because the iron chelation capacity of the compound is time-dependent (approx. 10-15 min). However, the experiments can be carried out in the absence of DTPA.
0SOD				SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H₂O₂, making it accessible to the Amplex UltraRed assay.
0HRP				HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H₂O₂ towards the fluorescent resorufin.
0AmR				AmR or Amplex UltraRed forms resorufin with H₂O₂ catalysed by horseradish peroxidase HRP.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1S	S_L(n)	S	CII	1S Succinate, S ( type S-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2Rot	S_L(n)	S	CII	1S;2Rot Succinate, S ( type S-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates). Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway.
3D	S_P	S	CII	1S;2Rot;3D Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4Ama	ROX			1S;2Rot;3D;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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Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

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.

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