SUIT-027 O2 ce-pce D065

high-resolution terminology - matching measurements at high-resolution

SUIT-027 O2 ce-pce D065

Description

Abbreviation: RET (respiratory control) of SUIT-026 AmR mt D064

Reference: A - SUIT-026

SUIT number: D063_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 to study the RET-initiated ROS production in [[Mitochondrial preparations| 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, 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)
ce1	ROUTINE			ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
1Dig	Dig			ce1;1Dig Optimum effective digitonin concentration for complete plasma membrane permeabilization.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1M.05	(M_L)			ce1;1Dig;1M Low concentration of malate, typically low concentration, does not saturate the N-pathway, and will allow us to find the minimum concentration to stimulate the anaplerosis.
2D	( M_P)			ce1;1Dig;1M;2D OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3M	M_P	N	CI	ce1;1Dig;1M;2D;3M NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4P	PM_P	N	CI	ce1;1Dig;1M;2D;3M;4P NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5G	PGM_P	N	CI	ce1;1Dig;1M;2D;3M;4P;5G NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
6Ama	ROX			ce1;1Dig;1M;2D;3M;4P;5G;6Ama 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.
The addition of cytochrome c is recommended for this SUIT protocol.
This protocol serves as a respiratory 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 protocol.

- 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 additing an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.

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 B, Find

MitoPedia methods: Fluorometry