Difference between revisions of "SUIT-026 O2 mt D063"

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SUIT-026 O2 mt D063

Description

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

Reference: B - SUIT-026

SUIT number: D063_1S;2Rot;3D;3c;4Ama

O2k-Application: O2

MitoPedia: SUIT - Protocol for the respiratory control of SUIT-026 AmR mt D064

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

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

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)
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.
3c	S_P	S	CII	1S;2Rot;3D;3c Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). 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 (OXPHOS capacity P 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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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

This protocol serves as a respiratory control (since fluorescence dyes may inhibit components of the ET system) and quality control for the sample for SUIT-026 AmR mt D064.

+ Short protocol.

+ The addition of cytochrome c to assess the mt outer membrane integrity provides a quality control of the sample, which cannot be performed in the protocol with Amplex UltraRed.

- 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-026 AmR mt D064: Protocol to evaluate the RET production of ROS.
SUIT-006 O2 mt D022: CCP mtprep for S-pathway.

References

MitoPedia concepts: SUIT protocol, SUIT B, Find

MitoPedia methods: Fluorometry

@@ Line 9: / Line 9: @@
 ::: '''[[SUIT protocol pattern]]:'''  1S;2Rot;3D;3c;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 [[Reverse_electron_flow_from_CII_to_CI| RET]]-initiated ROS production in [[Mitochondrial preparations| mitochondrial preparations ([[Isolated mitochondria|isolated mitochondria]] and [[Tissue homogenate|tissue homogenate]] and [[Permeabilized cells|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.
+SUIT-026 O2 mt D063 has been designed to serve as a respiratory control of the [[SUIT-026 AmR mt D064]] (protocol to study the [[Reverse_electron_flow_from_CII_to_CI| RET]]-initiated ROS production in [[Mitochondrial preparations|mitochondrial preparations]] ([[Isolated mitochondria|isolated mitochondria]], [[Tissue homogenate|tissue homogenate]] and [[Permeabilized cells|permeabilized cells]] which are already permeabilized when they are added to the chamber).
 __TOC__
@@ Line 19: / Line 17: @@
 == 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 serves as a respiratory control (since fluorescence dyes may inhibit components of the ET system) and quality control for the sample for [[SUIT-026 AmR mt D064]].
-:::* 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.
+:::+ The addition of cytochrome ''c'' to assess the [[Cytochrome_c#Performance_and_data_analysis|mt outer membrane integrity]] provides a quality control of the sample, which cannot be performed in the protocol with Amplex UltraRed.
 :::- This protocol does not provide information about all the coupling control states (LEAK, OXPHOS and ET). However, it is possible to create a [[Export DL-Protocol User (*.DLPU)|DLPU]] by additing an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.
 == Compare SUIT protocols ==