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

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

O2k-Application: O2

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, Gnaiger E (last update 2019-09-24)

Representative traces

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.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1S	Succinate (S)
2Rot	Rotenone (Rot)
3D	ADP (D)
3c	Cytochrome c (c)
4Ama	Antimycin A (Ama)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT B, Find

MitoPedia methods: Fluorometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
-|abbr=RET (Respiratory control)
+|abbr=RET (respiratory control) of [[SUIT-026 AmR mt D064]]
-|description=[[File:SUIT-026 AmR mt D064.png|400px]]
+|description=[[File:1S;2Rot;3D;3c;4Ama.png|400px]]
-|info='''A''' - '''[[SUIT-026]]'''
+|info='''B''' - '''[[SUIT-026]]''' - Protocol for the respiratory control of [[SUIT-026 AmR mt D064]]
-|application=AmR
+|application=O2
-|SUIT number=D064_1S;2Rot;3D;4Ama
+|SUIT number=D063_1S;2Rot;3D;3c;4Ama
 }}
-::: '''[[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 [[Reverse_electron_flow_from_CII_to_CI| 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.
+{{Template:SUIT text D063}}
+__TOC__
+ Communicated by  [[Komlodi T]], [[Gnaiger E]] (last update 2019-09-24)
+== Representative traces ==
-__TOC__
+[[File:SUIT-026 O2 mt D063 O2 trace.png|600px]]
- Communicated by  [[Iglesias-Gonzalez J]] and [[Gnaiger E]] (last update 2019-06-26)
 {{Template:SUIT-026 O2 mt D063}}
 == Strengths and limitations ==
-:::* The conditions under 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 protocol does not allow to obtain all the coupling states of the sample.
 :::+ Short protocol.
-:::+ Rotenone provides an excellent control step for RET.
+:::+ 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 ==
+::::* [[SUIT-026 AmR mt D064]]: Protocol to evaluate the RET production of ROS.
 ::::* [[SUIT-006 O2 mt D022]]: CCP mtprep for S-pathway.
-::::* [[SUIT-026 AmR mt D064]]: Protocol to evaluate the RET production of ROS.
+== Chemicals and syringes ==
+{{Template:Chemicals SUIT-026 O2 mt D063}}
+::: Suggested stock concentrations are shown in the specific DL-Protocol.
@@ Line 33: / Line 40: @@
 {{MitoPedia concepts
-|mitopedia concept=SUIT protocol, SUIT A, Find
+|mitopedia concept=SUIT protocol, SUIT B, Find
 }}
 {{MitoPedia methods