SUIT-027 O2 ce-pce D065: Difference between revisions

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SUIT-027 O2 ce-pce D065

Description

Abbreviation: Anaplerotic pathway

Reference: A - SUIT-027

SUIT number: D065_1S;2Rot;3D;4Ama

O2k-Application: AmR

MitoPedia: SUIT - Protocol for analysis of the anaplerotic pathway

SUIT protocol pattern: ce1;1Dig;1M;2D;3M;4P;5G

Anaplerotic pathway.

Communicated by  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).

Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

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

Compare SUIT protocols

References

MitoPedia concepts: SUIT protocol, SUIT B, Find

MitoPedia methods: Fluorometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
-|abbr=RET (respiratory control) of [[SUIT-026 AmR mt D064]]
+|abbr=Anaplerotic pathway
-|description=[[File:SUIT-026 AmR mt D064.png|400px]]
+|description=[[File:SUIT-027 O2 ce-pce D065.png|400px]]
-|info='''A''' - '''[[SUIT-026]]'''
+|info='''A''' - '''[[SUIT-027]]'''
 |application=AmR
-|SUIT number=D063_1S;2Rot;3D;4Ama
+|SUIT number=D065_1S;2Rot;3D;4Ama
 }}
-::: '''[[MitoPedia: SUIT]]''' - Protocol for the respiratory control of [[SUIT-026 AmR mt D064]]
+::: '''[[MitoPedia: SUIT]]''' - Protocol for analysis of the anaplerotic pathway
-::: '''[[SUIT protocol pattern]]:'''  1S;2Rot;3D;4Ama
+::: '''[[SUIT protocol pattern]]:'''  ce1;1Dig;1M;2D;3M;4P;5G
-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.
+Anaplerotic pathway.
 __TOC__
-  Communicated by  [[Komlodi T]], [[Iglesias-Gonzalez J]] and [[Gnaiger E]] (last update 2019-06-26)
+  Communicated by  [[Iglesias-Gonzalez J]] and [[Gnaiger E]] (last update 2019-06-26)
 {{Template:SUIT-027 O2 ce-pce D065}}
 == 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 [[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-006 O2 mt D022]]: CCP mtprep for S-pathway.
-::::* [[SUIT-026 AmR mt D064]]: Protocol to evaluate the RET production of ROS.
 == References ==