Difference between revisions of "SUIT-027 O2 ce-pce D065"

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SUIT-027 O2 ce-pce D065

Description

Abbreviation: Malate anaplerosis

Reference: A - SUIT-027 - Protocol for analysis of the malate anaplerotic pathway

SUIT number: D065_ce1;1Dig;1M;2D;3M;4P;5G

O2k-Application: O2

The SUIT-027 O2 ce-pce D065 is focused on the analysis of the malate-anaplerotic pathway control state in mitochondrial preparations (isolated mitochondria, permeabilized cells, homogenate, and permeabilized fibers). Malate alone does not support respiration if oxaloacetate is not metabolized further in the absence of acetyl-CoA. The careful titration of malate is required to analyze the activity of the mt-isoform of NADP⁺- or NAD(P)⁺-dependent malic enzyme (mtME). If these enzymes are present in the sample one should be able to reach with malate alone a high respiratory activity comparable to the NADH-linked pathway control states with more classical combinations of substrates (e.g. PM or GM).

Communicated by  Cardoso LHD, Gnaiger E, Garcia-Souza LF (last update 2019-07-01)

Representative traces

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

+ Multiple titrations with malate provide information on:

The presence of malate anaplerotic pathway due to mitochondrial malic enzyme in the sample studied.
Malate concentration that does not stimulate malate anaplerotic pathway, and therefore could be used to study F-pathway without N-pathway contribution in samples with mt malic enzyme.

- This protocol does not analyze coupling control.

- Different pathway control states are not analyzed in this protocol.

- The DLP for SUIT-027 O2 ce-pce D065 is provided without the antimycin A addition step. However, it is possible to create a DLPU by adding an Event&Mark for the antimycin A titration (6Ama) after glutamate (5G) to obtain ROX.

Compare SUIT protocols

SUIT-007: Protocol to analyze glutamate anaplerotic pathway.
SUIT-002 and SUIT-025: A low concentration of malate is initially used to assess F-pathway without N-pathway contribution. Further, a higher concentration of malate is added to analyze N-pathway.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1Dig	Digitonin (Dig)

Step	Chemical(s) and link(s)	Comments
1M.05	Malate (M)
2D	ADP (D)
3M	Malate (M)
4P	Pyruvate (P)
5G	Glutamate (G)
6Ama	Antimycin A (Ama)

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

References

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Fluorometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
-|abbr=RET (respiratory control) of [[SUIT-026 AmR mt D064]]
+|abbr=Malate anaplerosis
-|description=[[File:SUIT-026 AmR mt D064.png|400px]]
+|description=[[File:Ce1;1Dig;1M;2D;3M;4P;5G;6Ama.png|500px]]
-|info='''A''' - '''[[SUIT-026]]'''
+|info='''A''' - '''[[SUIT-027]]''' - Protocol for analysis of the malate anaplerotic pathway
-|application=AmR
+|application=O2
-|SUIT number=D063_1S;2Rot;3D;4Ama
+|SUIT number=D065_ce1;1Dig;1M;2D;3M;4P;5G
 }}
-::: '''[[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 [[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.
+{{Template:SUIT text D065}}
+__TOC__
+ Communicated by  [[Cardoso LHD]], [[Gnaiger E]], [[Garcia-Souza LF]] (last update 2019-07-01)
+== Representative traces ==
-__TOC__
+[[File:D065 O2 traces.png|600px]]
- Communicated by  [[Komlodi T]], [[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.
+:::+ Multiple titrations with [[malate]] provide information on:
-:::* The addition of cytochrome ''c'' is recommended for this SUIT protocol.
+:::# The presence of malate anaplerotic pathway due to mitochondrial [[malic enzyme]] in the sample studied.
-:::* This protocol serves as a respiratory control for [[SUIT-026 AmR mt D064]].
+:::# Malate concentration that does not stimulate malate anaplerotic pathway, and therefore could be used to study [[F-pathway]] without [[N-pathway]] contribution in samples with mt malic enzyme.
-:::+ Rotenone provides an excellent control step for RET owing to its inhibitory effect on RET leading to decreased ROS formation.
+:::- This protocol does not analyze [[Coupling control state|coupling control]].
-:::+ Short protocol.
+:::- Different [[pathway control states]] are not analyzed in this 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.
+:::- The DLP for SUIT-027 O2 ce-pce D065 is provided without the [[antimycin A]] addition step. However, it is possible to create a [[Export DL-Protocol User (*.DLPU)|DLPU]] by adding an Event&Mark for the antimycin A titration (6Ama) after glutamate (5G) to obtain ROX.
 == Compare SUIT protocols ==
-::::* [[SUIT-006 O2 mt D022]]: CCP mtprep for S-pathway.
+::::* [[SUIT-007]]: Protocol to analyze glutamate anaplerotic pathway.
-::::* [[SUIT-026 AmR mt D064]]: Protocol to evaluate the RET production of ROS.
+::::* [[SUIT-002]] and [[SUIT-025]]: A low concentration of malate is initially used to assess F-pathway without N-pathway contribution. Further, a higher concentration of malate is added to analyze N-pathway.
+== Chemicals and syringes ==
+{{Template:Chemicals ce}}
+{{Template:Chemicals SUIT-027}}
+::: Suggested stock concentrations are shown in the specific DL-Protocol.
 == References ==
+{{#ask:[[Category:Publications]] [[Additional label::SUIT-027 O2 ce-pce D065]] [[Additional label::SUIT-027]]
+| ?Was published in year=Year
+| ?Has title=Reference
+| ?Mammal and model=Organism
+| ?Tissue and cell=Tissue;cell
+| format=broadtable
+| limit=5000
+| offset=0
+| sort=Was published in year
+| order=descending
+}}
 {{MitoPedia concepts
-|mitopedia concept=SUIT protocol, SUIT B, Find
+|mitopedia concept=SUIT protocol, SUIT A, Find
 }}
 {{MitoPedia methods