Difference between revisions of "SUIT-027"

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SUIT-027

Description

Reference: A: Malate anaplerotic pathway

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

SUIT-027 is focused on the analysis of the malate anaplerotic pathway control state in mt-preparations (isolated mitochondria, tissue homogenate, permeabilized cells and permeabilized fibers). Malate alone does not support respiration of mt-preparations if oxaloacetate cannot be metabolized further in the absence of a source of acetyl-CoA. However, if malate anaplerotic pathway control state is present (e.g. mt-malic enzyme (mtME)), then malate alone supports mt-respiration. SUIT-027 includes a careful titration of malate which is required to analyze the malate anaplerotic pathway control state.

Communicated by Cardoso LHD, Garcia-Souza LF and Doerrier C (last update 2020-04-30)

Specific SUIT protocols

SUIT-027 O2 ce-pce D065 for permeabilized cells

Steps and respiratory states

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
3M	M_P	N	CI	ce1;1Dig;1M;2D;3M NADH-linked substrates (type N-pathway to Q). Malate titration in the presence of saturating [ADP] allows the evaluation of malate anaplerotic pathways.
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

+ Multiple titrations with malate provide information on:

The presence of malate anaplerotic pathway (e.g. mt-malic enzyme) in the studied sample.
Malate concentration which does not stimulate malate anaplerotic pathway, and therefore could be used to study F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways (see SUIT-002).

- This SUIT protocol does not analyze coupling control.

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

Compare SUIT protocols

SUIT-007: SUIT protocol to analyze glutamate anaplerotic pathway.
SUIT-002: SUIT-002 allows the evaluation of pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) whereas in SUIT-027 only malate anaplerotic pathway and N-pathway in OXPHOS state can be evaluated. 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 FN-pathway.
SUIT-025: SUIT-025 allows the evaluation of different ET-pathway states (F, F(N), FN, FNS and S) in OXPHOS state. 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 FN-pathway.

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended

MitoPedia methods: Respirometry

@@ Line 5: / Line 5: @@
 }}
 ::: '''[[SUIT protocol pattern]]:''' ce1;1Dig;1M;2D;3M;4P;5G
-SUIT-027 is focused on the analysis of the [[Malate anaplerotic pathway control state|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 in the absence of acetyl-CoA. The careful titration of malate is required to analyze the activity of the mt-isoform of NADP<sup>+</sup>- or NAD(P)<sup>+</sup>-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).
+SUIT-027 is focused on the analysis of the [[Malate anaplerotic pathway control state|malate anaplerotic pathway control state]] in [[mitochondrial preparations|mt-preparations]] (isolated mitochondria, tissue homogenate, permeabilized cells and permeabilized fibers). [[Malate]] alone does not support respiration of mt-preparations if oxaloacetate cannot be metabolized further in the absence of a source of acetyl-CoA. However, if malate anaplerotic pathway control state is present (e.g. [[Malic enzyme|mt-malic enzyme (mtME)]]), then malate alone supports mt-respiration. SUIT-027 includes a careful titration of malate which is required to analyze the malate anaplerotic pathway control state.
 __TOC__
-  Communicated by [[Cardoso LHD]] and [[Garcia-Souza LF]] (last update 2019-07-01)
+  Communicated by [[Cardoso LHD]], [[Garcia-Souza LF]] and [[Doerrier Carolina|Doerrier C]] (last update 2020-04-30)
 == Specific SUIT protocols ==
@@ Line 21: / Line 20: @@
 == Strengths and limitations ==
 :::+ Multiple titrations with [[malate]] provide information on:
-:::# The presence of malate anaplerotic pathway due to mitochondrial [[malic enzyme]] in the studied sample.
+:::# The presence of malate anaplerotic pathway (e.g. [[malic enzyme|mt-malic enzyme]]) in the studied sample.
-:::# 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.
+:::# Malate concentration which does not stimulate malate anaplerotic pathway, and therefore could be used to study [[F-pathway]] in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways (see [[SUIT-002]]).
-:::- This protocol does not analyze [[Coupling control state|coupling control]].
+:::- This SUIT protocol does not analyze [[Coupling control state|coupling control]].
-:::- Different [[pathway control states]] are not analyzed in this protocol.
+:::- Different [[pathway control states]] are not analyzed in this SUIT protocol.
 == Compare SUIT protocols ==
-::::* [[SUIT-007]]: Protocol to analyze glutamate anaplerotic pathway.
+::::* [[SUIT-007]]: SUIT 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.
+::::* [[SUIT-002]]: SUIT-002 allows the evaluation of pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) whereas in SUIT-027 only malate anaplerotic pathway and N-pathway in OXPHOS state can be evaluated. 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 FN-pathway.
+::::* [[SUIT-025]]: SUIT-025 allows the evaluation of different ET-pathway states (F, F(N), FN, FNS and S) in OXPHOS state. 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 FN-pathway.
 == References ==