SUIT-027

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SUIT-027

Description

1M;2D;3M;4P;5G-.png

Abbreviation: Malate anaplerosis

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

Ce1;1Dig;1M;2D;3M;4P;5G;6Ama.png D065 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

1M;2D;3M;4P;5G;6Ama.png


Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1M.05 (ML) 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 ( MP) ce1;1Dig;1M;2D


3M MP N CI ce1;1Dig;1M;2D;3M
4P PMP N CI ce1;1Dig;1M;2D;3M;4P
5G PGMP N CI ce1;1Dig;1M;2D;3M;4P;5G
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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Strengths and limitations

+ Multiple titrations with malate provide information on:
  1. The presence of malate anaplerotic pathway (e.g. mt-malic enzyme) in the studied sample.
  2. 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