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 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+- 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 and Garcia-Souza LF (last update 2019-07-01)

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 due to mitochondrial malic enzyme in the studied sample.
  2. 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.


Compare SUIT protocols

  • SUIT-007: Protocol to analyze glutamate anaplerotic pathway.
  • SUIT-002: 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.

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry