SUIT-027 O2 ce-pce D065

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SUIT-027 O2 ce-pce D065

Description

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

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

D065 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

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

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 (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 sample studied.
  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.
- 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