SUIT-007

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SUIT-007

Description

1G;2D;3M;4U-.png

Abbreviation: Glutamate anaplerosis

Reference: A: Glutamate anaplerotic pathway

SUIT protocol pattern: 1G;2D;2c;3M;4U;5Ama

The SUIT-007 is focused on the glutamate anaplerotic pathway. If glutamate dehydrogenase is present in the sample, when glutamate alone is added, it will be converted to α-ketoglutarate in an anaplerotic reaction, supporting respiration. After malate is added, it is possible to analyse the NADH Electron transfer-pathway state with glutamate and malate as substrates in the OXPHOS and ET coupling control states.


Communicated by Cardoso LH, Komlodi T, Doerrier C and Gnaiger E (last update 2019-02-25)

Specific SUIT protocols

Ce1;1Dig;1G;2D;2c;3M;4U;5Ama.png D030 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

1G;2D;3M;4U-.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1G GL (N) CI 1G
2D GP (N) CI 1G;2D
2c GcP (N) CI 1G;2D;2c
  • Addition of cytochrome c yields a test for integrity of the mtOM. Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (P, OXPHOS-capacity with saturating [ADP]).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
3M GMP N CI 1G;2D;2c;3M
4U GME N CI 1G;2D;2c;3M;4U
5Ama ROX 1G;2D;2c;3M;4U;5Ama
  • 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

  • Protocol focused on analysis of glutamate anaplerotic pathway/glutaminolysis.
+ The use of glutamate alone allows to analyse the glutamate anaplerotic pathway control state.
+ Short duration of the experiment.
+ Glutamate is easier to prepare compared to pyruvate.
+ CIV activity can be measured after this protocol.
- Analysis of N-OXPHOS capacity (instead of NS-OXPHOS capacity) does not provide the best estimate of maximum mitochondrial respiratory capacity.
- This protocol does not address questions regarding the convergence of pathways at the Q-junction.
- Addition of cytochrome c after glutamate alone (1G;2D;2c) is not as appropriated to evaluate integrity of the mitochondrial external membrane as it would be with GM. However, with addition of cytochrome c only after malate (e.g. 1G;2D;3M;3c), it would not be possible to compare GP and GMP if there are damages to the mitochondrial external membrane, therefore it is suggested to add cytochrome c after ADP (1G;2D;2c).
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.


Compare SUIT protocols

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry