SUIT-007 O2 ce-pce D030

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SUIT-007 O2 ce-pce D030

Description

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

Abbreviation: Glutamate anaplerotic pathway

Reference: A pce: permeabilized cells - SUIT-007

SUIT number: D030_ce1;1Dig;1G;2D;2c;3M;4U;5Ama

O2k-Application: O2

MitoPedia: SUIT
SUIT protocol pattern:ce1;1Dig;1G;2D;2c;3M;4U-
SUITbrowser: Glutamine/glutamate

The SUIT-007 O2 pce D030 is focused on the glutamate anaplerotic pathway. If glutamate dehydrogenase is present in the sample when glutamate is added alone, 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. In this protocol, non-permeabilized cells are added in the chamber, and ROUTINE respiration is measured. The cells are further permeabilized with digitonin inside the O2k chamber.

Communicated by Cardoso LH, Komlodi T and Gnaiger E (last update 2019-07-03)
MitoPedia: SUIT

Steps and respiratory states

Ce1;1Dig;1G;2D;2c;3M;4U;5Ama.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 ROX ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
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 the analysis of glutamate anaplerotic pathway/glutaminolysis in permeabilized cells (ce-pce).
+ The use of glutamate alone allows to analyze 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.


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References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry