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
- SUIT-007 O2 ce-pce D030 for permeabilized cells
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
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- Pathway control
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.
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