From Bioblast
Description
Abbreviation: N(GM)
Reference: A pce: permeabilized cells - SUIT-007
SUIT number: D030_ce1;1Dig;1G;2D;2c;3M;4U;5Ama
O2k-Application: O2
The SUIT-007 O2 pce D030 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 Ξ±-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.
- SUIT finder: Glutamate anaplerotic pathway
- SUIT-category: N(GM)
- SUIT protocol pattern: diametral ce1;1Dig;1G;2D;2c;3M;4U;5Ama
Communicated by Cardoso LH, Komlodi T and Gnaiger E (last update 2019-02-20)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
1Dig | REN | ce1;1Dig
|
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
|
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
|
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- Coupling control
- Pathway control
- Β» Electron transfer pathway
- Β» Fatty acid oxidation pathway control state, F
- Β» NADH electron transfer-pathway state, N
- Β» Succinate pathway control state, S
- Β» NS-pathway control state, NS
- Β» Glycerophosphate pathway control state, Gp
- Β» Complex IV single step, CIV
- Β» Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- Β» Glutamate, G
- Β» Glycerophosphate, Gp
- Β» Malate, M
- Β» Octanoylcarnitine, Oct
- Β» Pyruvate, P
- Β» Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- Protocol focused on analysis of glutamate anaplerotic pathway/glutaminolysis in permeabilized cells (pce).
- + 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.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
Compare SUIT protocols
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry