SUIT-007: Difference between revisions
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:::* Protocol focused on analysis of glutamate anaplerotic pathway/glutaminolysis. | :::* Protocol focused on analysis of glutamate anaplerotic pathway/glutaminolysis. | ||
:::+ The use of glutamate alone allows to analyse the glutamate anaplerotic pathway control state. | :::+ The use of glutamate alone allows to analyse the glutamate anaplerotic pathway control state. | ||
:::+ Short duration of the experiment. | :::+ Short duration of the experiment. | ||
:::+ Glutamate is easier to prepare compared to pyruvate. | :::+ Glutamate is easier to prepare compared to pyruvate. |
Revision as of 22:10, 20 February 2019
Description
Abbreviation: N(GM)
Reference: A: Glutamate anaplerotic pathway ยปVersions
- SUIT-category: N(GM)
- SUIT protocol pattern: diametral 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 ฮฑ-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, Doerrier C and Gnaiger E (last update 2019-01-22)
Specific SUIT protocols
- SUIT-007 O2 pce D030 for permeabilized cells
Steps and respiratory states
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
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- ยป Electron transfer pathway
- ยป Fatty acid oxidation pathway control state, F
- ยป NADH electron transfer-pathway state, N
- ยป Succinate pathway control state, S
- ยป NS-pathway control state, NS
- ยป Glycerophosphate pathway control state, Gp
- ยป Complex IV single step, CIV
- ยป Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- ยป Glutamate, G
- ยป Glycerophosphate, Gp
- ยป Malate, M
- ยป Octanoylcarnitine, Oct
- ยป Pyruvate, P
- ยป Succinate, S
- Main fuel substrates
- Glossary
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
- SUIT-006: Coupling control protocol, can be performed with GM.
References
MitoPedia concepts: MiP concept, SUIT protocol, Recommended
MitoPedia methods:
Respirometry