SUIT-007: Difference between revisions
From Bioblast
No edit summary |
No edit summary |
||
Line 32: | Line 32: | ||
:::* Protocol focused on analysis of glutamate dehydrogenase anaplerotic pathway/glutaminolysis. | :::* Protocol focused on analysis of glutamate dehydrogenase anaplerotic pathway/glutaminolysis. | ||
:::+ The use of glutamate alone allows to analyse the glutamate dehydrogenase anaplerotic pathway control state. | :::+ The use of glutamate alone allows to analyse the glutamate dehydrogenase anaplerotic pathway control state. | ||
:::+ The addition of cytochrome c after ADP makes it possible to analyse both G<sub<P</sub> and GM<sub<P</sub>, enabling to compare the results even when there is loss of cytochrome c due to damage to the mitochondrial external membrane. | |||
:::+ 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. | ||
Line 37: | Line 38: | ||
:::- Analysis of N-OXPHOS capacity (instead of NS-OXPHOS capacity) does not provide the best estimate of maximum mitochondrial respiratory capacity. | :::- 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. | :::- 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 G<sub<P</sub> and GM<sub<P</sub> 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. | :::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler. | ||
Revision as of 17:01, 25 January 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
Communicated by Cardoso LH, Doerrier C and Gnaiger E (last update 2019-01-22)
DLP applications
- SUIT-007 O2 pce D030 for permeabilized cells
References
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 dehydrogenase anaplerotic pathway/glutaminolysis.
- + The use of glutamate alone allows to analyse the glutamate dehydrogenase anaplerotic pathway control state.
- + The addition of cytochrome c after ADP makes it possible to analyse both G<sub<P and GM<sub<P, enabling to compare the results even when there is loss of cytochrome c due to damage to the mitochondrial external membrane.
- + 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 G<sub<P and GM<sub<P 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.
MitoPedia concepts:
MiP concept,
SUIT protocol,
Recommended
MitoPedia methods:
Respirometry