Difference between revisions of "SUIT-007"
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=[[Glutamate-anaplerotic pathway control state|Glutamate anaplerosis]] | ||
|description=[[File:1G;2D;3M;4U | |description=[[File:1G;2D;3M;4U-.png|300px]] | ||
|info='''A:''' '''Glutamate anaplerotic pathway''' | |info='''A:''' '''[[Glutamate-anaplerotic pathway control state|Glutamate anaplerotic pathway]]''' | ||
}} | }} | ||
::: '''[[SUIT protocol pattern]]:''' 1G;2D;2c;3M;4U;5Ama | |||
::: '''[[SUIT protocol pattern]]:''' | The SUIT-007 is focused on the [[Glutamate-anaplerotic pathway control state|glutamate anaplerotic pathway]]. If [[Glutamate dehydrogenase|glutamate dehydrogenase]] is present in the sample, when glutamate alone is added, it will be converted to Ξ±-ketoglutarate in an [[Anaplerosis|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 [[Electron transfer pathway]] [[Coupling control state|coupling control states]]. | ||
Β | |||
__TOC__ | __TOC__ | ||
Β Communicated by [[Cardoso LH]], [[Doerrier C]] and [[Gnaiger E]] (last update 2019- | Β Communicated by [[Cardoso LH]], [[Komlodi T]], [[Doerrier C]] and [[Gnaiger E]] (last update 2019-02-25) | ||
== | == Specific SUIT protocols == | ||
[[File:Ce1;1Dig;1G;2D;2c;3M;4U;5Ama.png|400px]] | |||
[[File:D030_O2_traces.png|500px]] | |||
::::* [[SUIT-007 O2 ce-pce D030]] for permeabilized cells | |||
{{Template:SUIT-007}} | {{Template:SUIT-007}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::* Protocol focused on analysis of glutamate | :::* Protocol focused on analysis of glutamate anaplerotic pathway/glutaminolysis. | ||
:::+ The use of glutamate alone allows to analyse the glutamate | :::+ 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. | ||
Line 37: | Line 26: | ||
:::- 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. | ||
== Compare SUIT protocols == | == Compare SUIT protocols == | ||
::::* [[SUIT-006]]: [[Coupling-control protocol]], can be performed with GM. | |||
== References == | |||
{{#ask:[[Category:Publications]] [[Instrument and method::O2k-Protocol]] [[Additional label::SUIT-007]] | |||
|?Was published in year=Year | |||
|?Has title=Reference | |||
|?Mammal and model=Organism | |||
|?Tissue and cell=Tissue;cell | |||
|format=broadtable | |||
|limit=5000 | |||
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|sort=Was published in year | |||
|order=descending | |||
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{{MitoPedia concepts | {{MitoPedia concepts |
Latest revision as of 11:30, 3 June 2020
Description
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 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 Electron transfer pathway 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) |
---|---|---|---|---|
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
- 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.
- + 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