SUIT-007 O2 ce-pce D030: Difference between revisions
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|SUIT number=D030_ce1;1Dig;1G;2D;2c;3M;4U;5Ama | |SUIT number=D030_ce1;1Dig;1G;2D;2c;3M;4U;5Ama | ||
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::: '''[[SUITbrowser]]:''' [[Glutamate-anaplerotic pathway control state|Glutamine/glutamate]] | ::: '''[[SUITbrowser]]:''' [[Glutamate-anaplerotic pathway control state|Glutamine/glutamate]] | ||
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== Compare SUIT protocols == | == Compare SUIT protocols == | ||
::::* [[SUIT-006]]: [[Coupling-control protocol]], can be performed with GM. | ::::* [[SUIT-006]]: [[Coupling-control protocol]], can be performed with GM. | ||
== Chemicals and syringes == | |||
{{Template:Chemicals ce}} | |||
{{Template:Chemicals SUIT-007}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == |
Latest revision as of 09:00, 17 September 2020
Description
Abbreviation: Glutamate anaplerotic pathway
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 is added alone, 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 Electron transfer pathway coupling control states. In this protocol, non-permeabilized cells are added in the chamber, and ROUTINE respiration is measured. The cells are further permeabilized with digitonin inside the O2k chamber.
Communicated by Cardoso LH, Komlodi T and Gnaiger E (last update 2019-07-03)
Representative traces
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
|
- 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 the analysis of glutamate anaplerotic pathway/glutaminolysis in permeabilized cells (ce-pce).
- + The use of glutamate alone allows to analyze 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.
Compare SUIT protocols
- SUIT-006: Coupling-control protocol, can be performed with GM.
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1Dig | Digitonin (Dig) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1G | Glutamate (G) | |
2D | ADP (D) | |
2c | Cytochrome c (c) | |
3M | Malate (M) | |
4U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
5Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry