SUIT-006 O2 mt D047: Difference between revisions
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ย Communicated by [[Cardoso | ย Communicated by [[Cardoso LHD]], [[Iglesias-Gonzalez J]], [[Komlodi T]] and [[Gnaiger E]] (last update 2019-06-28) ย | ||
{{Template:SUIT-006 O2 mt PM}} | {{Template:SUIT-006 O2 mt PM}} |
Revision as of 14:10, 1 August 2019
Description
Abbreviation: CCP mt PM
Reference: A SUIT-006 - is a coupling control protocol in the N-pathway for isolated mitochondria, tissue homogenate and cells permeabilized before addition to the O2k chamber.
SUIT number: D047_1PM;2D;2c;3Omy;4U;5Ama
O2k-Application: O2
- SUIT protocol pattern: 1PM;2D;2c;3Omy;4U;5Ama
SUIT-006 O2 mt D047 is designed to study different coupling control states (L(n) - P - L(Omy) - E) at N(PM) pathway control state. The use of oligomycin is optional, however, it provides important information when endogenous adenylates are present and ATPases are active in the sample. This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately.
Communicated by Cardoso LHD, Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-06-28)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M)
|
---|---|---|---|---|
1PM | PML(n) | N | CI | 1PM
|
2D | PMP | N | CI | 1PM;2D
|
2c | PMcP | N | CI | 1PM;2D;2c
|
3Omy | PML(Omy) | N | CI | 1PM;2D;2c;3Omy
|
4U | PME | N | CI | 1PM;2D;2c;3Omy;4U
|
5Ama | ROX | 1PM;2D;2c;3Omy;4U;5Ama
|
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- 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
- This protocol is indicated when the aim is to analyse coupling control in mitochondrial preparations in NADH-pathway using PM as substrates.
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - This protocol analyses the coupling control in NADH-pathway (with PM), however, it is less specific than a similar protocol containing the complex II inhibitor malonic acid, that would allow to analyse the NADH-pathway without any contribution of the S-pathway. A DLPU can be created, with the addition of an event for malonic acid right before the addition of the N-linked substrates.
- - Oligomycin may lead to underestimation of ET capacity. This may be prevented by the addition of low concentrations of oligomycin, which should be previously tested.
- - Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors, if used.
Compare SUIT protocols
- SUIT-001 O2 mt D001: Extended protocol with SUIT-006 without oligomycin as the initial coupling control module, for mitochondrial preparations.
- SUIT-003: Coupling control protocol for non-permeabilized cells (ce), counterpart to SUIT-006 for mt-preparations (mtprep).
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry