SUIT-006 O2 mt D022: Difference between revisions
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|info='''A''': Protocol for coupling control assessment in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) '''[[SUIT-006]]''' | |info='''A''': Protocol for coupling control assessment in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) '''[[SUIT-006]]''' | ||
|application=O2 | |application=O2 | ||
|SUIT number=D022_1SRot;2D;2c;4U;5Ama | |SUIT number=D022_1SRot;2D;2c;3(Omy);4U;5Ama | ||
}} | }} | ||
::: '''[[MitoPedia: SUIT]]'''ย ย | ::: '''[[MitoPedia: SUIT]]'''ย ย |
Revision as of 16:38, 28 March 2019
Description
Abbreviation: S
Reference: A: Protocol for coupling control assessment in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) SUIT-006
SUIT number: D022_1SRot;2D;2c;3(Omy);4U;5Ama
O2k-Application: O2
- MitoPedia: SUIT
- SUIT-category: S
- SUIT protocol pattern: linear coupling control protocol 1SRot;2D;2c;4U;5Ama
SUIT-006 O2 mt D022 is a coupling control protocol for mitochondrial preparations (tissue homogenate, isolated mitochondria and permeabilized cells). Different coupling control states are assessed (L(n) - P - E) at succinate pathway control state. The use of rotenone, that inhibits complex I, provides a specific coupling control protocol for S-pathway, avoiding reverse electron transfer (RET). The use of oligomycin is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated LEAK respiration measured in the abscence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately.
Communicated by Cardoso LH, Komlodi T, Huete-Ortega M and Doerrier C (last update 2019-03-28)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1SRot | SL | S | CII | 1SRot
|
2D | SP | S | CII | 1SRot;2D
|
2c | ScP | S | CII | 1SRot;2D;2c
|
(3Omy) | ScL(Omy) | 1SRot;2D;2c;(3Omy)
| ||
4U | SE | S | CII | 1SRot;2D;2c;4U
|
5Ama | ROX | 1SRot;2D;2c;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 recommended when the aim is to analyse coupling control in mtprep (see MitoPedia: Sample preparations) at Succinate pathway control state.
- + The combination of succinate with rotenone, that inhibits complex I, provides the best coupling control protocol specific for S-pathway, avoiding reverse electron transfer (RET).
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - This protocol does not include internal ET-pathway control steps.
- - Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors.
Compare SUIT protocols
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
Krumschnabel 2014 Methods Enzymol | 2014 | Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. https://doi.org/10.1016/B978-0-12-416618-9.00009-1 | Mouse | Nervous system |
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