From Bioblast
Description
File:CCP for mt preparations.png
Abbreviation: N, S, F, Gp
Reference: A: Coupling control protocol, mtprep Β»Versions
- SUIT-category: N, S, F, Gp
- SUIT protocol pattern: linear coupling control protocol 1X;2D;3Omy;4U-
The SUIT-006 protocol is a coupling control protocol for mitochondrial preparations. Depending on the aims of the researcher, different combinations of substrates can be used to assess coupling control at a defined electron transfer-pathway state. The use of oligomycin is optional, however, it provides important information when endogenous adenylates are present, a situation that may lead to overestimated LEAK respiration measured before ADP addition.
Communicated by Cardoso LH, Gnaiger E (last update 2019-01-21)
Specific SUIT protocols
- SUIT-006 O2 pce D029 for N-pathway with permeabilized cells. Omy may be added in order to not overestimate LEAK respiration, that might be overestimated in 1PM if there are endogenous adenylates.
- SUIT-006 O2 mt D022 for S-pathway with isolated mitochondria.
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet26.10 MgG data analysis | 2021-09-16 | Measurement of mitochondrial ATP production using Magnesium Green: DL-Protocols and data analysis with DatLab 7.4 | ||
Cardoso 2021 BEC MgG | 2021 | Cardoso LHD, Doerrier C, Gnaiger E (2021) Magnesium Green for fluorometric measurement of ATP production does not interfere with mitochondrial respiration. Bioenerg Commun 2021.1. https://doi.org/10.26124/bec:2021-0001 | Mouse | Heart |
Komlodi 2021 BEC AmR-O2 | 2021 | KomlΓ³di T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004 | Saccharomyces cerevisiae | Other cell lines |
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 |
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1X | XL(n) | 1X
| ||
2D | XP | 1X;2D
| ||
2c | XcP | 1X;2D;2c
| ||
3Omy | XLOmy | 1X;2D;2c;3Omy
| ||
4U | XE | 1X;2D;2c;3Omy;4U
| ||
5Ama | ROX | 1X;2D;2c;3Omy;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
- This protocol is indicated when the aim is to analyse coupling control in mtprep (see MitoPedia: Sample preparations) at a defined electron transfer-pathway state.
- Different combinations of substrates and inhibitors (X) can be used depending on the specific aims, e.g.: PGM, PM, GM, MnaPM or other combinations for N-pathway control state; S or RotS for S-pathway control state.
- CIV activity can be measured as an extension.
- + The combinations of substrates for one ET-pathway state with inhibitors for other pathways provide the best coupling control protocols specific for one ET-pathway state, e.g.: MnaPM for N-pathway control state, RotS for S-pathway control state.
- + Short duration of the experiment.
- - 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, if applied (e.g., rotenone & succinate, SRot-pathway control state).
- - LEAK respiration in the absence of Omy may be overestimated due to ATP turnover stimulated by endogenous adenylates. This can be evaluated by SUIT 1X;2D;3Omy;4U;-. In mouse brain homogenate with succinate & rotenone, Ln and LOmy are identical (Krumschnabel et al 2014).
Compare SUIT protocols
- SUIT-001: Extended protocol with SUIT-006 as the initial coupling control module.
- SUIT-004: Shortened version of SUIT-001, also with SUIT-006 as the initial coupling control module.
- SUIT-003: Coupling control protocol for non-permeabilized cells (ce), counterpart to SUIT-006 for mt-preparations (mtprep).
MitoPedia concepts:
MiP concept,
SUIT protocol,
Recommended
MitoPedia methods:
Respirometry,
Fluorometry