Difference between revisions of "SUIT-012 O2 mt D027"
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|SUIT number=D027_1PM;2D;2c;3G;4U;5Ama | |SUIT number=D027_1PM;2D;2c;3G;4U;5Ama | ||
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{{Template:SUIT text D027}} | {{Template:SUIT text D027}} | ||
__TOC__ | __TOC__ | ||
Communicated by [[Huete-Ortega M]], | Communicated by [[Huete-Ortega M]], [[Gnaiger E]] (last update 2019-06-06) | ||
== Representative traces == | |||
[[File:D027 O2 traces.png|600px]] | |||
{{Template:SUIT-012 O2 mt D027}} | {{Template:SUIT-012 O2 mt D027}} | ||
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== Compare SUIT protocols == | == Compare SUIT protocols == | ||
::::* [[SUIT-008]]: 1PM;2D;3G;4S;5U-; longer version that also analyses S-pathway. | |||
::::* [[SUIT-006]]: 1X;2D;3Omy;4U-; coupling control protocol with only one combination of substrates (''e.g.'' 1PM;2D;3Omy;4U-). | |||
== Chemicals and syringes == | |||
{{Template:Chemicals SUIT-012}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == |
Latest revision as of 00:12, 13 April 2021
Description
Abbreviation: N CCP mtprep
Reference: A Linear coupling control with NADH-linked substrates for isolated mitochondria, tissue homogenate and permeabilized cells- SUIT-012
SUIT number: D027_1PM;2D;2c;3G;4U;5Ama
O2k-Application: O2
The SUIT-012 O2 mt D027 protocol specifically focuses on assessing the coupling control (L- P- E) with NADH-linked substrates (PM and PGM). Addition of G enables evaluating the contribution of this substrate in NADH-supported OXPHOS respiration, which may be relevant in the presence of glutamate-anaplerotic pathway control state. SUIT-012 can be extended with the CIV assay module.
Communicated by Huete-Ortega M, Gnaiger E (last update 2019-06-06)
Representative traces
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
|
3G | PGMP | N | CI | 1PM;2D;2c;3G
|
4U | PGME | N | CI | 1PM;2D;2c;3G;4U
|
5Ama | ROX | 1PM;2D;2c;3G;4U;5Ama
|
Step | Respiratory state | Pathway control | ET-Complex | Comment |
---|---|---|---|---|
## AsTm | AsTmE | CIV | CIV | |
## Azd | CHB |
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- + This protocol allows to evaluate the function of the TCA cycle without the involvement of the complex II ( S-pathway). It can be useful to understand the contribution and the activity of the dehydrogenases.
- + Mitochondrial outer membrane integrity can be measured with the addition of cytochrome c. Application of the cytochrome c test early in the protocol ensures comparability of all states in case of any effect of cytochrome c.
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module, which can prolong the experimental time with ~30 min.
- + GM and PM yield typically identical fluxes in human skeletal muscle fibres. However, PM is the superior alternative to GM: the fraction of the N-pathway is lower and of the S-pathway is higher with GM compared to PM (GMP is inhibited by the CII inhibitor malonic acid to a larger extent than PMP). PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since an impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is an advantage compared to SUIT-011 for diagnosis of N-capacity.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
Compare SUIT protocols
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PM | Pyruvate (P) and Malate (M) | |
2D | ADP (D) | |
2c | Cytochrome c (c) | |
3G | Glutamate (G) | |
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