Difference between revisions of "SUIT-012 O2 mt D027"
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{{MitoPedia | {{MitoPedia | ||
|abbr=N | |abbr=N CCP mtprep | ||
|description=[[File:1PM;2D;3G;4U;5Ama.png|300px]] | |description=[[File:1PM;2D;3G;4U;5Ama.png|300px]] | ||
|info='''A''' Linear coupling control with NADH-linked substrates for isolated mitochondria, tissue homogenate and permeabilized cells- '''[[SUIT-012]]''' | |info='''A''' Linear coupling control with NADH-linked substrates for isolated mitochondria, tissue homogenate and permeabilized cells- '''[[SUIT-012]]''' | ||
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}} | }} | ||
::: '''[[MitoPedia: SUIT]]''' | ::: '''[[MitoPedia: SUIT]]''' | ||
::: '''[[SUIT protocol pattern]]:''' 1PM;2D;2c;3G;4U- | |||
::: '''[[SUIT protocol pattern]]:''' | |||
The SUIT-012 O2 mt D027 protocols specifically focus on assessing the linear coupling control ([[LEAK-respiration|''L'']]-[[Oxidative phosphorylation| ''P'']]-[[ET-capacity| ''E'']]) with NADH-linked substrates ([[PM pathway control state|PM]]) in [[Mitochondrial preparations| mitochondrial preparations]] such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species and tissues. Addition of [[Glutamate|G]] in NADH-supported [[Oxidative phosphorylation| OXPHO]]S enables evaluating the [[glutamate anaplerotic pathway control state]]. SUIT-012 O2 mt D027 can be extended with the CIV assay module. | The SUIT-012 O2 mt D027 protocols specifically focus on assessing the linear coupling control ([[LEAK-respiration|''L'']]-[[Oxidative phosphorylation| ''P'']]-[[ET-capacity| ''E'']]) with NADH-linked substrates ([[PM pathway control state|PM]]) in [[Mitochondrial preparations| mitochondrial preparations]] such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species and tissues. Addition of [[Glutamate|G]] in NADH-supported [[Oxidative phosphorylation| OXPHO]]S enables evaluating the [[glutamate anaplerotic pathway control state]]. SUIT-012 O2 mt D027 can be extended with the CIV assay module. | ||
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__TOC__ | __TOC__ | ||
Communicated by [[Huete-Ortega M]], [[Iglesias-Gonzalez J]] and [[Gnaiger E]] (last update 2019- | Communicated by [[Huete-Ortega M]], [[Iglesias-Gonzalez J]] and [[Gnaiger E]] (last update 2019-06-06) | ||
{{Template:SUIT-012 O2 mt D027}} | {{Template:SUIT-012 O2 mt D027}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::+ This protocol allows to evaluate the function of the TCA cycle without the involvement of the [[complex II]] ([[Succinate pathway control state| S-pathway]]). It can be useful to understand the contribution and the activity of the dehydrogenases. | :::+ This protocol allows to evaluate the function of the TCA cycle without the involvement of the [[complex II]] ([[Succinate pathway control state| 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. | :::+ 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. | :::+ Reasonable duration of the experiment. | ||
:::+ This protocol can be extended with the Complex IV module, which can prolong the experimental time with ~30 min. | :::+ This protocol can be extended with the Complex IV module, which can prolong the experimental time with ~30 min. |
Revision as of 11:41, 6 June 2019
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
- MitoPedia: SUIT
- SUIT protocol pattern: 1PM;2D;2c;3G;4U-
The SUIT-012 O2 mt D027 protocols specifically focus on assessing the linear coupling control (L- P- E) with NADH-linked substrates (PM) in mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species and tissues. Addition of G in NADH-supported OXPHOS enables evaluating the glutamate anaplerotic pathway control state. SUIT-012 O2 mt D027 can be extended with the CIV assay module.
Communicated by Huete-Ortega M, Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-06)
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
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry