SUIT-008 O2 mt D026: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=Q-junction mtprep | ||
|description=[[File:1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.png| | |description=[[File:1PM;2D;2c;3G;4S;5U;6Rot;7Ama;8AsTm;9Azd.png|600px]] | ||
|info='''A:ย SUIT protocol for mt; mt: isolated mitochondria and tissue homogenate - '''[[SUIT-008]]''' | |info='''A:ย SUIT protocol for mt; mt: isolated mitochondria and tissue homogenate - '''[[SUIT-008]]''' | ||
|SUIT number=D026_1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9Azd | |SUIT number=D026_1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9Azd | ||
|application=O2 | |application=O2 | ||
}} | }} | ||
: | {{Template:SUIT text D026}} | ||
ย | |||
__TOC__ | |||
Communicated by [[Doerrier C]] and [[Gnaiger E]] (last update 2019-06-06) | |||
== Representative traces == | |||
[[File:D026 O2 traces.png|600px]] | |||
{{Template:SUIT-008}} | {{Template:SUIT-008}} | ||
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:::+ The presence of PGM and S establishes fully operative TCA cycle activity. | :::+ The presence of PGM and S establishes fully operative TCA cycle activity. | ||
:::+ This protocol allows to analyse the convergence of pathways at the Q-junction (N, NS, S). | :::+ This protocol allows to analyse the convergence of pathways at the Q-junction (N, NS, S). | ||
:::+ Outer mitochondrial membrane integrity can be evaluated by the addition of cytochrome ''c'' ([[Cytochrome c control factor |cytochrome ''c'' test]]). The early addition of cytochrome ''c''ย in the protocol ensures comparability of all states in case of any effect of cytochrome ''c'. | :::+ Outer mitochondrial membrane integrity can be evaluated by the addition of cytochrome ''c'' ([[Cytochrome c control factor |cytochrome ''c'' test]]). The early addition of cytochrome ''c''ย in the protocol ensures comparability of all states in case of any effect of cytochrome ''c''. | ||
:::+ 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 S-pathway contribution is higher with GM compared to PM. PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is an advantage compared to [[SUIT-011]] for the diagnosis of N-capacity. | :::+ 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 S-pathway contribution is higher with GM compared to PM. PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is an advantage compared to [[SUIT-011]] for the diagnosis of N-capacity. | ||
:::+ Reasonable duration of the experiment. | :::+ Reasonable duration of the experiment. | ||
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== Compare SUIT protocols == | == Compare SUIT protocols == | ||
::::* [[SUIT-014]]: 1GM;2D;3P;4S;5U;6Rot-; similar version starting with GM, and then adding P. Used in combination with SUIT-008 in [[Lemieux_2017_Sci_Rep|Lemieux 2017]] for [[Permeabilized muscle fibers|pfi]]. | |||
::::* [[SUIT-004]]: SUIT-004 protocol provide a quick assessment of the linear coupling control (''L''-''P''-''E'') with NADH linked-substrates (PM) and the pathway control in ET state (N, NS, S pathways). | ::::* [[SUIT-004]]: SUIT-004 protocol provide a quick assessment of the linear coupling control (''L''-''P''-''E'') with NADH linked-substrates (PM) and the pathway control in ET state (N, NS, S pathways). | ||
::::* [[SUIT-011]]: SUIT-011 protocol are designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS capacity with GMS as NS-linked substrates). ย | ::::* [[SUIT-011]]: SUIT-011 protocol are designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS capacity with GMS as NS-linked substrates). ย | ||
== Chemicals and syringes == | |||
{{Template:Chemicals SUIT-008}} | |||
{{Template:Chemicals CIV}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == |
Latest revision as of 10:56, 17 September 2020
Description
Abbreviation: Q-junction mtprep
Reference: A: SUIT protocol for mt; mt: isolated mitochondria and tissue homogenate - SUIT-008
SUIT number: D026_1PM;2D;2c;3G;4S;4D;5U;6Rot;7Ama;8AsTm;9Azd
O2k-Application: O2
The SUIT-008 O2 mt D026 protocol can be used with 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 organisms and tissues. The protocol is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity. It also serves as a diagnostic tool for the activity of the glutamate dehydrogenase and its linked pathways, which could be relevant in some pathologies. SUIT-008 O2 mt D026 can be easily extended with the CIV assay module.
Communicated by Doerrier C and 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
|
4S | PGMSP | NS | CI&II | 1PM;2D;2c;3G;4S
|
5U | PGMSE | NS | CI&II | 1PM;2D;2c;3G;4S;5U
|
6Rot | SE | S | CII | 1PM;2D;2c;3G;4S;5U;6Rot
|
7Ama | ROX | 1PM;2D;2c;3G;4S;5U;6Rot;7Ama
|
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
- 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
- + NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
- + The presence of PGM and S establishes fully operative TCA cycle activity.
- + This protocol allows to analyse the convergence of pathways at the Q-junction (N, NS, S).
- + Outer mitochondrial membrane integrity can be evaluated by the addition of cytochrome c (cytochrome c test). The early addition of cytochrome c in the protocol ensures comparability of all states in case of any effect of cytochrome c.
- + 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 S-pathway contribution is higher with GM compared to PM. PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since impairment of N-pathway capacity can be compensated partially by activation of the S-pathway. This is an advantage compared to SUIT-011 for the diagnosis of N-capacity.
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module.
- - F-pathway is not analysed.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
Compare SUIT protocols
- SUIT-014: 1GM;2D;3P;4S;5U;6Rot-; similar version starting with GM, and then adding P. Used in combination with SUIT-008 in Lemieux 2017 for pfi.
- SUIT-004: SUIT-004 protocol provide a quick assessment of the linear coupling control (L-P-E) with NADH linked-substrates (PM) and the pathway control in ET state (N, NS, S pathways).
- SUIT-011: SUIT-011 protocol are designed to study physiologically relevant maximum mitochondrial respiratory capacity (OXPHOS capacity with GMS as NS-linked substrates).
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) | |
4S | Succinate (S) | |
5U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
6Rot | Rotenone (Rot) | |
7Ama | Antimycin A (Ama) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
## AsTm | Ascorbate (As) and TMPD (Tm) | |
## Azd | Azide (Azd) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry