SUIT-004 O2 pfi D010: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=RP1-short pfi | ||
|description=[[File:1PM;2D;2c;3U;4S;5Rot.png| | |description=[[File:1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd.png|450px]] | ||
|info='''[[SUIT-004]]''' - Short protocol linked to [[SUIT-001 O2 pfi D002]] '''SUIT RP1''' (human skeletal muscle) | |||
|info= | |||
|application=O2 | |application=O2 | ||
|SUIT number=D010_1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd | |||
}} | }} | ||
: | {{Template:SUIT text D010}} {{Template:Setting oxygen pfi}} | ||
: | |||
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__TOC__ | __TOC__ | ||
Communicated by [[Doerrier C]], [[Gnaiger E]] (last update 2019-06-05) | |||
== Representative traces == | |||
[[File:SUIT 004.png|600px]] | |||
{{Template:SUIT-004}} | {{Template:SUIT-004}} | ||
Line 31: | Line 18: | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::* A succinate concentration of >10 mM may be required for saturating S<sub>''E''</sub> capacity. | :::* A succinate concentration of >10 mM may be required for saturating S<sub>''E''</sub> capacity. | ||
ย | ::::* CIV assessment is skipped if a prolonged protocol is not practical due to time limitation. | ||
:::+ Linear coupling control (''L''-''P''-''E'') with | ::::# '''RP1-6Ama''' may be skipped since correction of the initial states by ROX measured in RP1-6 Ama may represent an over-correction, and inhibition takes a very long time in several cases ([[Pesta 2012 Methods Mol Biol]]). | ||
:::+ Pathway control in ET state ( | :::+ Linear coupling control ([[LEAK respiration|''L'']]-[[Oxidative phosphorylation| ''P'']]-[[ET capacity| ''E'']]) with NADPH-linked substrate substrates ([[PM-pathway control state|PM]]). | ||
:::+ Pathway control in ET state ([[NADH_Electron_transfer-pathway_state|N]], [[NS-pathway control state| NS]], [[Succinate pathway control state| S]]). | |||
:::+ This protocol can be extended with the Complex IV module. | :::+ This protocol can be extended with the Complex IV module. | ||
ย | :::+ Short experimental time. | ||
:::- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT- | :::- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-004 in favor of reducing the time consumed during the experiment. | ||
== Compare SUIT protocols == | == Compare SUIT protocols == | ||
:::* [[SUIT-001 O2 pfi D002]]: A comparable but more comprehensive protocol comprising additional substrate states. | |||
:::* Harmonized with [[SUIT-005 O2 pfi D011]]. | |||
:::'''Specific cases''' | :::'''Specific cases''' | ||
::::* [[Isolated mitochondria |imt]], ROX: NS(PM)01_imt,1PM,2D(c),3U,4S,5Rot,6Ama | ::::* [[Isolated mitochondria |imt]], ROX: NS(PM)01_imt,1PM,2D(c),3U,4S,5Rot,6Ama | ||
::::* [[Permeabilized muscle | ::::* [[Permeabilized muscle fibers |pfi]], high O<sub>2</sub>, no ROX: NS(PM)01_pfiO2,1PM,2D(c),3U,4S,5Rot | ||
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== Chemicals and syringes == | |||
{{Template:Chemicals SUIT-004}} | |||
{{Template:Chemicals CIV}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
: | == References == | ||
{{#ask:[[Category:Publications]] [[Additional label::SUIT-004 O2 pfi D010]] | |||
| ?Was published in year=Year | |||
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{{MitoPedia concepts | {{MitoPedia concepts |
Latest revision as of 23:49, 12 April 2021
Description
Abbreviation: RP1-short pfi
Reference: SUIT-004 - Short protocol linked to SUIT-001 O2 pfi D002 SUIT RP1 (human skeletal muscle)
SUIT number: D010_1PM;2D;2c;3U;4S;5Rot;6Ama;7AsTm;8Azd
O2k-Application: O2
The SUIT-004 O2 pfi D010 protocol provides information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS and S) can be evaluated by using this SUIT protocol. The SUIT-004 O2 pfi D010 can be extended with the CIV assay module. Permeabilized muscle fibers are sensitive to oxygen supply due to limited diffusion of oxygen to the fiber bundle core. To counteract this limitation, hyperoxic conditions (400-250 ยตM O2) must be employed. To set the optimal oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.
Communicated by Doerrier C, Gnaiger E (last update 2019-06-05)
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
|
3U | PME | N | CI | 1PM;2D;3U
|
4S | PMSE | NS | CI&II | 1PM;2D;3U;4S
|
5Rot | SE | S | CII | 1PM;2D;3U;4S;5Rot
|
6Ama | ROX | 1PM;2D;3U;4S;5Rot;6Ama
|
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
- A succinate concentration of >10 mM may be required for saturating SE capacity.
- CIV assessment is skipped if a prolonged protocol is not practical due to time limitation.
- RP1-6Ama may be skipped since correction of the initial states by ROX measured in RP1-6 Ama may represent an over-correction, and inhibition takes a very long time in several cases (Pesta 2012 Methods Mol Biol).
- + Linear coupling control (L- P- E) with NADPH-linked substrate substrates (PM).
- + Pathway control in ET state (N, NS, S).
- + This protocol can be extended with the Complex IV module.
- + Short experimental time.
- - The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-004 in favor of reducing the time consumed during the experiment.
Compare SUIT protocols
- SUIT-001 O2 pfi D002: A comparable but more comprehensive protocol comprising additional substrate states.
- Harmonized with SUIT-005 O2 pfi D011.
- Specific cases
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PM | Pyruvate (P) and Malate (M) | |
2D | ADP (D) | |
2c | Cytochrome c (c) | |
3U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
4S | Succinate (S) | |
5Rot | Rotenone (Rot) | |
6Ama | 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