SUIT-020 O2 mt D032: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=Q-junction additivity and respiratory control for membrane potential | ||
|description=[[File:1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama.png| | |description=[[File:1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama.png|500px]] | ||
|info='''A''': | |info='''A''': short protocol for determination of O<sub>2</sub> flux in mitochondrial preparations (isolated mitochondria, permeabilized cells, and tissue homogenate)-'''[[SUIT-020]]''' | ||
|application=O2 | |application=O2 | ||
|SUIT number=D032_1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama | |SUIT number=D032_1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama | ||
}} | }} | ||
::: '''[[SUIT protocol pattern]]:''' orthogonal 1PM;2D;2c;3G;4S;5Rot;6Omy;7U- | |||
::: '''[[SUIT protocol pattern]]:''' orthogonal 1PM;2D;2c;3G;4S;5Rot;6Omy;7U | Β | ||
This is a protocol to investigate the [[NADH Electron transfer-pathway state| N-]] and [[NS-pathway control state| NS-pathway]] control state | {{Template:SUIT text D032}} | ||
Originially this is a protocol for simultaneous determination of O<sub>2</sub> flux and mitochondrial membrane potential on isolated mitochondria and tissue homogenate. It is can be used as a control experiment for [[SUIT-020 Fluo mt D033]] without using the fluorescence dye | This is a protocol to assess the additivity between[[NADH Electron transfer-pathway state| N-]]pathway and [[Succinate pathway control state|S-]] pathway in the Q-junction as well as investigate the [[NADH Electron transfer-pathway state| N-]] and [[NS-pathway control state| NS-pathway]] control state in mitochondrial preparations. It can serve as a diagnostic tool for the activity of glutamate dehydrogenase and its linked pathway.Β Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the required concentration of Omy has to be assessed by the [https://bioblast.at/index.php/OligomycinΒ Omy titration test]. The Complex III inhibitor Antimycin A (Ama) blocks the respiration.Β Β | ||
Originially this is a protocol for simultaneous determination of O<sub>2</sub> flux and mitochondrial membrane potential on isolated mitochondria and tissue homogenate. It is can be used as a control experiment for [[SUIT-020 Fluo mt D033]] without using the fluorescence dye to evaluate its effect on the O<sub>2</sub> flux as a control. Β | |||
__TOC__ | __TOC__ | ||
Β Communicated by [[Huete-Ortega M]], | Β Communicated by [[Huete-Ortega M]], [[Komlodi T]] and [[Gnaiger E]]Β (last update 2019-06-06) | ||
Β | |||
== Representative traces == | |||
[[File:SUIT-020 O2 mt D032 DL-File.png|600px]] | |||
{{Template:SUIT-020}} | {{Template:SUIT-020}} | ||
== Strengths and limitations == | == Strengths and limitations == | ||
:::+ You | :::* NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity. | ||
:::+ This protocol can be used as a control for [[SUIT-020 Fluo mt D033]]. | :::* Comparison of GM- with PM-capacity yields important information on N-pathway respiratory control upstream of CI. | ||
:::+ CIV activity and Cytochrome c test can be performed. | :::* A succinate concentration of >10 mM may be required for saturating SE capacity. | ||
:::- [[Oligomycin| Oligomycin]] concentration has to be determined. Higher | :::* The fluorescence dye can be added after cytochrome c to test its effect on the respiration. | ||
:::+ You obtain information in a single protocol about the [[NS-pathway control state| NS-]], [[Succinate pathway control state| S]] and [[NADH Electron transfer-pathway state| N-]] pathway control state. | |||
:::+ This protocol can be used as a control for [[SUIT-020 Fluo mt D033]] to test the effect of the fluorescence dyes on the respiration. | |||
:::+ CIV activity determination and Cytochrome c test can be performed. Β | |||
:::- [[Oligomycin| Oligomycin]] concentration has to be determined. Higher concentrations of Oligomycin may inhibit the [[Electron transfer pathway| ET state]]. | |||
:::- Careful washing is required after the experiment to avoid carry-over of the inhibitors and uncoupler. | |||
== Compare SUIT protocols == | |||
:::* [[SUIT-001]];[[SUIT-001 O2 mt D001]]for mitochondrial preparations: a complex protocol to get information not only about the NS(PGM) in the OXPHOS state, but also in ET state and about the FGp-pathways. | |||
:::* [[SUIT-004]];[[SUIT-004 O2 pfi D010]]: provides information on NS(PM) pathway in the ET state without the contribution of G. | |||
:::* [[SUIT-008]];[[SUIT-008 O2 pfi D014]] and [[SUIT-008 O2 ce-pce D025]]: provide information on the NS(PGM) pathway in the OXPHOS and ET-states, but without the addition of Omy. | |||
:::* [[SUIT-011]]: provides information about the NS(GM) pathway in the OXPHOS and ET-states, but without the addition of Omy. | |||
:::* [[SUIT-012]]: provides information about the N(PGM) pathway in the OXPHOS and ET-states without the contribution of S-pathway and without the addition of Omy. | |||
:::* [[SUIT-014]]: a protocol similar to [[SUIT-021 O2 mt D035]], but with the addition of P in the OXPHOS state before S. Omy is not added in the protocol. | |||
== | == Chemicals and syringes == | ||
: | {{Template:Chemicals SUIT-020 O2 mt D032}} | ||
::: | ::: Suggested stock concentrations are shown in the specific DL-Protocol. | ||
== References == | == References == |
Latest revision as of 15:11, 17 September 2020
Description
Abbreviation: Q-junction additivity and respiratory control for membrane potential
Reference: A: short protocol for determination of O2 flux in mitochondrial preparations (isolated mitochondria, permeabilized cells, and tissue homogenate)-SUIT-020
SUIT number: D032_1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama
O2k-Application: O2
- SUIT protocol pattern: orthogonal 1PM;2D;2c;3G;4S;5Rot;6Omy;7U-
SUIT-020 O2 mt D032 is design to assess the additivity between N-pathway and S- pathway in the Q-junction as well as investigate the N- and NS-pathway control state in mitochondrial preparations. It can serve as a diagnostic tool for the activity of glutamate dehydrogenase and its linked pathway. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Oligomycin has to be titrated in small amounts to avoid overtitration and inhibition of the flux in the ET-state. The Complex III inhibitor Antimycin A (Ama) blocks the respiration. Originially this is a protocol for simultaneous determination of O2 flux and mitochondrial membrane potential on isolated mitochondria and tissue homogenate. It is can be used as a control experiment for SUIT-020 Fluo mt D033 without using the fluorescence dye to evaluate its effect on the O2 flux as a control. This is a protocol to assess the additivity between N-pathway and S- pathway in the Q-junction as well as investigate the N- and NS-pathway control state in mitochondrial preparations. It can serve as a diagnostic tool for the activity of glutamate dehydrogenase and its linked pathway. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the required concentration of Omy has to be assessed by the Omy titration test. The Complex III inhibitor Antimycin A (Ama) blocks the respiration. Originially this is a protocol for simultaneous determination of O2 flux and mitochondrial membrane potential on isolated mitochondria and tissue homogenate. It is can be used as a control experiment for SUIT-020 Fluo mt D033 without using the fluorescence dye to evaluate its effect on the O2 flux as a control.
Communicated by Huete-Ortega M, Komlodi T 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
|
5Rot | SP | S | CII | 1PM;2D;2c;3G;4S;5Rot
|
6Omy | SL(Omy) | 1PM;2D;2c;3G;4S;5Rot;6Omy
| ||
7U* | SE | S | CII | 1PM;2D;2c;3G;4S;5Rot;6Omy;7S
|
8Ama | ROX | 1PM;2D;2c;3G;4S;5Rot;6Omy;7S;8Ama
|
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
- 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
- NS-OXPHOS capacity provides a physiologically relevant estimate of maximum mitochondrial respiratory capacity.
- Comparison of GM- with PM-capacity yields important information on N-pathway respiratory control upstream of CI.
- A succinate concentration of >10 mM may be required for saturating SE capacity.
- The fluorescence dye can be added after cytochrome c to test its effect on the respiration.
- + You obtain information in a single protocol about the NS-, S and N- pathway control state.
- + This protocol can be used as a control for SUIT-020 Fluo mt D033 to test the effect of the fluorescence dyes on the respiration.
- + CIV activity determination and Cytochrome c test can be performed.
- - Oligomycin concentration has to be determined. Higher concentrations of Oligomycin may inhibit the ET state.
- - Careful washing is required after the experiment to avoid carry-over of the inhibitors and uncoupler.
Compare SUIT protocols
- SUIT-001;SUIT-001 O2 mt D001for mitochondrial preparations: a complex protocol to get information not only about the NS(PGM) in the OXPHOS state, but also in ET state and about the FGp-pathways.
- SUIT-004;SUIT-004 O2 pfi D010: provides information on NS(PM) pathway in the ET state without the contribution of G.
- SUIT-008;SUIT-008 O2 pfi D014 and SUIT-008 O2 ce-pce D025: provide information on the NS(PGM) pathway in the OXPHOS and ET-states, but without the addition of Omy.
- SUIT-011: provides information about the NS(GM) pathway in the OXPHOS and ET-states, but without the addition of Omy.
- SUIT-012: provides information about the N(PGM) pathway in the OXPHOS and ET-states without the contribution of S-pathway and without the addition of Omy.
- SUIT-014: a protocol similar to SUIT-021 O2 mt D035, but with the addition of P in the OXPHOS state before S. Omy is not added in the protocol.
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) | |
5Rot | Rotenone (Rot) | |
6Omy | Oligomycin (Omy) | |
7U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
8Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry