SUIT-038 O2 mt D091: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=FAO control & M kinetics | ||
|description= | |description=[[File:1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U;9Rot;10Ama.png|400px]] | ||
[[File:1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U;9Rot;10Ama.png|400px]] | |info='''A''' - '''[[SUIT-038]]''' - '''[[Fatty_acid_oxidation_pathway_control_state|F-pathway]] and [[Malic_enzyme|malate anaplerosis]] - control protocol''' | ||
|info='''A''' - '''[[SUIT-038]]''' - ''' | |||
|application=O2 | |application=O2 | ||
|SUIT number=D091_1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U;9Rot;10Ama | |SUIT number=D091_1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U;9Rot;10Ama | ||
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__TOC__ | __TOC__ | ||
ย Communicated by [[ | ย Communicated by [[Grings M]], [[Cecatto C]] and [[Cardoso LHD]] (last update 2023-04-12) | ||
== Representative traces == | == Representative traces == | ||
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== Strengths and limitations == | == Strengths and limitations == | ||
:::* SUIT- | :::* SUIT-038 provides a control protocol without fatty acids, to in combination with [[SUIT-036]] and [[SUIT-037]] study fatty acid oxidation (FAO) in a large variety of species, tissues and cell types. These protocols in combination allow: | ||
:::: 1. to | :::: 1. to assess FAO carefully avoiding overestimation by endogenous substrates or malate anaplerosis. | ||
:::: 2. to | :::: 2. to compare FAO with palmitoylcarnitine or octanoylcarnitine. | ||
:::: 3. to | :::: 3. to analyse malate anaplerosis with malate kinetics | ||
::: | :::: 4. to analyse OXPHOS respiration and ET-capacity with various pathways (F, N, S, Gp) | ||
:::* 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. | ||
:::+ SUIT- | :::+ SUIT-038 allows the depletion of endogenous substrates with ADP (1D). | ||
:::+ Pathway control in OXPHOS (N, NS, NSGp pathways) and in ET state (NSGp and SGp) can be observed. | |||
:::+ Pathway control in OXPHOS | |||
:::+ In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, ''P''/''E'' (8Gp/9U) at high ET capacity can be calculated. | :::+ In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, ''P''/''E'' (8Gp/9U) at high ET capacity can be calculated. | ||
:::+ This protocol can be extended with the Complex IV module. | :::+ This protocol can be extended with the Complex IV module. | ||
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:::- S-pathway in ET state is not obtained (it is obtained in [[SUIT-001]]). | :::- S-pathway in ET state is not obtained (it is obtained in [[SUIT-001]]). | ||
:::- Lengthy duration of the experiment. | :::- Lengthy duration of the experiment. | ||
:::- We do not generally | :::- We do not generally recommend the addition of already permeabilized cells, but we recommend to perform the permeabilization of the cell plasma membrane in the chambers (see [[SUIT-002 O2 ce-pce D007]]).ย ย | ||
== Compare SUIT protocols == | == Compare SUIT protocols == |
Revision as of 12:22, 12 April 2023
Description
Abbreviation: FAO control & M kinetics
Reference: A - SUIT-038 - F-pathway and malate anaplerosis - control protocol
SUIT number: D091_1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U;9Rot;10Ama
O2k-Application: O2
The SUIT-038 O2 mt D091 protocol is focused on the analysis of the malate anaplerotic pathway control state 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, tissues and cell types. Malate alone does not support respiration if oxaloacetate is not metabolized further in the absence of acetyl-CoA. The careful titration of malate is required to analyze the activity of the mt-isoform of NADP- or NAD(P)-dependent malic enzyme (mtME). If these enzymes are present in the sample one should be able to reach with malate alone a high respiratory activity comparable to the NADH-linked pathway control states with more classical combinations of substrates (e.g. PM or GM). Moreover, the pathway control in OXPHOS state ((N), N, NS, NSGp pathways) and in ET state (NSGp and SGp) can be evaluated by using this SUIT protocol.
Communicated by Grings M, Cecatto C and Cardoso LHD (last update 2023-04-12)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1D | ROX | 1D
| ||
2M.1 | 1D;2M.1 | |||
2H2O | 1D;2M.1;2H2O
| |||
3c | 1D;2M.1;2H2O;2c
| |||
3M.2 | 1D;2M.1;2H2O;2c;3M.2
| |||
3M.5 | 1D;2M.1;2H2O;2c;3M.2;3M.5
| |||
3M1 | 1D;2M.1;2H2O;2c;3M.2;3M.5;3M1
| |||
3M2 | 1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2
| |||
4P | PMP | N | CI | 1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P
|
5G | PGMP | N | CI | 1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G
|
6S10 | PGMSP | NS | CI&II | 1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10
|
6S50 | PGMSP | NS | CI&II | 1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50
|
7Gp | PGMSGpP | NSGp | CI&II&GpDH | 1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp
|
8U | PGMSGpE | NSGp | CI&II&GpDH | 1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U
|
9Rot | SGpE | SGp | CII&GpDH | 1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U;9Rot
|
10Ama | ROX | 1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U;9Rot;10Ama
|
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- 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
- SUIT-038 provides a control protocol without fatty acids, to in combination with SUIT-036 and SUIT-037 study fatty acid oxidation (FAO) in a large variety of species, tissues and cell types. These protocols in combination allow:
- 1. to assess FAO carefully avoiding overestimation by endogenous substrates or malate anaplerosis.
- 2. to compare FAO with palmitoylcarnitine or octanoylcarnitine.
- 3. to analyse malate anaplerosis with malate kinetics
- 4. to analyse OXPHOS respiration and ET-capacity with various pathways (F, N, S, Gp)
- A succinate concentration of >10 mM may be required for saturating SE capacity.
- + SUIT-038 allows the depletion of endogenous substrates with ADP (1D).
- + Pathway control in OXPHOS (N, NS, NSGp pathways) and in ET state (NSGp and SGp) can be observed.
- + In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, P/E (8Gp/9U) at high ET capacity can be calculated.
- + This protocol can be extended with the Complex IV module.
- - S-pathway in ET state is not obtained (it is obtained in SUIT-001).
- - Lengthy duration of the experiment.
- - We do not generally recommend the addition of already permeabilized cells, but we recommend to perform the permeabilization of the cell plasma membrane in the chambers (see SUIT-002 O2 ce-pce D007).
Compare SUIT protocols
- SUIT-001 O2 mt D001 (RP1): Harmonized SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized).
- SUIT-002 O2 ce-pce D007: SUIT-002 for non-permeabilized cells. Permeabilization of the cell plasma membrane occurs in the chambers through digitonin addition. Therefore, SUIT-002 O2 ce-pce D007 protocol provides information of non-permeabilized cell respiration (ce) and permeabilized cell respiration (pce).
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1D | ADP (D) | |
2M.1 | Malate (M) | |
2H2O | ||
2c | Cytochrome c (c) | |
3M.2 | Malate (M) | |
3M.5 | Malate (M) | |
3M1 | Malate (M) | |
3M2 | Malate (M) | |
4P | Pyruvate (P) | |
5G | Glutamate (G) | |
6S10 | Succinate (S) | |
6S50 | Succinate (S) | |
7Gp | Glycerophosphate (Gp) | |
8U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
9Rot | Rotenone (Rot) | |
10Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.