SUIT-037 O2 mt D090: Difference between revisions
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:::+ SUIT-037 allows the depletion of endogenous substrates with ADP (1D). | :::+ SUIT-037 allows the depletion of endogenous substrates with ADP (1D). | ||
:::+ The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. ย | :::+ The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. ย | ||
:::+ F-pathway ( | :::+ F-pathway (3Pal-2M.1) can be compared to FN-pathway (5P) in OXPHOS state. | ||
:::+ Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed. | :::+ Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed. | ||
:::+ | :::+ Multiple pathways converging into Q (FNSGp) are assessed in OXPHOS and ET states. Therefore, ''P''/''E'' (8Gp/9U) at high ET capacity can be calculated. | ||
:::- Lengthy duration of the experiment. | :::- Lengthy duration of the experiment. | ||
:::- | == Compare SUIT protocols == | ||
ย | |||
::::* [[SUIT-036_O2_mt_D089]]: Similar protocol, using palmitoylcarnitine instead of octanoylcarnitine. | |||
ย | |||
::::* [[SUIT-038 O2 mt D091]]: Control protocol, without acylcarnitine. | |||
::::* [[SUIT-002 O2 mt D005]]: Reference protocol SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized). Also allows evaluation of FAO without overestimation by using low malate concentration, besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and ET-state (FNSGp, SGp pathways). | |||
::::* [[SUIT- | ::::* [[SUIT-025_O2_mt_D057]]: Also allows evaluation of FAO without overestimation by using low malate concentration, besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS pathways). | ||
::::* [[SUIT- | ::::* [[SUIT-017_O2_mt_D046]]: For samples without mitochondrial malic enzyme activity, allows to assess FAO with octanoylcarnitine. Besides this, it is possible to analyse FN and FNS-linked respiration in OXPHOS state, and FNS- and S-linked respiration in and ET-state. | ||
== Chemicals and syringes == | == Chemicals and syringes == |
Revision as of 13:42, 12 April 2023
Description
Abbreviation: FAO(Oct) & M kinetics
Reference: A - SUIT-037 - F-pathway and malate anaplerosis
SUIT number: D090_1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot;11Ama
O2k-Application: O2
The SUIT-037 O2 mt D090 protocol provides a common reference for comparison of respiratory control of 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. SUIT-037 O2 mt D090 is specially designed to give information on F-pathway with octanoylcarnitine as a FAO substrate in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. The careful titration of malate is required to analyze the activity of the mt-isoform of NADP- or NAD(P)-dependent malic enzyme (mtME). Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp 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 | |||
3Oct | OctMP | F | FAO | 1D;2M.1;3Oct
|
3c | OctMcP | F | FAO | 1D;2M.1;3Oct;3c
|
4M.2 | OctMP | F(N) | FAO | 1D;2M.1;3Oct;3c;4M.2
|
4M.5 | OctMP | F(N) | FAO | 1D;2M.1;3Oct;3c;4M.2;4M.5
|
4M1 | OctMP | F(N) | FAO | 1D;2M.1;3Oct;3c;4M.2;4M.5;4M1
|
4M2 | OctMP | F(N) | FAO | 1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2
|
5P | OctPMP | FN | FAO&CI | 1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P
|
6G | OctPGMP | FN | FAO&CI | 1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G
|
7S10 | OctPGMSP | FNS | FAO&CI&II | 1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10
|
7S50 | OctPGMSP | FNS | FAO&CI&II | 1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50
|
8Gp | OctPGMSGpP | FNSGp | FAO&CI&II&GpDH | 1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp
|
9U | OctPGMSGpE | FNSGp | FAO&CI&II&GpDH | 1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U
|
10Rot | SGpE | SGp | CII&GpDH | 1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot
|
11Ama | ROX | 1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot;11Ama
|
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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-037 in combination with SUIT-036 and SUIT-038 provides a basis to 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-037 allows the depletion of endogenous substrates with ADP (1D).
- + The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
- + F-pathway (3Pal-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
- + Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed.
- + Multiple pathways converging into Q (FNSGp) are assessed in OXPHOS and ET states. Therefore, P/E (8Gp/9U) at high ET capacity can be calculated.
- - Lengthy duration of the experiment.
Compare SUIT protocols
- SUIT-036_O2_mt_D089: Similar protocol, using palmitoylcarnitine instead of octanoylcarnitine.
- SUIT-038 O2 mt D091: Control protocol, without acylcarnitine.
- SUIT-002 O2 mt D005: Reference protocol SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized). Also allows evaluation of FAO without overestimation by using low malate concentration, besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and ET-state (FNSGp, SGp pathways).
- SUIT-025_O2_mt_D057: Also allows evaluation of FAO without overestimation by using low malate concentration, besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS pathways).
- SUIT-017_O2_mt_D046: For samples without mitochondrial malic enzyme activity, allows to assess FAO with octanoylcarnitine. Besides this, it is possible to analyse FN and FNS-linked respiration in OXPHOS state, and FNS- and S-linked respiration in and ET-state.
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1D | ADP (D) | |
2M.1 | Malate (M) | |
3Oct | Octanoylcarnitine (Oct) | |
3c | Cytochrome c (c) | |
4M.2 | Malate (M) | |
4M.5 | Malate (M) | |
4M1 | Malate (M) | |
4M2 | Malate (M) | |
5P | Pyruvate (P) | |
6G | Glutamate (G) | |
7S10 | Succinate (S) | |
7S50 | Succinate (S) | |
8Gp | Glycerophosphate (Gp) | |
9U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
10Rot | Rotenone (Rot) | |
11Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.