Difference between revisions of "SUIT-036 O2 mt D089"
Line 52: | Line 52: | ||
== References == | == References == | ||
{{Labeling | |||
|additional=FAT4BRAINSUIT | |||
}} |
Revision as of 15:23, 13 April 2023
Description
Abbreviation: FAO(Pal) & M kinetics
Reference: A - SUIT-036 - F-pathway and malate anaplerosis
SUIT number: D089_1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot;11Ama
O2k-Application: O2
The SUIT-036 O2 mt D089 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-036 O2 mt D089 is specially designed to give information on F-pathway with palmitoylcarnitine 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 | |||
3Pal | PalMP | F | FAO | 1D;2M.1;3Pal
|
3c | PalMcP | F | FAO | 1D;2M.1;3Pal;3c
|
4M.2 | PalMP | F(N) | FAO | 1D;2M.1;3Pal;3c;4M.2
|
4M.5 | PalMP | F(N) | FAO | 1D;2M.1;3Pal;3c;4M.2;4M.5
|
4M1 | PalMP | F(N) | FAO | 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1
|
4M2 | PalMP | F(N) | FAO | 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2
|
5P | PalPMP | FN | FAO&CI | 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P
|
6G | PalPGMP | FN | FAO&CI | 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G
|
7S10 | PalPGMSP | FNS | FAO&CI&II | 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10
|
7S50 | PalPGMSP | FNS | FAO&CI&II | 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50
|
8Gp | PalPGMSGpP | FNSGp | FAO&CI&II&GpDH | 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp
|
9U | PalPGMSGpE | FNSGp | FAO&CI&II&GpDH | 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U
|
10Rot | SGpE | SGp | CII&GpDH | 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot
|
11Ama | ROX | 1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot;11Ama
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- SUIT-036 in combination with SUIT-037 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-036 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-037 O2 mt D090: Similar protocol, using octanoylcarnitine instead of palmitoylcarnitine.
- 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). Allows evaluation of FAO with octanoylcarnitine, also 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: Allows evaluation of FAO with octanoylcarnitine, also without overestimation by using low malate concentration. Besides this, provides information on pathway control in OXPHOS state (F, F(N), FN, FNS pathways).
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1D | ADP (D) | |
2M.1 | Malate (M) | |
3Pal | Palmitoylcarnitine (Pal) | |
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.
References
Labels:
FAT4BRAINSUIT