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SUIT-006
Description
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Abbreviation: N, S, F, Gp
Reference: A »Versions
- SUIT-category: N, S, F, Gp
- SUIT protocol pattern: linear coupling control protocol 1SI;2D;3U;-
DLP applications
References
Steps and respiratory states
Step
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State
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Pathway
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Q-junction
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Comment - Events (E) and Marks (M)
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1X
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XL(n)
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|
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1X
- X: combination of substrates and inhibitors chosen according to the aims.
- Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
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2D
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XP
|
|
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1X;2D
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2c
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XcP
|
|
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1X;2D;2c
- OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
- Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (OXPHOS capacity P with saturating [ADP]).
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3Omy
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XLOmy
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|
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1X;2D;2c;3Omy
- Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
- The addition of oligomycin is optional depending on the experimental question to resolve. If it is known that Ln has the same values for LOmy for a kind of sample and substrates, this step may be skipped. If the carrier of oligomycin (EtOH) is used instead, it is possible to evaluate whether it affects OXPHOS. In these cases, LEAK state is not reached again.
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4U
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XE
|
|
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1X;2D;2c;3Omy;4U
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5Ama
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ROX
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|
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1X;2D;2c;3Omy;4U;5Ama
- Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).
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- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- » Coupling control state
- » ET capacity
- » OXPHOS capacity
- » LEAK respiration
- Pathway control
- » Electron transfer pathway
- » Fatty acid oxidation pathway control state, F
- » NADH electron transfer-pathway state, N
- » Succinate pathway control state, S
- » NS-pathway control state, NS
- » Glycerophosphate pathway control state, Gp
- » Complex IV single step, CIV
- » Anaplerotic pathway control state
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Glossary
- » List of SUIT states
- » SUIT concept
Strengths and limitations
- This protocol is indicated when the aim is to analyse coupling control in mt preparations.
- Different combinations of substrates and inhibitors can be used depending on the specific aims, e.g.: PM, GM, MnaPM or other combinations for N-pathway control state; S or RotS for S-pathway control state.
- + The combinations of substrates for one ET-pathway state with inhibitors for other pathways provide the best coupling control protocols specififc for one ET-pathway state, e.g.: MnaPM for N-pathway control state, RotS for S-pathway control state.
- + Short duration of the experiment.
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
- - CIV activity is not measured, to save experimental time.
Compare SUIT protocols
MitoPedia concepts:
MiP concept,
SUIT protocol,
Recommended
MitoPedia methods:
Respirometry