SUIT-006 O2 mt D022

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SUIT-006 O2 mt D022

Description

Abbreviation: S

Reference: A SUIT-006

SUIT number: D022_1SRot;2D;2c;3U;4Ama

O2k-Application: O2

MitoPedia: SUIT

SUIT-category: S

SUIT protocol pattern: linear coupling control protocol 1SRot;2D;2c;3U;4Ama

SUIT-006 O2 mt D022 is a coupling control protocol for for mitochondrial preparations (tissue homogenate and isolated mitochondria). Different coupling control states are assessed (L(n) - P - E) at succinate pathway control state. The use of rotenone, that inhibits complex I, provides a specific coupling control protocol for S-pathway, avoiding reverse electron transfer (RET).

Communicated by Cardoso LH, Komlodi T, Huete-Ortega M and Doerrier C (last update 2019-03-05) 

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1SRot	SL	S	CII	1SRot Succinate, S ( type S-pathway to Q). Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway. Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	SP	S	CII	1SRot;2D Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	ScP	S	CII	1SRot;2D;2c Succinate, S ( type S-pathway to Q). 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]).
(3Omy)	ScL(Omy)			1SRot;2D;2c;(3Omy) Omy addition is skipped in SUIT-006 O2 mt D022 Succinate, S ( type S-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
4U	SE	S	CII	1SRot;2D;2c;4U Succinate, S ( type S-pathway to Q). Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency.
5Ama	ROX			1SRot;2D;2c;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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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 mtprep (see MitoPedia: Sample preparations) at Succinate pathway control state.

+ The combination of succinate with rotenone, that inhibits complex I, provides the best coupling control protocol specific for S-pathway, avoiding reverse electron transfer (RET).

+ Reasonable duration of the experiment.

+ This protocol can be extended with the Complex IV module.

- This protocol does not include internal ET-pathway control steps.

- Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors.

Compare SUIT protocols

• SUIT-006: Extended protocol with SUIT-006 as the initial coupling control module.
• SUIT-003: Coupling control protocol for non-permeabilized cells (ce), counterpart to SUIT-006 for mt-preparations (mtprep).

References

	Year	Reference	Organism	Tissue;cell
Krumschnabel 2014 Methods Enzymol	2014	Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. https://doi.org/10.1016/B978-0-12-416618-9.00009-1	Mouse	Nervous system

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry