SUIT-031 O2 mt D075

high-resolution terminology - matching measurements at high-resolution

SUIT-031 O2 mt D075

Description

Abbreviation: PM+S+Rot

Reference: A to analyse O₂ flux in the NS- pathway control state on isolated mitochondria- SUIT-031

SUIT number: D075_1mt;1PM;2D;2c;3S;4Rot;5U;6Ama

O2k-Application: O2

This is a protocol to investigate oxygen flux N- and NS-pathway and S-pathway control state in the OXPHOS state and S-pathway control state in the ET state in isolated mitochondria, tissue homogenate and cells permeabilized before addition in the O2k-chamber. SUIT-031 Q mt D072 and SUIT-031 Q ce-pce D074 are designed to measure simultaneously O₂ flux and the Q-redox state in isolated mitochondria and permeabilized cells.

Communicated by Komlodi T (last update 2021-12-02) 

Representative traces

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1PM	PML(n)	N	CI	1PM NADH-linked substrates (type N-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	PMP	N	CI	1PM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	PMcP	N	CI	1PM;2D;2c NADH-linked substrates (type N-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]).
3S	PMSP	NS	CI&II	1PM;2D;2c;3S Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4Rot	SP	S	CII	1PM;2D;2c;3S;4Rot Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5U	SE	N	CII	1PM;2D;2c;3S;4Rot;5U 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. Noncoupled electron transfer state, ET state, with ET capacity E.
6Ama	ROX			1PM;2D;2c;3S;4Rot;5U;6Ama 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

• SUIT-031 O2 mt D075 in combination with SUIT-006 O2 mt D022 provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:

1. to obtain reference values.

2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.

3. to screen specific defects.

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

+ Reasonable duration of the experiment.

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

Compare SUIT protocols

• SUIT-001 O2 mt D001 is an extended coupling-control proton in the N-pathway and contribution of G-, S-, Oct- and Gp-pathways in the ET state for isolated mitochondria, tissue homogenate and cells permeabilized before addition to the O2k-chamber.
• SUIT-004 is designed to provide a quick assessment of the linear coupling control (L- P- E) with NADH-linked substrates (PM) and the contribution of the S-pathway to the ET state (N, NS, S).
• SUIT-006 O2 mt D047 is a coupling-control protocol in the N-pathway for isolated mitochondria, tissue homogenate and cells permeabilized before addition to the O2k-chamber.
• SUIT-008 is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1PM	Pyruvate (P) and Malate (M)
2D	ADP (D)
2c	Cytochrome c (c)
3S	Succinate (S)
4Rot	Rotenone (Rot)
5U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler.
6Ama	Antimycin A (Ama)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

	Year	Reference	Organism	Tissue;cell
MiPNet24.12 NextGen-O2k: Q-Module	2021-10-29	NextGen-O2k: Q-Module manual

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry