SUIT-020 O2 mt D032

high-resolution terminology - matching measurements at high-resolution

SUIT-020 O2 mt D032

Description

Abbreviation: NS(PGM)

Reference: A: SUIT-020 short protocol for determination of O₂ flux in mitochondrial preparations (isolated mitochondria and tissue homogenate).

SUIT number: D032_1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama

O2k-Application: O2

SUIT-category: NS(PGM)

SUIT protocol pattern: orthogonal 1PM;2D;2c;3G;4S;5Rot;6Omy;7U;8Ama

This is a protocol to investigate the N- and NS-pathway control state on isolated mitochondria and tissue homogenate. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Oligomycin has to be titrated in small amounts to avoid overtitration and inhibition of the flux in the ET-state. The Complex III inhibitor Antimycin A (Ama) blocks the respiration. Originially this is a protocol for simultaneous determination of O₂ flux and mitochondrial membrane potential on isolated mitochondria and tissue homogenate. It is can be used as a control experiment for SUIT-020 Fluo mt D033 without using the fluorescence dye to evaluate its effect on the O₂ flux as a control.

Communicated by Huete-Ortega M, Komlodi T and Gnaiger E  (last update 2019-02-20)
 

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). 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]). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3G	PGMP	N	CI	1PM;2D;2c;3G NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4S	PGMSP	NS	CI&II	1PM;2D;2c;3G;4S 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.
5Rot	SP	S	CII	1PM;2D;2c;3G;4S;5Rot Succinate, S ( type S-pathway to Q). Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway.
6Omy	SL(Omy)			1PM;2D;2c;3G;4S;5Rot;6Omy Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
7U*	SE	S	CII	1PM;2D;2c;3G;4S;5Rot;6Omy;7S 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.
8Ama	ROX			1PM;2D;2c;3G;4S;5Rot;6Omy;7S;8Ama 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).

Step	Respiratory state	Pathway control	ET-Complex	Comment
## AsTm	AsTmE	CIV	CIV
## Azd	CHB

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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

+ You obtain information in a single protocol about the NS-, S and N- pathway control state.

+ This protocol can be used as a control for SUIT-020 Fluo mt D033.

+ CIV activity determination and Cytochrome c test can be performed.

- Oligomycin concentration has to be determined. Higher concentrations of Oligomycin may inhibit the ET state.

Compare SUIT protocols

• SUIT-001;SUIT-001 O2 mt D001for isolated mitochondria and tissue homogenate: a complex protocol to get information not only about the NS(PGM) in the OXPHOS state, but also in ET state and about the FGp-pathways.
• SUIT-004;SUIT-004 O2 pfi D010: provides information on NS(PM) pathway in the ET state without the contribution of G.
• SUIT-008;SUIT-008 O2 pfi D014 and SUIT-008 O2 pce D025: provide information on the NS(PGM) pathway in the OXPHOS and ET-states, but without the addtion of Omy.
• SUIT-011: provides information about the NS(GM) pathway in the OXPHOS and ET-states, but without the addtion of Omy.
• SUIT-012: provides information about the N(PGM) pathway in the OXPHOS and ET-states without the contribution of S-pathway and without the addition of Omy.
• SUIT-014: a protocol similar to SUIT-020 O2 mt D035, but with the addition of P in the OXPHOS state before S. Omy is not added in the protocol.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry