SUIT-014

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SUIT-014

Description

Abbreviation: NS(PGM)

Reference: A  »Versions

SUIT-category: NS(PGM)

SUIT protocol pattern: diametral 1GM;2D;3P;4S;5U;6Rot-

The SUIT-014 protocols are designed for cells or tissue types that display a preference for GM over PM to support NADH-linked respiration. Adding pyruvate in the OXPHOS state enables to check if NADH-linked respiration can be further stimulated to determine N-pathway P capacity. SUIT-014 protocols provide an assessment of the linear coupling control (L- P- E) with NADH linked-substrates (PGM) and the control in ET state (N, NS, S), covering the contribution of two pathways which are most important in the mitochondria of many species, tissues and cell types. SUIT-014 can be extended with the CIV assay module.

Communicated by Cardoso LH, Doerrier C, Huete-Ortega M and Gnaiger E (last update 2019-01-30)

Specific SUIT protocols

• SUIT-014 O2 pfi D042 for permeabilized fibers

References

	Year	Reference	Organism	Tissue;cell
Lemieux 2017 Sci Rep	2017	Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840. doi:10.1038/s41598-017-02789-8	Mouse	Heart

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1GM	GML(n)	N	CI	1GM 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	GMP	N	CI	1GM;2D NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	GMcP	N	CI	1GM;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.
3P	PGMP	N	CI	1GM;2D;2c;3P NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4S	PGMSP	NS	CI&II	1GM;2D;2c;3P;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.
5U	PGMSE	NS	CI&II	1GM;2D;2c;3P;4S;5U Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. 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.
6Rot	SE	S	CII	1GM;2D;2c;3P;4S;5U;6Rot Succinate, S ( type S-pathway to Q). Noncoupled electron transfer state, ET state, with ET capacity E.
7Ama	ROX			1GM;2D;2c;3P;4S;5U;6Rot;7Ama 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

Compare SUIT protocols

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 

MitoPedia methods: Respirometry