Template:SUIT-39

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1D	ROX			1D ADP is added to stimulate consumption of endogenous fuel-substrates. Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.
2M.1				1D;2M.1 Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
3Pal	PalM_P	F	FAO	1D;2M.1;3Pal Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration.
3c	PalMc_P	F	FAO	1D;2M.1;3Pal;3c Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration. 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]).
4M2	PalM_P	F(N)	FAO	1D;2M.1;3Pal;3c;4M2 Respiratory stimulation of the FAO-pathway, F, by fatty acid FA in the presence of malate M. Malate is a type N substrate (N), required for the F-pathway. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the F-pathway capacity is overestimated, if there is an added contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration. High concentration of malate, typically 2 mM, saturates the N-pathway. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5P	PalPM_P	FN	FAO&CI	1D;2M.1;3Pal;4M2;5P Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
6G	PalPGM_P	FN	FAO&CI	1D;2M.1;3Pal;4M2;5P;6G Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
7S	PalPGMS_P	FNS	FAO&CI&II	1D;2M.1;3Pal;4M2;5P;6G;7S Respiratory stimulation by simultaneous action of the F-pathway, N-pathway, and S-pathway, with convergent electron flow in the FNS-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
8U	PalPGMS_E	FNS	FAO&CI&II	1D;2M.1;3Pal;4M2;5P;6G;7S;8U Respiratory stimulation by simultaneous action of the F-pathway, N-pathway, and S-pathway, with convergent electron flow in the FNS-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F. 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.
9Rot	S_E	S	CII	1D;2M.1;3Oct;4M2;5P;6G;7S;8U;9Rot Succinate, S ( type S-pathway to Q). Noncoupled electron transfer state, ET state, with ET capacity E.
10Ama	ROX			1D;2M.1;3Oct;4M2;5P;6G;7S;8U;9Rot;10Ama 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).