SUIT-027 O2 ce-pce D065

high-resolution terminology - matching measurements at high-resolution

SUIT-027 O2 ce-pce D065

Description

Abbreviation: Anaplerotic pathway

Reference: A - SUIT-027

SUIT number: D065_ce1;1Dig;1M;2D;3M;4P;5G;6Ama

O2k-Application: O2

MitoPedia: SUIT - Protocol for analysis of the anaplerotic pathway

SUIT protocol pattern: ce1;1Dig;1M;2D;3M;4P;5G-

This protocol is focused on the analysis of the Malate anaplerotic pathway control state in mitochondrial preparations (isolated mitochondria, permeabilized cells, homogenate and permeabilized fibers). Malate alone does not support respiration if the oxaloacetate is not metabolized further in the absence of acetyl-CoA. The careful titration of malate is required to analyse the activity of the mt-isoform of NADP⁺- or NAD(P)⁺-dependent malic enzyme (mtME). If these enzymes are present in our sample we should be able to reach a high respiratory activity comparable to the NADH linked pathway control states with more classical combinations of substrates (e.g. PM or GM).

Communicated by  Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-26)

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
ce1	ROUTINE			ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
1Dig	Dig			ce1;1Dig Optimum effective digitonin concentration for complete plasma membrane permeabilization.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1M.05	(M_L)			ce1;1Dig;1M Low concentration of malate, typically low concentration, does not saturate the N-pathway, and will allow us to find the minimum concentration to stimulate the anaplerosis.
2D	( M_P)			ce1;1Dig;1M;2D OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3M	M_P	N	CI	ce1;1Dig;1M;2D;3M NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4P	PM_P	N	CI	ce1;1Dig;1M;2D;3M;4P NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
5G	PGM_P	N	CI	ce1;1Dig;1M;2D;3M;4P;5G NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
6Ama	ROX			ce1;1Dig;1M;2D;3M;4P;5G;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).