SUIT-027 O2 ce-pce D065

high-resolution terminology - matching measurements at high-resolution

SUIT-027 O2 ce-pce D065

Description

Abbreviation: Malate anaplerosis

Reference: A - SUIT-027 - Protocol for analysis of the malate anaplerotic pathway

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

O2k-Application: O2

The SUIT-027 O2 ce-pce D065 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 oxaloacetate is not metabolized further in the absence of acetyl-CoA. The careful titration of malate is required to analyze the activity of the mt-isoform of NADP⁺- or NAD(P)⁺-dependent malic enzyme (mtME). If these enzymes are present in the sample one should be able to reach with malate alone 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  Cardoso LHD, Gnaiger E, Garcia-Souza LF (last update 2024-09-19)

Representative traces

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	REN			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).

Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

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

+ Multiple titrations with malate provide information on:

The presence of malate anaplerotic pathway due to mitochondrial malic enzyme in the sample studied.
Malate concentration that does not stimulate malate anaplerotic pathway, and therefore could be used to study F-pathway without N-pathway contribution in samples with mt malic enzyme.

- This protocol does not analyze coupling control.

- Different pathway control states are not analyzed in this protocol.

Compare SUIT protocols

SUIT-007: Protocol to analyze glutamate anaplerotic pathway.
SUIT-002 and SUIT-025: A low concentration of malate is initially used to assess F-pathway without N-pathway contribution. Further, a higher concentration of malate is added to analyze N-pathway.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1Dig	Digitonin (Dig)

Step	Chemical(s) and link(s)	Comments
1M.05	Malate (M)
2D	ADP (D)
3M	Malate (M)
4P	Pyruvate (P)
5G	Glutamate (G)
6Ama	Antimycin A (Ama)

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

References

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Fluorometry