SUIT-007 O2 ce-pce D030

high-resolution terminology - matching measurements at high-resolution

SUIT-007 O2 ce-pce D030

Description

Abbreviation: Glutamate anaplerotic pathway

Reference: A pce: permeabilized cells - SUIT-007

SUIT number: D030_ce1;1Dig;1G;2D;2c;3M;4U;5Ama

O2k-Application: O2

SUITbrowser: Glutamine/glutamate

The SUIT-007 O2 pce D030 is focused on the glutamate anaplerotic pathway. If glutamate dehydrogenase is present in the sample when glutamate is added alone, it will be converted to α-ketoglutarate in an anaplerotic reaction, supporting respiration. After malate is added, it is possible to analyse the NADH Electron transfer-pathway state with glutamate and malate as substrates in the OXPHOS and Electron transfer pathway coupling control states. In this protocol, non-permeabilized cells are added in the chamber, and ROUTINE respiration is measured. The cells are further permeabilized with digitonin inside the O2k chamber.

Communicated by Cardoso LH, Komlodi T and Gnaiger E (last update 2019-07-03)

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)
1G	GL	(N)	CI	1G NL(n) if glutamate dehydrogenase is present in the sample studied (Glutamate-anaplerotic pathway control state). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	GP	(N)	CI	1G;2D NP if glutamate dehydrogenase is present in the sample studied (Glutamate-anaplerotic pathway control state). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	GcP	(N)	CI	1G;2D;2c 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.
3M	GMP	N	CI	1G;2D;2c;3M NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4U	GME	N	CI	1G;2D;2c;3M;4U NADH-linked substrates (type N-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.
5Ama	ROX			1G;2D;2c;3M;4U;5Ama 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).

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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

• Protocol focused on the analysis of glutamate anaplerotic pathway/glutaminolysis in permeabilized cells (ce-pce).

+ The use of glutamate alone allows to analyze the glutamate-anaplerotic pathway control state.

+ Short duration of the experiment.

+ Glutamate is easier to prepare compared to pyruvate.

+ CIV activity can be measured after this protocol.

- Analysis of N-OXPHOS capacity (instead of NS-OXPHOS capacity) does not provide the best estimate of maximum mitochondrial respiratory capacity.

- This protocol does not address questions regarding the convergence of pathways at the Q-junction.

Compare SUIT protocols

• SUIT-006: Coupling-control protocol, can be performed with GM.

Chemicals and syringes

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

Step	Chemical(s) and link(s)	Comments
1G	Glutamate (G)
2D	ADP (D)
2c	Cytochrome c (c)
3M	Malate (M)
4U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
5Ama	Antimycin A (Ama)

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

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry