SUIT-007

high-resolution terminology - matching measurements at high-resolution

SUIT-007

Description

Abbreviation: Glutamate anaplerosis

Reference: A: Glutamate anaplerotic pathway

SUIT protocol pattern: 1G;2D;2c;3M;4U-

The SUIT-007 is focused on the glutamate anaplerotic pathway. If glutamate dehydrogenase is present in the sample, when glutamate alone is added, it will be converted to α-ketoglutarate in an anaplerotic reaction, supporting respiration. After malate is added, it is possible to analyze the NADH Electron transfer-pathway state with glutamate and malate as substrates in the OXPHOS and Electron transfer pathway coupling control states.

Communicated by Cardoso LH, Komlodi T, Doerrier C and Gnaiger E (last update 2024-09-19)

Specific SUIT protocols

• SUIT-007 O2 ce-pce D030 for living cells (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs into the chambers through digitonin addition)

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1G	GL	(N)	CI	1G NL 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).

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

• Protocol focused on analysis of glutamate anaplerotic pathway/glutaminolysis.

+ 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.

- Addition of cytochrome c after glutamate alone (1G;2D;2c) is not as appropriated to evaluate integrity of the mitochondrial external membrane as it would be with GM. However, with addition of cytochrome c only after malate (e.g. 1G;2D;3M;3c), it would not be possible to compare G_P and GM_P if there are damages to the mitochondrial external membrane, therefore it is suggested to add cytochrome c after ADP (1G;2D;2c).

- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

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

References

MitoPedia concepts: MiP concept, SUIT protocol 

MitoPedia methods: Respirometry