SUIT-007 O2 ce-pce D030

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SUIT-007 O2 ce-pce D030

Description

Abbreviation: N(GM)

Reference: A

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

O2k-Application: O2

SUIT-category: N(GM)

SUIT protocol pattern: diametral ce1;1Dig;1G;2D;2c;3M;4U;5Ama

References

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 analysis of glutamate dehydrogenase anaplerotic pathway/glutaminolysis in permeabilized cells (pce).

+ The use of glutamate alone allows to analyse the glutamate dehydrogenase 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.

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

Compare SUIT protocols

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry