SUIT-003 O2 ce D039

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SUIT-003 O2 ce D039

Description

Abbreviation: ce

Reference: A: Coupling control in intact cells without oligomycin; ce: non-permeabilized cells -SUIT-003

SUIT number: D039_ce1;(ce2Omy);ce3U;ce4Rot;ce5Ama

O2k-Application: O2

MitoPedia: SUIT

SUIT-category: ce

SUIT protocol pattern: linear cell (ce) coupling control protocol ce1;(ce2Omy);ce3U;ce4Rot;ce5Ama

SUIT-003 O2 ce D039 has been designed to study the coupling control state of intact microalgal cells. The different capacities are tested in the sequence OXPHOS, LEAK-respiration and ET. Depending on the species of microalgae studied, the oligomycin or any other inhibitor of the ATP synthase or of the adenine nucleotide translocase might not have an effect and the LEAK-respiration cannot be achieved. Therefore, it is recommend to test the effect of these inhibitors in preliminary experiments for different species. Also, the inhibitory effect on the electron transfer system has to be studied to avoid an underestimation of the ET-capacity. This protocol has been optimised specifically for the microalgae species Chlamydomonas reinhardtii in which oligomycin does not have an effect.

Communicated by Huete-Ortega M and Gnaiger E (last update 2019-04-10)

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.
(ce2Omy)	L(Omy)			ce1;(ce2Omy) Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin. The addition of oligomycin, any other inhibitor of the ATP synthase or the adenine nucleotide translocase or its carriers (EtOH) is optional depending on the microalgal species studied. If we add only the carrier we do not reach LEAK state and we will evaluate if the EtOH alters the ROUTINE state.
ce3U	E			ce1;(ce2Omy);ce3U 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. Noncoupled electron transfer state, ET state, with ET capacity E.
ce4Rot	B(Rot) or ROX			ce1;(ce2Omy);ce3U;ce4Rot In microalgal cells, Rotenone inhibits Complex I (CI) but, if the internal type II NADH dehydrogenase remains active NADH-Q branch respiration, B(Rot), can continue Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.
ce5Ama	ROX			ce1;(ce2Omy);ce3U;ce4Rot;ce5Ama 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

• The SUIT-003 O2 ce D039 is a cell coupling control protocol. The total cell count (N_ce) is the sum of viable cells (N_vce) and dead cells (N_dce).
• CIV activity can also be measured after the cell viability test module.

+ Reasonable duration of the experiment.

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

- Depending on the species of microalgae studied oligomycin or any other inhibitor of the ATP synthase or of the adenine nucleotide translocase might not have an effect and therefore the LEAK-respiration cannot be achieved.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry