SUIT-003 O2 ce D039

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SUIT-003 O2 ce D039

Description

Ce1;(ce2Omy);ce3U;ce4Rot;ce5Ama.jpg

Abbreviation: CCP-ce microalgae

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


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 Electron transfer pathway. 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 2019-06-12)


Representative traces

D009 Traces algae.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;(ce2Omy);ce3U;ce4Rot;ce5Ama.jpg

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
ce4Rot ROX ce1;(ce2Omy);ce3U;ce4Rot
  • 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).


Questions.jpg


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Strengths and limitations

+ 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.

Compare SUIT protocols

Chemicals and syringes

Step Chemical(s) and link(s) Comments
(ce2Omy) Oligomycin (Omy) This step can be skipped
ce3U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
ce4Rot Rotenone (Rot)
ce5Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry