SUIT-024

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SUIT-024

Description

Ce1;1Dig;1PM;2T;2D;3Omy-.png

Abbreviation: ATPase (PM)

Reference: A: Determination of the presence of ATPases in mitochondrial preparations.

SUIT protocol pattern: ce1;1Dig;1PM;2T;2D;3Omy;4Ama

The SUIT-024 is designed to test the contamination of the sample with ATPases. When isolating mitochondria, contamination with other membranous organelles containing ATPases (e.g., plasma membrane and endoplasmic reticulum) can occur. In experiments with tissue homogenates and permeabilized cells, it is expected to have active ATPases in the respiration medium. When ATPases are active and residual endogenous adenylates are present in the sample, ATP can be consumed and converted to ADP, which will stimulate respiration. This may lead to an overestimation of LEAK respiration - L(n). Therefore, if the samples are contaminated with ATPases/adenylates, it might be necessary to assess LEAK respiration in the presence of ATPases inhibitors or oligomycin - L(Omy). This protocol can be used as a quality control of the mitochondrial preparation.

In the DatLab software, SUIT-024 DLP file is provided for ce-pce and the category N(PM). For using this protocol with other sample preparation or substrate/inhibitor combinations, a personalized DLPU can be created.


Communicated by Garcia-Souza LF, Cardoso LH (last update 2019-06-05)

Specific SUIT protocols

SUIT-024 O2 ce-pce D056

Ce1;1Dig;1PM;2T;2D;3Omy;4Ama.png D056 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;1Dig;1PM;2T;2D;3Omy;4Ama.png

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 ROX ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1PM PML(n) N CI 1PM
2T PML or PMP N CI 1PM;2T
  • NADH-linked substrates (type N-pathway to Q).
  • When ATPases are not present in the sample, the LEAK state is maintained. However, if ATPases are active in the sample and thus generate ADP, oxidative respiration coupled to oxidative phosphorylation is stimulated, and might reach OXPHOS respiration.
2D PMP N CI 1PM;2T;2D
3Omy PML(Omy) 1PM;2T;2D;3Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
4Ama ROX 1PM;2T;2D;3Omy;4Ama
  • 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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Strengths and limitations

  • This protocol allows to analyse the presence of ATPases in mitochondrial preparations and is useful as a quality control of the sample preparation.
+ Different combinations of substrates and inhibitors can be used depending on the specific aims, e.g.: PGM, PM, GM, MnaPM or other combinations for N-pathway control state; S or RotS for S-pathway control state.
+ LEAK respiration overestimation is prevented in the presence of Oligomycin.
+ Reasonable duration of the experiment.
- This protocol does not include internal ET-pathway control steps.
- CIV activity is not measured, to save experimental time.
- Careful washing is required after the experiment to avoid carry-over of oligomycin.

Compare SUIT protocols

References

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry