SUIT-024

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SUIT-024

Description

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

Abbreviation: ATPase (PM)

Reference: A: Determination of ATPase activity in mitochondrial preparations.

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

SUIT-024 is designed to quantify the ATPase activity of mitochondrial preparations by addition of ATP (step 2T). ATPase activity in isolated mitochondria suggests contamination of the preparation by non-mitochondrial membranes. Endogenous adenylates are phosphorylated to ATP in the LEAK state, which is recycled to ADP in isolated mitochondria contaminated by ATPases, which leads to an overestimation of LEAK respiration, when comparing L(n) and L(Omy). Tissue homogenates, permeabilized muscle fibers and permeabilized cells contain all cellular membranes with high ATPase activity. The present SUIT-024 DLP file shows an application with ce-pce in the pathway category N(PM). For using this protocol with other mitochondrial preparations or substrate/inhibitor combinations, a personalized DLPU can be created.

Communicated by Garcia-Souza LF, Cardoso LHD, Gnaiger E (last update 2020-04-17)

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).
  • In the absence of ATPase activity, the LEAK state is maintained. However, if ATPases are active and thus generate ADP, respiration coupled to phosphorylation is stimulated. Stimulation is limited up to OXPHOS capacity; therefore, higher ATPase activities cannot be determined.
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 determine ATPase acitivity in mitochondrial preparations. It is particularly useful as a quality control of the purity of isolated mitochondria.
+ Different combinations of substrates and inhibitors can be used depending on the specific aims, e.g.: PGM, PM, GM, MnaPM or other N-pathway substrate combinations; S or RotS for the S-pathway control state.
+ LEAK respiration is evaluated by inhibition of ATP synthase by Oligomycin.
+ Reasonable duration of the experiment.
- If ATPase activity is actually higher than OXPHOS capacity, then the maximum ATPase activity cannot be quantified.
- This protocol does not include Electron transfer pathway control steps.
- CIV activity is not measured, to save experimental time.
- Careful washing of the chamber 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