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SUIT-024 O2 ce-pce D056

From Bioblast


high-resolution terminology - matching measurements at high-resolution


SUIT-024 O2 ce-pce D056

Description

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

Abbreviation: N(PM)

Reference: Determination of the presence of ATPases in mitochondrial preparations - SUIT-024

SUIT number: D056_ce1;1Dig;1PM;2T;2D;3Omy;4Ama

O2k-Application: O2

SUIT-category: N(PM)
SUIT protocol pattern: Bidirectional linear ce1;1Dig;1PM;2T;2D;3Omy;4Ama

SUIT-024 O2 ce-pce D056 is a protocol for permeabilized cells, designed to quantify the ATPase activity in the sample. 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 contains 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 (last update 2019-05-14)

Representative traces

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 REN 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 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 Electron transfer 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

Chemicals and syringes

Step Chemical(s) and link(s) Comments
1Dig Digitonin (Dig)
Step Chemical(s) and link(s) Comments
1PM Pyruvate (P) and Malate (M)
2T ATP (T)
2D ADP (D)
3Omy Oligomycin (Omy)
4Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry