Oligomycin

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Oligomycin

Description

Oligomycin (Omy) is an inhibitor of ATP synthase by blocking its proton channel (Fo subunit), which is necessary for oxidative phosphorylation of ADP to ATP (energy production). The inhibition of ATP synthesis also inhibits respiration. In OXPHOS analysis, Omy is used to induce a LEAK respiration state of respiration (abbreviated as L(Omy) to differentiate from L(n), LEAK state in the absence of ADP).

Abbreviation: Omy

Reference: MiPNet09.12, BEC 2020.1, Doerrier 2018 Methods Mol Biol






MitoPedia topics: Inhibitor 

Application in HRR

Omy: Oligomycin (from Streptomyces diastatochromogenes; mixture of oligomycins A, B, and C), Sigma-Aldrich: O4876, store at -20 Β°C, CAS: 1404-19-9, M = 800 gΒ·mol-1
Oligomycin has frequently shown an inhibitory effect on ET capacity (Garcia-Souza 2018 Life Sciences Meeting 2018 Innsbruck AT, Doerrier 2018 Methods Mol Biol). We found that the use of lower oligomycin concentration in many mt-preparations and cell types allows measurement of LEAK respiration without or with only slight inhibition of ET respiration. Therefore, we recommend to test and replace in your specific model high oligomycin concentrations (2.5 Β΅M) for a lower oligomycin concentration (5-10 nM) (Doerrier 2018 Methods Mol Biol).


Preparation of 5 mM storage and stock solution (dissolved in ethanol abs.):
  1. Weigh 4 mg Oligomycin into a 2 mL glass vial.
  2. Dissolve in 1 mL ethanol.
  3. Store in glass vials (recommendation) at -20 Β°C.
  • Storage option:
  1. Divide into 0.2 mL portions in clear glass vials.
  2. Store the whole solution in 2 mL clear glass vial and use subsamples during experiments to avoid contamination of the storage solution.


Preparation of 0.01 mM stock solution (dissolved in ethanol abs.) for 2-mL O2k-chamber:
  1. Add 998 Β΅L ethanol into a 2 mL glass vial.
  2. Take 2 Β΅L of 5 mM Omy stock solution and add it into the glass vial with ethanol.
  3. Mix the solution and divide it into 0.2 mL portions or store in 2 mL glass vial and use subsamples during experiments to avoid contamination of the storage solution.
  4. Store in glass vials (recommendation) at -20 Β°C.
Β» O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (2-mL O2k-chamber): 1-2 Β΅L using a 10 Β΅L Hamilton syringe.
  • Final concentration: 5-10 nM (optimum oligomycin concentrations should be tested in each cell type and mt-preparation).


Preparation of 0.0025 mM stock solution (dissolved in ethanol abs.) for 0.5-mL O2k-chamber:
  1. Add 1999 Β΅L ethanol into a 2 mL glass vial.
  2. Take 1 Β΅L of 5 mM Omy stock solution and add it into the glass vial with ethanol.
  3. Mix the solution and divide it into 0.2 mL portions or store in 2 mL glass vial and use subsamples during experiments to avoid contamination of the storage solution.
  4. Store in glass vials (recommendation) at -20 Β°C.
Β» O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume (0.5-mL O2k-chamber): 1-2 Β΅L using a 10 Β΅L Hamilton syringe.
  • Final concentration: 5-10 nM (optimum oligomycin concentrations should be tested in each cell type and mt-preparation).


Problems with the use of oligomycin

  • Oligomycin has frequently been shown to lower subsequently measured ET capacity, a problem also reported in the O2k-Network Discussion forum. A paper by Ruas et al (2016) addressed this issue systematically and reports detailed results also on (unfortunately unsuccessful) suggested alternatives to oligomycin. A potentially simple solution for at least some cell types has been suggested by Garcia-Souza Luiz F, see here.
  • Several researchers have reported problems associated with the use of oligomycin which appear primarily related to difficulties with getting rid of the inhibitor after application in a respirometry chamber. In our experience, following rigorous washing procedures generally helps to avoid such problems: for more details, see MiPNet19.03_O2k-cleaning_and_ISS. Solutions suggested by our users in the O2k-Network Discussion forum can be found on the Discussion page
  • Oligomycin has been found ineffective as inhibitor of the ATP synthase in various strains of yeast, a problem we were made aware of by Gennaro Agrimi and which is briefly described and discussed here.

Omy titration test

  • Higher concentration of Omy can decrease the ET capacity owing to its uncoupling side effect. In order to evaluate the optimal Omy concentration, it is necessary to titrate Omy, starting at very low concentrations (in the nM range). In parallel, carrier control titrations, i.e. ethanol titrations, should be performed in the other chamber. Omy titration should be stopped when the maximum inhibition of respiration is reached. If the ET is already inhibited at this Omy concentrations, lower concentration of Omy is needed, which should be tested again with the same protocol. The test should be performed with the same protocol chosen for the experiments.

References

  • Estabrook_1961_Biochem Biophys Res Commun


>>Discussion
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