Oligomycin

From Bioblast
Jump to: navigation, search
Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         Oroboros O2k         MiP and biochemistry         Preprints and history



MitoPedia

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 state of respiration (abbreviated as L(Omy) to differentiate of L(n), LEAK state in the absence of ADP).

Abbreviation: Omy

Reference: MiPNet09.12


MitoPedia topics: Inhibitor 

Application in HRR

Oligomycin has frequently been 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 to measure LEAK respiration without or with slight inhibition of ET respiration. Therefore, we recommend to test and replace in your specific model the standard used oligomycin concentration (2.5 µM) for lower oligomycin concentration (5-10 nM) (Doerrier 2018 Methods Mol Biol).

Omy: Oligomycin (Oligomycin from Streptomyces diastatochromogenes; mixture of oligomycins A, B, and C); Sigma O 4876, 5 mg; store at -20 °C; FW = 800.
Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.


Preparation of 5 mM storage and stock solution (dissolved in 99.9% ethanol):
  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 °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.


O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume: 1 µL using a 10 µL syringe (2 mL O2k-Chamber).
  • Final concentration: 2 µg/mL (2.5 µM).



Preparation of 0.01 mM storage and stock solution (dissolved in 99.9% ethanol):
  1. Add 998 µL ethanol into a 2 mL glass vial.
  2. Take 2 µL 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 °C.


O2k manual titrations MiPNet09.12 O2k-Titrations
  • Titration volume: 1-2 µL titrations using a 10 µl syringe (2 mL O2k-Chamber).
  • 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 recent paper by Ruas et al. (2016) adressed this issue systematically and reports detailed results also on (unfortunately non-successfull) suggested alternatives to oligomycin. A potentially simple solution for at least some cell types has been suggested by Luiz Felipe Garcia e Souza, 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 hands following rigouros 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 uncuopling side effect. In order to adjust the optimal Omy concentration, it is necessary to run in parallel to our treatment experiment a control experiment on which we will add carrier control titrations,in this case pure ethanol. We start the Omy titration at very low concentrations [in nM range] and in the other chamber ethanol titrations are performed. The Omy titration should be stopped when the maximum inhibition of the respiration is reached. If the ET- state is already blocked at this Omy concentrations, lower concentration of Omy is needed, which should be tested again with the same protocol.

References

  • Estabrook_1961_Biochem Biophys Res Commun


>>Discussion