From Bioblast
- high-resolution terminology - matching measurements at high-resolution
Oligomycin
Description
Oligomycin (Omy) inhibits ATP synthase by blocking its proton channel, specifically the FO subunit. In coupled mitochondria, inhibition of ATP synthesis leads to hyperpolarization of the mtIM and a concurrent reduction of mitochondrial respiration. Omy is used to induce LEAK respiration L(Omy). LEAK respiration in the presence of fuel substrates is measured as L(Omy) distinct from L(n) in the absence of ADP.
Historically Omy was used at high concentrations (1 to 2 µg·mL-1 or 1.26 to 2.53 µM), whereas more recently (2016 onwards) Omy concentrations as low as 10 nM were found to be fully inhibitory to induce the LEAK state and less inhibitory in the ET state.
Abbreviation: Omy
Reference: MiPNet09.12, Vernerova 2021 Biomedicines, Zdrazilova 2022 PLOS ONE
Application in HRR
- Omy: Oligomycin (mixture of oligomycins A, B, C from Streptomyces diastatochromogenes; C45H74O11), Sigma-Aldrich: O4876, store at -20 °C, CAS: 1404-19-9, M = 791 g·mol-1
- Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.
- Preparation of 5 mM Omy concentrated stock solution
- 1. Add 1.25 mL EtOH into the original container of Omy (5 mg).
- 2. Mix the solution by gently shaking the original closed container by hand.
- 3. Store the whole solution in the original commercial container and use it for preparation of less concentrated stock solutions.
- 4. Store at -20 °C.
- Preparation of 0.01 mM Omy stock solution (2 mL chamber)
- 1. Add 998 µL EtOH into a 2 mL glass vial.
- 2. Use 2 µL of 5 mM Omy concentrated stock solution and add it into the glass vial with EtOH.
- 3. Mix the solution with a pipette.
- 4. Divide into 0.2 mL portions in small glass vials.
- 5. Store at -20 °C.
- Preparation of 0.0025 mM Omy stock solution (0.5 mL chamber)
- 1. Add 600 µL EtOH into a 2 mL glass vial.
- 2. Use 200 µL of 0.01 mM Omy stock solution and add it into the glass vial with EtOH.
- 3. Mix the solution with a pipette.
- 4. Divide into 0.2 mL portions in small glass vials.
- 5. Store at -20 °C.
O2k-Titrations
- » Careful titration of Omy is necessary to determine the optimal inhibitory concentration and should be tested in each cell type and mt-preparation
- Titration volume for both 2 mL and 0.5 mL O2k-chamber: 1-2 µL steps using a 10 µL Hamilton syringe.
- Experimental concentration: 5-10 nM steps
- » Careful titration of Omy is necessary to determine the optimal inhibitory concentration and should be tested in each cell type and mt-preparation
Inhibition of ET capacity by oligomycin
- The side effect of Omy at higher concentrations is inhibition of ET-capacity as detailed by Ruas et al (2016). In experiments measuring respiration in intact human platelets (Doerrier et al 2018, Sumbalova et al 2018), the addition of Omy at 2.5 µM only resulted in a modest 1.2-fold increase in oxygen flux from ROUTINE to apparent ET capacity, compared to a 2.7-fold increase observed in control group with just the solvent (EtOH). However, by reducing the concentration of Omy to 0.5 µM, the 2.7-fold increase was fully preserved, while the transition from ROUTINE to LEAK respiration remained unchanged. This issue is further discussed in talk page
Oligomycin titration test
- To find the optimal concentration of Omy it is necessary to perform an Omy titration test starting from very low concentrations (by 5-10 nM steps) to reach final concentration between 10-40 nM (further described in Vernerova et al 2021 and Zdrazilova et al 2022). Moreover, it is essential to run a control experiment with titrations of the carrier (EtOH) in a parallel O2k-chamber. Titration of Omy should cease once maximum inhibition of respiration is observed. If the ET state is inhibited at these concentrations of Omy in comparison to the control measurement, a lower concentration of Omy should be tested following the same protocol.
Contamination of O2k-chamber by oligomycin
- Various research groups reported to the O2k-network that Omy was hard to remove after application in an O2k-chamber. In our experience, following rigorous washing procedures generally helps to avoid such problems Di Marcello et al 2023. Solutions suggested by our users in the O2k-Network Discussion forum can be found on the Discussion page
Oligomycin and yeast
- 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.
References
- Estabrook_1961_Biochem Biophys Res Commun
MitoPedia topics: Inhibitor
>>Discussion
