2.6. Acyl‐CoA synthetase activity

Total cellular oleoyl‐CoA synthetase activity was determined by a radiometric assay (Füllekrug & Poppelreuther, 2016; Milger et al., 2006). For 30 min, 7–15 mg of muscle tissue was lysed on ice with 1% Triton X‐100, 130 mM KCl, 25 mM Tris–HCl, pH 7.4 and homogenized using a teflon pestle. Cell debris was sedimented by centrifugation at 4°C for 5 min at 10000 x g. 10 μL of the supernatant was incubated for 10 min at 30°C in 100 mM Tris pH 7.4, 5 mM MgCl₂, 200 μM dithiothreitol, 10 mM ATP, 0.2% Triton X‐100, 20 μM [¹⁴C]‐oleate (specific activity 10 Ci/moL, supplied by NEC #661) and 200 μM CoA. The incubation was terminated by the addition of Dole's solution (isopropanol‐n‐heptane‐H₂SO₄, 40:10:1) and unreacted oleate was extracted four times with n‐heptane. The remaining oleoyl‐CoA in the aqueous phase was quantified by scintillation counting with a Wallac 1414 liquid scintillation counter (Wallac Oy, Perkin Elmer).