Fig. 4.

ACOT9 activity is regulated by NADH and CoA. a Recombinant ACOT9 was incubated at fixed concentrations of C₃-CoA (50 μM) or C₁₄-CoA (10 μM) and varying concentrations of NADH (0–1,000 μM). The activity was measured using the standard spectrophotometric assay at 412 nm and the graph shows one representative experiment out of three for C₃-CoA (upper panel) and one out of two experiments for C₁₄-CoA (lower panel). The data are shown as the % remaining activity in the presence of NADH (mean ± SEM of 1–4 measurements). b Thioesterase activity was measured with soluble extracts of mitochondria isolated from BAT, kidney, and liver with 50 μM C₃-CoA or 25 μM C₁₄-CoA in the presence (light grey bars) or absence (dark grey bars) of 400 μM NADH. c Recombinant ACOT9 was incubated at fixed concentrations of C₃-CoA or (50 μM) and C₁₄-CoA (10 μM) in the presence of varying concentrations of CoA (0–200 μM) and activity was measured using the UV spectrophotometric assay. One representative experiment out of three is shown for C₃-CoA and one out of two for C₁₄-CoA. The values represent mean ± SEM of 2–5 measurements