Fluorometric Assay for ATX lysoPLD Activity

The
biochemical studies of ATX lysoPLD activity were performed with ATX.
Activity was measured by a coupled reaction with 1 U mL^–1 choline oxidase and 2 U mL^–1 horseradish peroxidase
(HRP) and 2 mM homovanillic acid (HVA) (all from Sigma-Aldrich). For
the assays, 14:0, 16:0, 18:1, and 20:0 LPC (Avanti Polar Lipids Inc.)
were incubated with 20 nM ATX, reaching a final volume of 100 μL
of the buffer, which contained 50 mM Tris, 0.01%, 50 mM CaCl₂, Triton X-100, pH 7.4. Steady-state choline release was measured
at 37 °C by HVA fluorescence at λ_ex/λ_em = 320/460 nm in Corning 96- or 384-well OptiPlate (Sigma-Aldrich)
and with a Pherastar plate reader (BMG Labtech). To determine the
IC₅₀ for the different inhibitors on ATX activity, the
velocity of the reaction was monitored for each compound as a function
of time and the linear phase of the kinetics was taken from 60 min
after the addition of ATX to the reaction buffer. The resulting fluorescence
intensity signal over time was used to model all inhibitor concentrations
simultaneously using the following formula, where v_max and v_min were fitted for
the minimum and maximum relative velocities, respectively, and c_i corresponds to the inhibitor
concentration for each assay^{20 (link)}