Figure 6.

Fluorimetric evidence of FAD synthesis. The FAD synthesis reaction was started by the addition of purified recombinant proteins (hFADS6 open circle or D238A-hFADS6 closed circle) and measured by the initial rate of fluorescence decrease (λ excitation = 450 nm and λ emission = 520 nm). V₀ was expressed as nmol min⁻¹ mg⁻¹ (a,b) and as a percentage of the V_max value (a’,b’) set arbitrarily to 100%. Data points are fitted according to the Michaelis–Menten equation with Grafit 3.0 software. (a) ATP concentration dependence. FAD synthesis rate, catalyzed by purified hFADS6 (open circle, 10 µg, 0.26 nmoL) or D238A-hFADS6 (closed circle, 4.2 µg, 0.11 nmoL), was fluorimetrically measured at 37 °C in 2 mL of 50 mM Tris⁄HCl pH 7.5, in the presence of 5 mM MgCl₂, 2 µM or 3 µM FMN, respectively, and of the given ATP concentrations. (b) FMN concentration dependence. FAD synthesis rate catalyzed by purified hFADS6 (open circle, 10 µg, 0.26 nmoL) or D238A-hFADS6 (closed circle, 3.9 µg, 0.10 nmoL) was fluorimetrically measured at 37 °C in 2 mL of 50 mM Tris⁄HCl pH 7.5, in the presence of 5 mM MgCl₂, 100 µM ATP, and of the given FMN concentrations.