FIGURE 6.

Chromatographic and enzymatic evidence of nuclear FAD synthesis. A, upper panel, typical HPLC chromatogram of neutralized perchloric acid extracts of isolated nuclei from rat liver showing flavin content (see “Experimental Procedures”). Lower panel, time course of FAD synthesis catalyzed by rat liver nuclei added to 2 μm FMN, 5 mm ATP, and 5 mm MgCl₂ in 50 mm Tris-Cl (pH 7.5) at 37 °C, in the absence (●) or in the presence (○) of 50 μm HgCl₂. B, the amount of FAD in ultrafiltered rat liver nuclei was enzymatically assayed by using the FAD detecting system (FADS D.S.), here schematized and described in greater detail under “Experimental Procedures.” Ultrafiltered nuclei (0.06 mg of protein) were incubated at 37 °C for 5 min in 100 μl of 50 mm Tris-HCl (pH 7.5) in the absence (dotted line, −FMN & ATP) or in the presence of the substrate pair 2 μm FMN and 1 mm ATP (straight lines). The effect of 5 mm MgCl₂ on FAD synthesis was also evaluated.