Research Article

A Regulatory Role of NAD Redox Status on Flavin Cofactor Homeostasis in S. cerevisiae Mitochondria

Figure 5

FAD pyrophosphatase in solubilized SCM. (a) SCM (0.05 mg protein), solubilized with TX100, were incubated under the experimental conditions described in Section 2. The reaction was started by FAD (3 μM) addition to the mitochondrial suspension and followed at 25°C. FAD emission spectra (at excitation wavelength 450 nm) were monitored at different incubation times. (b) FAD fluorescence decrease was continuously monitored at 450/520 nm under the same experimental condition described in (a) using intact (−TX100) or solubilized SCM (+TX100). (c) Extracts of the FAD hydrolysis reaction mixture were taken at different incubation times and analyzed via HPLC, as described in Section 2 with measurements of FAD, FMN, and Rf concentrations. The value obtained were plotted against the incubation time after FAD addition. FAD hydrolysis reaction was carried in the absence (panel (A)) or in the presence of AMP (1 mM) (panel (B)) or NaPPi (1 mM) (panel (C)). The inset in (panel (A)) is an enlargement of the first 60 min of the plot reported in the same panel.
612784.fig.005a
(a)
612784.fig.005b
(b)
612784.fig.005c
(c)