A Regulatory Role of NAD Redox Status on Flavin Cofactor Homeostasis in S. cerevisiae Mitochondria
Figure 4
Some features of -NAD+ cleavage. (a) The pH profile of the rate of -NAD+ (100 μM) cleavage, catalyzed by solubilized SCM (0.3 mg protein), was fluorimetrically measured as described in Section 2. Use was made of 100 mM acetate/acetic acid and 50 mM Tris/HCl buffering mixtures (supplemented with 5 mM MgCl2), whose pH was adjusted to the desired value. A specific calibration curve was obtained at each pH value as described in Section 2. The values are reported as percentage of the maximum rate (i.e., 1.6 nmol · min−1 · mg−1), arbitrarily set equal to 100%. (b) The dependence of the rate of -NAD+ cleavage on substrate concentration was measured using solubilized SCM (0.3 mg protein) at pH 6. (c) The rate of -NAD+ (200 μM) cleavage catalyzed by TX100-solubilized sphero and SCM (0.3 mg protein each) was measured at pH 6. (d) The sensitivity of -NAD+ (50 μM) cleavage (catalyzed by solubilized SCM, 0.3 mg protein) towards NaF, CaCl2, AMP (1 mM each), and nicotinamide (at the indicated concentrations) was measured at pH 6.
