Enzyme activity assay

Enzyme activity of NMNAT was measured in a continuous spectrophotometric coupled assay by monitoring the increase in absorbance of NADH at 340 nm, The reaction process is as follows Balducci et al. (1995):

The reaction solution contains 28 mM HEPES buffer (pH 7.4), 11.2 mM MgCl2, 16 nM semicarbazide-HCl, 0.046 mM ethanol, 1.5 mM ATP, and 0.03 mg/ml yeast alcohol dehydrogenase (Sigma, A7011), and NMNAT or variants. The reaction was initiated by adding NMN to a final concentration of 0.625 mM. All measurements were performed at 37 °C. The activity was calculated using the equation below.

Where C₀β-NADH, the extinction coefficient of β-NADH at 340 nm, is 6.22 (Zhai et al., 2006).