FIGURE 6.

Synthesis of ADP-ribosylagmatine and nicotinamide release by NarE and site-directed mutants. Affinity-purified proteins were incubated for 18 h at 30 °C in 50 mm potassium phosphate buffer with 0.1 mm [adenine-U-¹⁴C]NAD (0.05 μCi) and 20 mm agmatine as ADP-ribose acceptor (A) or with 0.1 mm [carbonyl-¹⁴C]NAD (0.05 μCi) (B) in a final volume of 0.3 ml. ADP-ribosylagmatine or ¹⁴C-nicotinamide were purified by AG1-X2 and quantified by scintillation counting. Data are expressed as ADP-ribosylagmatine formed or nicotinamide released per mg of mutant proteins. Data presented are the means ± S.D. of three independent assays, with each activity measurement performed in duplicate.