Figure 1.

Incubation of α-NAD⁺ with the ARHs and macrodomain proteins. (I) Time course of ADP-ribose production. (II) HPLC separation of reaction products. (III) HPLC separation of wild-type and mutant ARH3, ARH1, and Af1521.

Time course of ADP-ribose production. ARH3 (●), C6orf130 (Δ) and Af1521(○) ARH 1 (□), MacroD1 (▲) and MacroD2 (◊) (70 pmols) were incubated with α-NAD⁺ (20 nmols) as described in Methods. The data show the amount of ADP-ribose formed from the hydrolysis of α-NAD⁺ in 50μl of the 200μl reaction mixture and its separation on HPLC. The data represent the means ± SD of two experiments performed in duplicate and are representative of more than three time-course experiments (I).

Reaction products from the incubation of the ARHs and the macrodomain proteins with α-NAD⁺. ARHs and macrodomain proteins were incubated with α-NAD⁺ (50μM) in 50mM Tris pH 7.5, with 10mM MgCl₂ for ARH1 or ARH3, and without 10mM MgCl₂ for macrodomain proteins in 200μl of reaction mix for 1hr at 37°C before 50μl of the reaction products were separated by HPLC and monitored at 258nm as described in Methods. A. (●) α-NAD⁺, (10 nmol), B (■) ADP-ribose (20 nmol), C. ARH1 (13μg), D. ARH3 (1.2μg) E. MacroD2 (23μg), F. Af1521 (10μg). The data are examples of more than 5 chromatograms performed in duplicate (II). ARH3 (1μg, H,I), ARH1 (25μg, J,K) and Af1521 (10μg, L,M) wild-type and mutated protein were incubated with α-NAD⁺ (●) (100μM, ARH3, ARH1, 50μM, Af1521) in 50mM Tris pH 7.5, 10mM MgCl₂ in 200μl (ARHs), 225μl (Af1521) of reaction mix for one hour at 37°C before 150μl (ARHs) or 200μl (Af1521) of the reaction products were separated by HPLC, monitored at 258nm as described in Methods. Assays were done in triplicate. Data represent a single separation (III).