Purification and characterization of adenosine diphosphate ribose pyrophosphatase from human erythrocytes

Free ADP-ribose is a turnover product of NAD⁺, protein-bound polymeric and monomeric ADP-ribose, and cyclic ADP-ribose. But little is known about the specific cellular roles or metabolism of free ADP-ribose. ADPribose pyrophosphatase (EC 3.6.1.13), which hydrolyzes ADP-ribose into AMP and ribose-5'-phosphate, was purified from human erythrocytes. Purification was achieved to homogeneity by successive chromatographic steps, resulting in a final purification of 75,790-fold from the hemolysate. The purified enzyme showed a single band with the molecular weight of 34 kDa on SDS-PAGE both in the presence and absence of 2-mercaptoethanol. The molecular weight of the native enzyme calculated by gel filtration was 68 kDa, indicating that the active enzyme is a dimer of identical subunits. The enzyme requiring Mg²⁺ showed highest activity toward ADP-ribose, and about 4070% activities with IDP-ribose, ADP-mannose and GDP-mannose. The enzyme showed a K(m) of 169 ± 11 μM for ADP-ribose, broad pH optimum around pH 9.5, and pI of 5.1. ADP was a potent noncompetitive inhibitor with a K(i) of 16 ± 1.2 μM. These results suggest that our enzyme is unique, and different from the other ADP-ribose pyrophosphatases reported. ADP-ribose pyrophosphatase may play an important role in the regulation of intracellular steady-state of free ADP-ribose.