Polymerization of ADP-ribose pyrophosphatase: Conversion mechanism of Mg²⁺-dependent ADP-ribose pyrophosphatase into Mg²⁺- independent form

ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose (ADPR) into AMP and ribose-5′-phosphate. It is classified into two groups, Mg ²⁺-dependent and Mg²⁺-independent ADPRase, depending on its Mg²⁺ requirement. Here, we purified Mg²⁺-dependent ADPRase from rabbit liver and examined what factors affect Mg²⁺ requirement. 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 ADPRase is a dimer made up of two identical subunits. Mg ²⁺-dependent ADPRase with the highest ADPR affinity had a K _m of 160±10 μM and a pH optimum of around pH 9.5. Treatment of the purified ADPRase with heated cytosol fractions at 37°C for 3 h caused some changes in the chemical properties of the enzyme, including an increase in molecular weight, a decrease in solubility, and a loss of Mg ²⁺-dependency. The molecular weight of the cytosol-treated ADPRase measured by gel filtration was over 420 kDa, suggesting, for the first time, that ADPRase could be polymerized by undefined cytoplasmic factors, and that polymerization is accompanied by changes in the solubility and metal ion dependency of the enzyme.