Studies on nucleotide pyrophosphatase I. Partial purification and properties of a sheep liver enzyme that catalyzes the hydrolysis of dinucleotides

Abstract

A partially purified sheep liver enzyme that hydrolyzed dinucleotides at the pyrophosphate bond was obtained by solubilizing the 18,000g sediment with n-butanol and fractionating the solubilized enzyme with acetone. The enzyme activity when measured using FAD as substrate, (FAD → FMN + AMP), was optimal at pH 9.7 and temperatures between 30°-36° and at 60°. The rate of release of FMN with time occurred with an initial lag of 30 sec, a linear increase for 1 min, and a subsequent irregular rate. In the presence of orthophosphate (P_i; 10μM), FMN was released at an uniformly continuous and enhanced rate. ³²P_i was not incorporated into the substrate or products. Sodium arsenate counteracted the effects of P_i. The apparent K_m and V_max were 0.133mM and 100 units; and 0.133mM and 200 units, in the absence and presence of P_i, respectively. The temperature optimum was 42° in the presence of P_i. Negative cooperative interactions observed at low concentrations of FAD were abolished by the addition of P_i. The inhibition by AMP was sigmoid and P_i abolished this sigmoidal response. The enzyme hydrolyzed in addition to FAD, NAD⁺ and NADP⁺. Nucleoside triphosphates were potent inhibitors of the enzyme activity. The partial inhibition of the enzyme by o-phenanthroline and by p-hydroxymercuribenzoate could be reversed by Fe²⁺ ions and by reduced glutathione, respectively.