Erythrocytic Nucleoside Diphosphokinase: II. ISOLATION AND KINETICS

Abstract

Abstract Nucleoside diphosphokinase (NDP kinase) was identified in high concentrations in human erythrocytes, and a procedure was developed for the isolation of this enzyme. The best preparations had about 1400-fold greater specific activity (65 units per mg of protein) than that of hemolysates. Like crystalline yeast NDP kinase and partially purified NDP kinase preparations from various tissues, the erythrocytic enzyme is relatively nonspecific with regard to nucleotide substrates. Although there are differences in the kinetic constants, the di- and triphosphate nucleotides which contain natural purine and pyrimidine bases, and ribose or deoxyribose, and in addition a number of nucleotides which contain purine or pyrimidine analogues all serve as substrates. A kinetic analysis was performed which included initial velocity and alternative substrate studies, and study of inhibition by 5'-monophosphate nucleotides. The results of these studies were consistent with the concept that the erythrocytic NDP kinase follows a ping-pong mechanism. This permitted the postulation that the reactive intermediate is the phosphorylated enzyme.