Allosteric Properties of Nucleoside Diphosphatase and Its Identity with Thiamine Pyrophosphatase

Abstract A procedure has been described for the purification of nucleoside diphosphatase from bovine liver microsomes. The purified enzyme is shown, by analysis in the ultracentrifuge and by polyacrylamide disc gel electrophoresis, to be nearly homogeneous. The enzyme has an s20,w of 4.9 S. Thiamine pyrophosphatase was purified together with nucleoside diphosphatase. Evidence has been obtained which indicates that the nucleoside diphosphates and thiamine pyrophosphate are hydrolyzed by a single enzyme, although the pH activity curves were not identical with the two types of substrates. Both enzyme activities were markedly enhanced by the presence of adenosine triphosphate. The effect of ATP was more pronounced at low substrate concentrations and disappeared as the substrate level increased. ATP was not consumed during the reactions. The effect of ATP was exerted without a measurable lag when it was added during the course of the reaction, and the stimulatory effect was lost immediately when ATP was removed from the reaction mixture. ATP protected the enzyme against heat inactivation, and gel filtration experiments showed ATP to be bound to the enzyme protein. Inosine triphosphate, guanosine triphosphate, and deoxyadenosine triphosphate showed the same degree of stimulatory effect as ATP.