Interaction of 6-azauridine-5'-diphosphate with Escherichia coli polynucleotide phosphorylase

Abstract

The activity of enzyme preparations of E. coli polynucleotide phosphorylase was estimated by following the exchange of P32-orthophosphate with nucieoside-5[image]-diphos-phates. The exchange was evaluated by means of paper electrophoresis which made a rapid and complete separation of p32-orthophosphate from nucleoside-5''-diphosphates possible. Although the exchange of labeled phosphate with uridine-5[image]-diphosphate (I) catalyzed by the E. coli polynucleotide phosphorylase was very intense, no exchange took place with 6-azauridine-5[image]-diphosphate (II). In the presence of II the exchange of labeled phosphate with I and adenosine-5[image]-diphosphate (III) was inhibited. The inhibition of P32-orthophosphate exchange studied in preparations of various degrees of purity showed that the incorporation of the labeled phosphate into I as well as III decreased linearly with increasing concentration of II in the incubation mixture, and at the ratio I or HI to H equal to 1:2 the incorporation of P32-orthophosphate was fully inhibited. The inhibition cannot be explained on the basis of interaction of II with Mg ions since if the optimum concentration of I is tripled a considerable exchange of p32-orthophosphate still takes place.