Purification and properties of polynucleotide phosphorylase from Escherichia coli.

A method for the purification of polynucleotide phosphorylase from Escherichia coli has been developed. The purified enzyme has a specific activity 700-fold higher than the crude extract. When enzyme fractions obtained at different stages of the purification were assayed by phosphorolysis of polyadenylic acid (poly A) or 32P-orthophosphate exchange with the S’diphosphates of adenosine, uridine, cytidine, guanosine, and inosine, the degree of purification at each step was approximately the same. Various factors affecting the exchange reactions between labeled orthophosphate and different ribonucleoside diphosphates were examined. An activating factor which enhances the rate of the ADP-Pi exchange reaction 3to 6-fold was isolated during the purification of the enzyme. It affects only the exchange reaction and not the phosphorolysis of poly A. It was found to be susceptible to proteolytic enzyme action, but not to RNase degradation. The effect of the dinucleoside monophosphate ApA on the polymerization and exchange reactions was studied. At low Mg++ concentration, it could overcome the lag period in the polymerization of ADP and also stimulate the rate of the ADP-Pi exchange reaction. The results presented in this report support the hypothesis that the polymerization and exchange reactions are catalyzed by the same enzyme. Their relation to the mechanism of the exchange reaction is discussed.