Pyrimidine nucleoside phosphorylases of rat liver. Separation by ion exchange chromatography and studies of the effect of cytidine or uridine administration.

By chromatography on diethylaminoethyl Sephadex, three enzyme fractions with pyrimidine nucleoside phosphorylase activity have been separated from extracts of normal or regenerating liver of rats. Two of these probably represent isoenzymes of uridine phosphorylase. Both are phosphate-dependent and both exhibit maximal activities toward uridine, deoxyuridine, and thymidine at pH values of 7.9 to 8.2, 6.4 to 6.8, and 5.6 to 5.8, respectively. One isoenzyme is of nuclear origin, whereas the other is predominantly of cytoplasmic origin. At the pH optimum of the respective substrates, as well as at physiological pH, both isoenzymes catalyze the phosphorolysis of uridine at a more rapid rate than the phosphorolysis of deoxyuridine and are much less active toward thymidine. The third enzyme, thymidine phosphorylase, is mainly of cytoplasmic origin. It is also phosphate-dependent; it is most active toward deoxyuridine at pH 5.8 and toward thymidine at pH 5.4, but shows no activity toward uridine or deoxycytidine. It has more deoxyuridinethan thymidine-cleaving activity over the pH range studied, but is probably the enzyme mainly responsible for catalyzing the phosphorolysis of thymidine in liver cells. Under the standard conditions of assay the specific activities of the nuclear and cytoplasmic uridine phosphorylases were found to be increased about 3-fold when assayed in the presence of uridine, deoxyuridine, or thymidine 6 hours after the injection of uridine. Six hours after the injection of cytidine the specific activities of both enzymes with each of the three substrates were increased about Z-fold. Concomitantly, the specific activity of thymidine phosphorylase when assayed with deoxyuridine or thymidine was not changed by uridine injection but was decreased after the injection of cytidine.