Purification and Properties of Human Erythrocyte Pyrimidine 5′-nucleotidase

A 250,000-fold purification of pyrimidine 5′-nucleotidase from human erythrocytes has been achieved using a combination of DEAE-cellulose chromatography, ammonium sulfate fractionation, gel filtration, and isoelectric focusing. Polyacrylamide disc and starch gel electrophoreses of the purified material show two strong protein bands. On starch gel these bands exhibited pyrimidine 5′-nucleotidase activity. Two faint protein bands devoid of enzyme activity were also found in the case of polyacrylamide electrophoresis. The enzyme has a pH optimum at 7.5 and is most stable between pH 6 and 7.5. The enzyme has a pI of 5.0 and a molecular weight of 28,000 by gel filtration. The Kmof the purified enzyme was 10 μ M, compared to 40 μ M when measured in hemolysate. The higher Kmin the hemolysate is due to the presence of an inhibitor. Inorganic phosphate was shown to be a competitive inhibitor of pyrimidine 5′-nucleotidase and inorganic phosphate in the hemolysate may be responsible for increasing the Kmof the enzyme for the substrate cytidine monophosphate.