Purification and properties of human erythrocyte pyrimidine 5'-nucleotidase.

Abstract

A 250,000-fold purification of pyrimidine 5''-nucleotidase from human erythrocytes was 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 showed 2 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 had a pH optimum at 7.5 and was most stable between pH 6 and 7.5. The enzyme had a pI [isoelectric point] of 5.0 and a MW of 28,000 by gel filtration. The Km of the purified enzyme was 10 .mu.M, compared to 40 .mu.M when measured in hemolysate. The higher Km in the hemolysate was 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 Km of the enzyme for the substrate cytidine monophosphate.