Partial Purification and Zinc Dependence of Human Red Cell Pyrimidine-5'-Nucleotidase

Abstract

Human red cell pyrimidine-5'-nucleotidase (EC 3.1.3.5) was partially purified from the blood of normal subjects by ion-exchange and affinity chromatography. Red cells were lysed in 50 mmol/l Tris-Cl buffer at pH 7.5 containing 1.0 mmol/l dithiothreitol and 0.5 mmol/l EDTA. The lysate was centrifuged and introduced onto a column of Sephadex A-50. After washing, the pyrimidine-5'-nucleotidase activity was eluted from the column with a NaCl gradient from 0 to 200 mmol/l in Tris buffer at pH 7.5. The pyrimidine-5'-nucleotidase was then desalted on Sephadex G-25 and introduced onto a UDP agarose column with a Tris buffer at pH 6.5 containing 150 mmol/l NaCl. This partial purification resulted in an approximately 80,000-fold increase in enzyme concentration. The K(m) for the partially purified enzyme was 0.32 mmol/l for UMP, 0.16 mmol/l for CMP and 0.11 mmol/l for OMP with a pH maximum of 7.5. This partially purified pyrimidine-5'-nucleotidase was then dialyzed in 50 mmol/l Tris-Cl buffer at pH 7.5 with 0.01 mmol/l CaCl(2) and NaCl against 2 X 10^-3 mol/l 1,10-phenanthroline for 24 h at 4 °C. This incubation resulted in a 73% decrease in enzyme activity which could be restored by the addition of zinc into the mixture, but not by the addition of other divalent metal ions.

Molecular aspects of erythroenzymopathies associated with hereditary hemolytic anemia

“…Beutler et a1 [90] found that P5N is not limited to red cells but also exists in the rat brain and kidney. This enzyme was partially purified from human red cells [91,92] and has a molecular weight of 28,000 [91].…”