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.
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