Partial purification and zinc dependence of human red cell pyrimidine-5'-nucleotidase.

R. S. Markin, C. R. Angle, M. S. Swanson, S. J. Stohs

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3 Scopus citations


Human red cell pyrimidine-5'-nucleotidase (EC 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 Km 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 CaCl2 and NaCl against 2 X 10(-3) mol/l 1,10-phenanthroline for 24 h at 4 degrees C. This incubation resulted in 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.

Original languageEnglish (US)
Pages (from-to)134-138
Number of pages5
Issue number2
StatePublished - 1983
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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