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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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 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
JournalEnzyme
Volume30
Issue number2
DOIs
StatePublished - 1983

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5'-Nucleotidase
Tromethamine
Purification
Zinc
Cells
Buffers
Uridine Monophosphate
Cytidine Monophosphate
Affinity chromatography
Enzymes
Uridine Diphosphate
Dithiothreitol
Enzyme activity
Washing
Edetic Acid
Sepharose
Metal ions
Ion Exchange Chromatography
Ion exchange
Blood

ASJC Scopus subject areas

  • Biochemistry

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Partial purification and zinc dependence of human red cell pyrimidine-5'-nucleotidase. / Markin, R. S.; Angle, C. R.; Swanson, M. S.; Stohs, S. J.

In: Enzyme, Vol. 30, No. 2, 1983, p. 134-138.

Research output: Contribution to journalArticle

Markin, R. S. ; Angle, C. R. ; Swanson, M. S. ; Stohs, S. J. / Partial purification and zinc dependence of human red cell pyrimidine-5'-nucleotidase. In: Enzyme. 1983 ; Vol. 30, No. 2. pp. 134-138.
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