Abstract
Pyrimidine 5′-nucleotidase catalyzes the hydrolysis of pyrimidine 5′-monophosphates to the respective nucleosides. The enzyme activity was determined using HPLC assay by a chromatografic separation on a 100×4.6 mm column containing 3 μm reversed-fase C-18 stationary phase with a 20×2.1 gard column containing 5 μm reverse-phase. The mobile phase was 100mM KH2PO4 solution pH 6.0 containing 6% methanol for the determination of cytidine and uridine or 14% methanol for the determination of thymidine. Products were identified from their retention times and quantified by standards. The complete chromatogram was developed within 3 min. In order to ascertain on the presence in the erythrocytes of multiple forms of the enzyme, as observed by others (1), crude hemolysate was passed through a TSK-DEAE 5PW column. The elution performed with a linear gradient from 0 to 0.2 M of KCl revealed two peaks of activity by using both CMP and TMP as substrates. However preliminary results indicated no difference in the susceptibility of both substrates as it could be expected from the observations made by other investigators (1). Characterizaton of the purified enzyme preparation (4400 fold) showed a molecular weight of 30,000, a pH optimum around 7.5 and an isoelectric point at 5.1. Kinetic analysis conducted using CMP as the substrate showed a Km value of 30 μM
(1) Hirono et all (1987) Br. J. Haemat. 65, 35-41
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Amici, A., Santini, F. & Magni, G. 81 ERYTHROCYTE PYRIMIDINE NUCLEOTIDE METABOLISM 5′-NUCLOTIDASE HPLC ASSAY AND OBSERVATIONS ON THE ENZYME BEHAVIOR. Pediatr Res 24, 124 (1988). https://doi.org/10.1203/00006450-198807000-00105
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DOI: https://doi.org/10.1203/00006450-198807000-00105