HOMOGENEOUS PYRIMIDINE NUCLEOTIDASE FROM HUMAN ERYTHROCYTES - ENZYMATIC AND MOLECULAR-PROPERTIES

A pyrimidine nucleotidase with unique specificity has been obtained fo r the first time as an homogeneous protein from the cytosolic fraction of human erythrocytes. Both conventional chromatography and f.p.l.c. techniques have been used in the purification procedure. The final enz yme preparation gave a single protein band of M(r) = 23 500 on SDS/PAG E under both reducing and non-reducing conditions. The native enzyme w as eluted at M(r) = 45 000 in gel filtration chromatography on Superos e 12, suggesting a dimeric structure. Amino acid analysis was consiste nt with an acidic isoelectric point and revealed the presence of six h alf-cystine and two methionine residues per subunit. The enzyme was ac tive on a variety of pyrimidine nucleoside monophosphates, being most active on the 3'-monophosphates. K-m values for 3'dUMP, 3'UMP, 5'dUMP, 5'UMP, 5-fluoro-2'dUMP, ranged from 192 mu M to 1.15 mM. The enzyme a ctivity was inhibited by both reaction products, orthophosphate, and t he nucleoside formed. Product inhibition studies suggested an Ordered Uni-Bi mechanism for the reaction. The enzyme required Mg2+ for its ac tivity, while heavy metals cations were strongly inhibitory. The enzym e activity was inhibited by metal chelating agents and it was sensitiv e to thioreactive reagents. The isoelectric point was 5.4 and the opti mum activity pH was 6.5.