Homogeneous pyrimidine nucleotidase from human erythrocytes: enzymic and molecular properties.
A pyrimidine nucleotidase with unique specificity has been obtained for 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 enzyme preparation gave a single protein band of M(r) = 23,500 on SDS/PAGE under both reducing and non-reducing conditions. The native enzyme was eluted at M(r) = 45,000 in gel filtration chromatography on Superose 12, suggesting a dimeric structure. Amino acid analysis was consistent with an acidic isoelectric point and revealed the presence of six half-cystine and two methionine residues per subunit. The enzyme was active on a variety of pyrimidine nucleoside monophosphates, being most active on the 3'-monophosphates. Km values for 3'dUMP, 3'UMP, 5'dUMP, 5'UMP, 5-fluoro-2'dUMP, ranged from 192 microM to 1.15 mM. The enzyme activity was inhibited by both reaction products, orthophosphate, and the nucleoside formed. Product inhibition studies suggested an Ordered Uni-Bi mechanism for the reaction. The enzyme required Mg2+ for its activity, while heavy metals cations were strongly inhibitory. The enzyme activity was inhibited by metal chelating agents and it was sensitive to thioreactive reagents. The isoelectric point was 5.4 and the optimum activity pH was 6.5.
Amici
A
A
Istituto di Biochemica, Facoltà di Medicina e Chirurgia, Università di Ancona, Italy.
Emanuelli
M
M
Ferretti
E
E
Raffaelli
N
N
Ruggieri
S
S
Magni
G
G
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000737:chemistry; D000596:Amino Acids / Q000032:analysis; D004795:Enzyme Stability; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010710:Phosphates / Q000032:analysis; D012680:Sensitivity and Specificity; D012995:Solubility; D013053:Spectrophotometry; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D000596:Amino Acids; D008670:Metals; D010710:Phosphates; D013438:Sulfhydryl Compounds; D015720:5'-Nucleotidase