IDENTIFICATION OF A PURINE-SPECIFIC 5′-NUCLEOTIDASE IN HUMAN ERYTHROCYTES: 19

Abstract

In human erythrocytes, a pyrimidine 5′-nucleotidase has been characterized (Paglia & Valentine, Curr. Top. Hematol 3:75, 1980) but the enzyme(s) responsible for the dephosphorylation of AMP and IMP had hitherto not been identified. Haemolysates in 10 mM Tris-maleate buffer pH 7, incubated with 1 mM [14C] IMP, produced [14C] inosine at the rate of approx. 2 μmol/h per g of Hb. The enzymic activity was purified around 3000-fold in 3 steps : (1) batchwise binding to DEAE cellulose, followed by washing with 10 mM Tris-maleate pH 6.5 to remove Hb; (2) column separation from acid phosphatase and pyrimidine 5′-nucleotidase by elution with a NaCl gradient in the same buffer, which released the purine 5′-nucleotidase at 0.2 M NaCl; (3) adsorption on blue-sepharose, followed by elution with 2 M NaCl. The enzyme was Mg++-dependent and displayed a broad pH optimum around pH 7. It was maximally active with IMP and hydrolysed GMP at 75 % and AMP at 10 % of the rate of IMP. Km for the three nucleotides was around 0.1 mM. At 0.25 mM AMP, the enzyme activity was 80 % inhibited by 5 mM Pi and stimulated 8-fold by 5 mM 2,3-bis-P-glycerate, half maximal stimulation being obtained at 0.5 mM of this compound. In contrast with rat liver cytosolic 5′-nucleotidase, the erythrocyte enzyme was not stimulated by ATP or GTP. Supported by FRSM.