Studies on Human Erythrocyte Nucleotide Metabolism. I. Monisotopic Methodologies

Methods were developed to assay the activities of ribosephosphate pyrophosphokinase (RPK, PRPP synthetase, E.C. 2.7.6.1) and adenine phosphoribosyltransferase (APRT, AMP pyrophosphorylase, E.C. 2.4.2.7) in hemolysates of human erythrocytes. RPK activity was determined by measuring the production of phosphoribosyl pyrophosphate (PRPP) and the formation of adenosine mononucleotide (AMP), the two products of the reaction. The K_m for adenosine triphosphate (ATP) was 0.12 mM with the PRPP method and 0.1 mM with the AMP method. The K_m for ribose-5-phosphate (R-5-P) was 0.14 mM with the former method and 0.033-0.042 mM with the latter. The pH optimum with the PRPP method was between 7.6 and 8.2. The mean RPK activity of hemolysates of normal mature erythrocytes was 30.5 ± 3.78 µmoles of PRPP or 30.6 ± 3.82 µmoles of AMP produced/hr per 10¹⁰ erythrocytes at 37° C. Although RPK activity of normal mature erythrocytes did not appear to be markedly age dependent, activity was substantially increased in reticulocyte-rich blood. APRT activity was assayed by measuring the AMP produced in a system linked to the generation of oxidized nicotinamide adenine dinucleotide (NAD). The activity of normal mature erythrocytes was 3.38 ± 0.37 µmoles of AMP formed/hr per 10¹⁰ erythrocytes at 37° C. Enzyme activity was significantly elevated in reticulocyte-rich blood and in the erythrocytes of a patient with the Lesch-Nyhan syndrome. These methods obviated the need for isotopic techniques and were readily applicable to small samples of blood.