Inhibition of Human Purine Nucleoside Phosphorylase

Inhibition of human purine nucleoside phosphorylase was studied by measuring the conversion of 14C-hypoxanthine to 14C-inosine, both by intact erythrocytes (in vitro) and by a partially purified enzyme preparation from these cells. In contrast to previous investigations, no exogenous nucleoside ribosyl donor was used with the intact cells. With the isolated enzyme, ribose 1-phosphate was used as the ribosyl donor. The inhibition constants of purines and purine analogues obtained from studies with intact cells agreed well with those from the purified enzyme. Hypoxanthine, xanthine, and guanine were bound well to the isolated enzyme (Ki values of 1 to 4 x 10-5 m). Methylation of these compounds at any one of the ring nitrogens or at the oxygen on position 6 markedly decreased this binding. The Ki values for 6-mercaptopurine and 6-thioguanine were of the order of 10-4 m. In contrast to the oxypurines, methylation of these thiopurines did not markedly reduce their binding to the enzyme. Adenine, 2,6-diaminopurine, allopurinol, oxoallopurinol, and 6-thioxanthine were poorly bound to the enzyme. With intact erythrocytes, inhibition by 6-methylthiopurine ribonucleoside was observed only when an exogenous nucleoside ribosyl donor was used. With the isolated enzyme, 6-methylthiopurine ribonucleoside was a very poor inhibitor of both the synthetic and phosphorolytic reactions. These results support the hypothesis that nucleoside transport is inhibited by the ribonucleoside of 6-methylthiopurine.