Molecular Pharmacology, Vol 10, 657-668, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

Oxipurinol-Associated Inhibition of Pyrimidine Synthesis in Human Lymphoblasts

¹ Divisions of Rheumatology and Hematology, Department of Medicine, University of California, San Diego, La Jolla, California 92037

The administration of allopurinol or its major metabolic oxidation product, oxipurinol, to man consistently results in inhibition not only of uric acid synthesis but also of pyrimidine synthesis. Molecular correlates of the latter inhibition produced by oxipurinol have been studied in permanent human lymphoblast lines in tissue culture, which appear to be satisfactory models for the study of this drug effect. Incubation of intact lymphoblasts with oxipurinol resulted within 90 min in significant inhibition of incorporation of [¹⁴C]orotic acid but not [³H]uridine into cold acid-precipitable counts. This inhibition of pyrimidine synthesis was correlated with the generation of an orotidylic acid decarboxylase inhibitor which, in cell-free extracts, required 5-phosphoribosyl 1-pyrophosphate and oxipurinol for its formation and could be generated in extracts of cells deficient in hypoxanthine-guanine phosphoribosyltransferase. Stabilization of both orotidylic acid decarboxylase and orotate phosphoribosyltransferase also resulted from incubation of lymphoblasts with oxipurinol. The production of the stabilized activities appeared to coincide temporally, in substrate requirements, and in reversibility with the generation of the inhibitor of orotidylic acid decarboxylase activity, suggesting a common molecular basis for these effects. In addition, oxipurinol treatment resulted in an alteration in the quaternary structure of the bifunctional enzyme complex containing both activities, with an increase in molecular weight from 41,000 to 108,000 as estimated by gel filtration studies. In the higher molecular weight material both increased stability and inhibition of orotidylic acid decarboxy1ase were demonstrated. These findings are in accord with the suggestion that ribonucleotides of oxipurinol result in inhibition of pyrimidine synthesis through alterations in the activity, stability, and quaternary structure of the enzyme complex. A concentration-dependent increase in the specific activities of orotidylic acid decarboxylase and orotate phosphoribosyltransferase has been demonstrated in both lymphoblasts and fibroblasts incubated with oxipurinol. In lymphoblasts this increase in enzyme activity occurred within 2-3 hr and was not prevented by treatment of the cells with cycloheximide. Stabilization of the enzymes during extraction from the cell or direct activation of the enzyme activities appear to be more likely explanations for the increased enzyme activities than induction of new enzyme synthesis or inhibition of normal enzyme catabolism. No differences were found between normal and hypoxanthine-guanine phosphoribosyltransferase-deficient lymphoblast (or fibroblast) lines in activities of either orotate phosphoribosyltransferase or orotidylic acid decarboxylase.