A simple and sensitive method for estimating the concentration and synthesis of 5-phosphoribosyl 1-pyrophosphate in red blood cells

Abstract

A method is presented for the determination of 5-phosphoribosyl 1-pyrophosphate (PRPP), which is based on the release of 14CO2 from [carboxyl-14C]-orotic acid by the consecutive action of orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. The assay is simpler and less time-consuming than most methods currently employed and is equally sensitive. The method proved to be suitable for measuring low concentrations of PRPP such as found in human erythrocytes and fibroblasts. An increased PRPP concentration was observed in erythrocytes from patients with partial or complete deficiency of hypoxanthine-guanine phospho-ribosyltransferase. frp, sp,e (but not all) gouty patients and from a patient with deficiency of purine nucleoside phosphorylase. PRPP synthetase activity was measured with a method similar to the assay for PRPP. In erythrocytes with an increased PRPP concentration, PRPP synthetase activity was found to be normal at both optimal and suboptimal substrate concentrations.