Determination of the intracellular concentration of 5-phosphoribosyl-1-pyrophosphate in cultured mammalian fibroblasts

The properties of an assay for the 5-phosphoribosyl-1-pyrophosphate (PRPP) content of cultured mammalian fibroblasts are described. The assay is based upon the PRPP-dependent release of 14CO 2 from [ carboxyl- 14C]orotic acid by a commercially available preparation of yeast orotidine-5′-monophosphate pyrophosphorylase and orotidine-5′-monophosphate decarboxylase. The advantages of the assay include the fact that it is based on the enzymatic recognition of PRPP, employs an irreversible reaction, and does not involve either the chromatographic separation of substrate and product or the purification of a phosphoribosyltransferase. The disadvantage of the assay is that the efficiency of PRPP measurement varies somewhat, in part because the yeast enzyme preparation contains 5′-nucleotidase activity. A calibration procedure is described which corrects for variation in efficiency both between and within experiments. This procedure seems to yield highly reliable estimates of PRPP content. The assay will readily detect 0.6 nmol, and the cell strain studied contained 7.76 ± 1.14 nmol of PRPP 10 7 cells .