A rapid and simple radioassay for inosinic acid: Pyrophosphate phosphoribosyltransferase in the presence of xanthine oxidase and vice versa

Abstract

A simple and rapid technique for measuring IMP:pyrophosphate phosphoribosyltransferase (HPRibTase) activity of rat intestinal homogenates, in the presence of xanthine oxidase, is described. By introducing 2.5 × 10 −5 m allopurinol (4-hydroxypyrazolo [3,4- d]pyrimidine) into the reaction mixture, the [8- 14C]hypoxanthine (Hx) is converted only to [8- 14C]inosinic acid (IMP). The xanthine oxidase activity is completely inhibited under this condition. When xanthine oxidase is not blocked, diversion of substrate to urate can invalidate assays of HPRibTase. Using [8- 14C]Hx as substrate, in the presence and absence of allopurinol, the activity of both HPRibTase and xanthine oxidase of the same tissue homogenate is determined. We have simplified the conventional chromatographic separation of the reactant products by spotting the reactant on DEAE cellulose paper followed by repeated washings with 4 m m ammonium formate solution. The unreacted radiosubstrate is washed off, and the [8- 14C]IMP or [8- 14C]uric acid formed remains adsorbed on the paper. The major advantages of this method are speed, reproducibility, sensitivity, ability to process many samples, and a low blank value. Our studies on the enzyme distribution along the intestinal villus have shown that while most of the HPRibTase activity is associated with rapidly multiplying crypt cells, the xanthine oxidase activity is more evenly distributed along the villus, and the activity is effected more by exongeneous effectors. The colon has the highest HPRibTase and lowest xanthine oxidase activity of all the intestinal mucosa cells. Small bowel mucosa is high both in xanthine oxidase and HPRibTase.