Hypoxanthine phosphoribosyltransferase: radiochemical assay procedures for the forward and reverse reactions

Simple and rapid radiochemical assay procedures for the forward (IMP synthesis) and reverse (IMP pyrophosphorolysis) reactions catalyzed by hypoxanthine phosphoribosyltransferase have been developed. Enzyme activity in the forward direction was assessed by measuring the amount of [8-14C]IMP formed from [8-14C]hypoxanthine following their separation by polyethyleneimine-cellulose TLC in methanol:water (1:1, v/v). [8-14C]IMP has been synthesized from [8-14C]hypoxanthine, using hypoxanthine phosphoribosyltransferase derived from human brain, with subsequent purification by elution from phenyl boronate-agarose. Enzyme activity in the reverse direction was assessed by measuring the amount of [8-14C]uric acid formed from the labeled IMP following their separation by polyethyleneimine-cellulose TLC in 0.2 M LiCl saturated with boric acid (pH 4.5):95% ethanol (1:1, v/v), the transferase reaction being coupled with excess xanthine oxidase and catalase to overcome the unfavorable equilibrium.