A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts.
A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H2O----adenosine + Pi (5'-nucleotidase); adenosine + H2O----inosine + NH3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.
D000228:Adenine Phosphoribosyltransferase / Q000032:analysis*; D000818:Animals; D002250:Carbon Radioisotopes; D010428:Pentosephosphates / Q000032:analysis*; D010430:Pentosyltransferases / Q000032:analysis*; D010754:Phosphoribosyl Pyrophosphate / Q000032:analysis*; D011854:Radiochemistry / Q000379:methods*; D051381:Rats; D011919:Rats, Inbred Strains; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D002250:Carbon Radioisotopes; D010428:Pentosephosphates; D010754:Phosphoribosyl Pyrophosphate; D010430:Pentosyltransferases; D000228:Adenine Phosphoribosyltransferase