Purification of Adenine Phosphoribosyltransferase by Affinity Chromatography

The purine salvage pathway enzyme adenine phosphoribosyltransferase (AMP: pyrophosphate phosphoribosyltransferase EC 2.42.7) has been purified to greater than 85% homogeneity from crude rat liver 100,000 × g supernatant in one step by affinity chromatography. The enzyme binds to an AMP-agarose column and is eluted off the column by 1 mM 5-phosphori-bosyl pyrophosphate with a 50 to 80% recovery. Enzyme kinetics indicate that the mechanism of the specific elution is due to competition of the product AMP and substrate 5-phosphoribosyl pyrophosphate for the same site on the enzyme.