Purification and kinetic studies of adenine phosphoribosyltransferase from human erythrocytes

Adenine phosphoribosyltransferase was purified 2200-fold from human erythrocytes. The purification steps involved (NH₄)₂SO₄ fractionation followed by Sephadex-G75 column chromatography, DEAE-Sephadex chromatography, and (NH₄)₂SO₄ precipitation. Partially purified enzyme was stabilized by 0.3 m (NH₄)₂SO₄, which also protected the enzyme from heat denaturation. The enzyme was partially inhibited by sodium ions which were partially antagonized by magnesium. Divalent cation is an absolute requirement for high enzyme activity. Ca²⁺, Mn²⁺, and Mg²⁺ supported high initial rates and Hg²⁺ completely inhibited the enzyme. The kinetic data with initial velocity and the product inhibition studies are consistent with a mechanism in which the synthesis of nucleotide proceeds as an ordered sequential reaction.