Bull semen nicotinamide adenine dinucleotide nucleosidase. I. Purification and properties of the enzyme.

A procedure is described for the purification of bull semen NAD nucleosidase. The enzyme was purified over 400-fold to a final specific activity of 9906 units per mg of protein. A spectrophotometric assay was developed in which the enzyme-catalyzed release of 3-acetylpyridine from d-acetylpyridine adenine dinucleotide was followed by measuring the increase in optical density at 232 mp. At pH 7.5 the Km value for 3-acetylpyridine adenine dinucleotide was determined to be 6.4 x 10m6 M. The spectrophotometric method was used to study the effect of pH on the Km and V,, of the enzyme-catalyzed reaction, and the stability of the enzyme at different temperatures. Sucrose density gradient studies showed the enzyme to have an estimated sedimentation coefficient of 3.1 S at 20’.