Purification, assay, and kinetic properties of sheep liver pyridoxal kinase

Pyridoxal kinase from sheep liver has been purified 1100-fold by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Sephadex G-150, and ADP-Sepharose. Polyacrylamide gel electrophoresis indicated the enzyme to be nearly homogeneous. Initial velocity studies were consistent with a sequential mechanism. The Michaelis constants for pyridoxal and ZnATP²⁻ complex are 160 and 31 μ , respectively. A new assay was developed in which [³H]pyridoxine was used as substrate. The product, [³H]pyridoxine 5′-P, was separated from the substrate with DEAE-cellulose disks. Determination of the Michaelis constants for pyridoxine and the ZnATP²⁻ complex by this new method gave values of 110 and 32 μ , respectively.