Purification and characterization of pyridoxal kinase from human erythrocytes.

Pyridoxal kinase has been purified 50,000-fold from human erythrocytes. The purification procedure included dextran-induced aggregation of red blood cells, ammonium sulphate fractionation of the haemolysate, DEAE-cellulose chromatography, hydroxyapatite chromatography. Sephadex G-100 gel filtration and omega-aminooctyl agarose chromatography. The enzyme preparation migrated as a single protein and activity band on analytical gel electrophoresis. Determination of the Michaelis constants for pyridoxal, pyridoxine and pyridoxamine using a new assay gave comparable values of 33 microM, 16 microM and 6.2 microM respectively. Various amines were shown as competitive inhibitors of pyridoxal kinase with respect to ATP. The inhibition order was: N-dansyl-1,8-diaminooctane greater than 1,8-diaminooctane greater than 1,6-diaminohexane greater than 1,4-diaminobutane greater than gamma-aminobutyric acid, whereas octane, hexane and butane were not inhibitors. Results suggest that the amino groups on the above inhibitors are essential for competitive inhibition at saturating concentrations of pyridoxal. It was also observed that increasing the chain length of the hydrophobic backbone of these competitive inhibitors can facilitate its action.