HUMAN LIVER ALDEHYDE DEHYDROGENASES - NEW METHOD OF PURIFICATION OF THE MAJOR MITOCHONDRIAL AND CYTOSOLIC ENZYMES AND REEVALUATION OF THEIR KINETIC-PROPERTIES

A new purification procedure, based on dye-adsorption and affinity chromatography, has been developed for the isolation of the two major ALDH isozymes from human liver: ALDH-1 (cytosolic, pI 5.2) and ALDH-2 (mitochondrial, pI 4.9). The procedure affords milligram quantities of ALDH-1 and -2 at 850- and 275-fold purifications, respectively, from 50 g of liver in two days. Kinetic parameters for acetaldehyde oxidation were determined with these purified enzymes, because there is a wide discrepancy in the absolute magnitude of these parameters in the biochemical literature. The Michaelis constants for ALDH-1 and -2, determined from initial velocities (for ALDH-1) and single reaction progress curves (for ALDH-2), are 180 +/- 10 mu M and 0.20 +/- 0.02 mu M, respectively (pH 7.5 and 9.5, saturating NAD(+) in both cases). This three orders of magnitude difference in K-m values is much greater than that reported previously in all but one study.