Human liver aldehyde dehydrogenases: new method of purification of the major mitochondrial and cytosolic enzymes and re-evaluation 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 microM and 0.20 +/- 0.02 microM, respectively (pH 7.5 and 9.5, saturating NAD+ in both cases). This three orders of magnitude difference in Km values is much greater than that reported previously in all but one study.
Rashkovetsky
L G
LG
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, MA 02115.
Maret
W
W
Klyosov
A A
AA
D000079:Acetaldehyde / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000201:enzymology*; D006801:Humans; D007526:Isoelectric Point; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D010084:Oxidation-Reduction
D000596:Amino Acids; D007527:Isoenzymes; D000444:Aldehyde Dehydrogenase; D000079:Acetaldehyde