Horse liver aldehyde dehydrogenase. Purification and characterization of two isozymes

Two isozymes of horse liver aldehyde dehydrogenase (aldehyde, NAD oxidoreductase (EC 1.2.1.3)), F1 and F2, have been purified to homogeneity using salt fractionation followed by ion exchange and gel filtration chromatography. The specific activities of the two isozymes in a pH 9.0 system with propionaldehyde as substrate were approximately 0.35 and 1.0 μmol of NADH/min/mg of protein for the F1 and F2 isozymes, respectively. The multiporosity polyacrylamide gel electrophoresis molecular weights of the F1 and F2 isozymes were approximately 230,000 and 240,000, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gave subunit molecular weight estimates of 52,000 and 53,000 for the F1 and F2 isozymes, respectively. The amino acid compositions of the two isozymes were found to be similar; the ionizable amino acid contents being consistent with the electrophoretic and chromatographic behavior of the two isozymes. Both isozymes exhibited a broad aldehyde specificity, oxidizing a wide variety of aliphatic and aromatic aldehydes and utilized NAD as coenzymes, but at approximately 300 fold higher coenzyme concentrations could use NADP. The F1 isozyme exhibited a very low K(m) for NAD (3 μM) and a higher K(m) for acetaldehyde (70 μM), while the F2 isozyme was found to have a higher K(m) for NAD (30 μM) and a low K(m) for acetaldehyde (0.2 μM). The two isozymes showed similar chloral hydrate and p chloromercuribenzoate inhibition characteristics, but the F1 isozyme was found to be several orders of magnitude more sensitive to disulfiram, a physiological inhibitor of acetaldehyde oxidation. Based on its disulfiram inhibition characteristics, it has been suggested that the F1 isozyme may be the primary enzyme for oxidizing the acetaldehyde produced during ethanol oxidation in vivo.