| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 87 === Cytosolic aldehyde dehydrogenase-1（ALDH1）and mitochondrial ALDH2 are the principal enzymes responsible for oxidation of acetaldehyde, a major metabolite of ethanol, in mammalian livers. Recombinant human ALDH1, ALDH2E, ALDH2K, and ALDH2E/K were isolated from E. coli transformed with plasmids of the expression vectors harboring cDNAs of the respective enzymes, and purified via DEAE—Sepharose and p-hydroxyacetophenone—Sepharose chromatographies to apparent homogeneity. The molecular weights of the ALDH1 and ALDH2 forms were determined to be ~55 kD. Catalytic efficiencies for oxidation of acetaldehyde were found in the following order: ALDH2E > ALDH2E/K > ALDH1 > ALDH2K. Using polyacrylamide isoelectric focusing, protein content ratios for bands of the tetrameric ALDH2E/K mixture were quantitated to be E4 : E3K : E2K2 : EK3 : K4 = 1 : 4 : 6 : 4 : 1 as well as ratios for the activity staining intensities, E4 : E3K : E2K2 : EK3 : K4 = 1 : 2 : 1 : 0 : 0. These results strongly support that (a) the E and K subunits can randomly combine to form homo- and heterotetrameric ALDH2 molecules; (b) dimers act as the forming unit for the tetrameric molecules; (c) both the EE-KK and EK-EK exist in the E2K2 molecules; (d) ALDH2 exhibits half-of-the-sites activity; (e) the K subunits function dominantly over loss of the enzyme activity in the dimer units. The proposed molecular model for the subunit composition and enzymatic activity of the recombonant tetrameric ALDH2E/K mixture appears to be in agreement with recent findings of the protein contents and activities of mitochondrial ALDH2 expressed in the human livers with homo- and heterozygous ALDH2 genotypes.
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