Atypical human liver alcohol dehydrogenase: the β2-Bern subunit has an amino acid exchange that is identical to the one in the β2-Oriental chain

AbstractThe ‘atypical’ human liver alcohol dehydrogenase dimer, homogeneous for β2-Bern chains, was isolated from human liver of Caucasian individuals. It is derived from an allelic variant at the ADH2 gene locus and exhibits a considerably higher specific activity and lower pH optimum than its ‘typical’ counterpart (isoenzyme β1β1) from the β1-chain predominant in Caucasians. Peptides were prepared by trypsin or CNBr cleavage, and were purified by exclusion chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Structural analysis of the peptides showed that β2-Bern differs at one position from β1. Thus, Arg-47 in β1 is substituted by His in β2-Bern. This exchange, compatible with a one-base mutation, explains all functional differences by altered interactions with the pyrophosphate moiety of the coenzyme. The difference is also structurally identical to that found for another atypical β2-subunit, the β2-Oriental type of major Asian occurrence, linking these two atypical forms of human alcohol dehydrogenase.Alcohol dehydrogenaseHuman liver isoenzymePrimary structureMutatio

Determination of human alcohol dehydrogenase and acetaldehyde dehydrogenase genotypes by single strand conformation polymorphism in discontinuous buffer electrophoresis

Under appropriate conditions single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products allows the detection of single base mutations in a given DNA fragment. We adapted this method for the routine determination of allele variants of human alcohol and acetaldehyde dehydrogenase without radioisotopic labeling. After PCR amplification of the selected exon, the DNA fragments were heat‐denatured and loaded on a polyacrylamide gel containing glycerol. For electrophoresis a discontinuous buffer system was used with sulfate as leading ion and borate as trailing ion. The DNA bands were revealed by silver staining. Acrylamide concentrations, ionic strength and electrophoresis temperature were systematically investigated for each DNA fragment. The polymorphisms detected by SSCP were identical to those found by hybridization with 32P‐labeled allele‐specific oligonucleotides. This method avoids the use of radioactivity, is less expensive and simpler than the allele‐specific oligonucleotide (ASO) methodology and thus particularly suited for routine analysis