Purification and molecular properties of mouse alcohol dehydrogenase isozymes

Abstract

Alcohol dehydrogenase isozymes from mouse liver (A2 and B2) and stomach (C2) tissues were purified to homogeneity using triazine-dye affinity chromatography. The enzymes were dimers with similar but distinct subunit sizes, as determined by SDS[sodium dodecylsulfate]/polyacrylamide gel electrophoresis: A, 43,000, B, 39,000 and C, 47,000. Zn analyses and 1,10-phenanthroline inhibition studies indicated that the A and C subunits each contained 2 atoms of Zn, with at least 1 being involved catalytically; the B subunit probably contained a single noncatalytic Zn atom. The isozymes exhibited widely divergent kinetic characteristics. A2 exhibited a Km value for ethanol of 0.15 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length; C2 also exhibited this broad specificity for alcohols but showed a Km value of 232 mM for ethanol. These isozymes also showed broad substrate specificities as aldehyde reductases. B2 showed no detectable activity as an aldehyde reductase for the aldehydes examined, and used ethanol as substrate only at high concentrations (> 0.5 M). The isozyme exhibited low Km and high Vmax values with medium-chain alcohols. Immunological studies showed that A2 was immunologically distinct from the B2 and C2 isozymes. In vitro molecular hybridization studies gave no evidence for association between the alcohol dehydrogenase subunits. The results confirmed genetic analyses which were consistent with at least 3 structural genes encoding alcohol dehydrogenase in the mouse and confirmed the role of the major liver isozyme (A2) in ethanol metabolism.