Purification and molecular properties of a Class II alcohol dehydrogenase (ADH-C2) from horse liver

An alcohol dehydrogenase (ADH) from horse liver was purified by ion-exchange and affinity chromatography. The enzyme (designated ADH-C2), is a dimer with a similar subunit size (47,300 mol. wt), as determined by SDS-polyacrylamide gel electrophoresis, to other mammalian ADHs. Zinc analyses and 1,10 phenanthroline inhibition studies indicated that each subunit contained 2 g atoms of zinc, with at least one involved catalytically. The enzyme exhibited similar kinetic properties to human pi-ADH and mouse ADH-C2, previously classified as class II ADHs [Vallee and Bazzone (1983) Isozymes, Vol. 8, pp. 219-244; Algar et al. (1983) Eur. J. Biochem. 137, 139-147] but differed in most respects from the extensively investigated horse Class I ADHs; EE, ES and SS. Horse ADH-C2 exhibited a Km value for ethanol of 42 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length. The enzyme was much less sensitive to pyrazole inhibition (by at least 3 orders of magnitude) as compared with the Class I ADHs.