Purification and molecular properties of a class II alcohol dehydrogenase (ADH-C 2) from horse liver

1. 1. An alcohol dehydrogenase (ADH) from horse liver was purified by ion-exchange and affinity chromatography. 2. 2. The enzyme (designated ADH-C 2), is a dimer with a similar subunit size (47,300 mol. wt), as determined by SDS-polyacrylamide gel electrophoresis, to other mammalian ADHs. 3. 3. Zinc analyses and 1,10 phenanthroline inhibition studies indicated that each subunit contained 2 g atoms of zinc, with at least one involved catalytically. 4. 4. The enzyme exhibited similar kinetic properties to human 71-ADH and mouse ADH-C 2, 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. 5. 5. Horse ADH-C 2 exhibited a K m value for ethanol of 42 mM and a broad substrate specificity, with K m values decreasing dramatically with an increase in chain length. 6. 6. The enzyme was much less sensitive to pyrazole inhibition (by at least 3 orders of magnitude) as compared with the Class I ADHs.