Stomach aldehyde dehydrosenase was structurally evaluated by analysis of pcptide fragments of the human enzyme and ¢ontparisons with corresponding parts from other characterized aldehyde dehydrogenases. The results establish a large part of the structure, confirming that the stomach enzyme Is identica; to the inducible or tumor, derived dimcric aldehyde dchydrogena~, In addition, species variations between identical sets of different aldehyde lind alcohol dchydr0genases reveal that stomach aldehyde dehydrogenas¢ exhibits a fairly rapid rate of evolutionary changes, similar to th:tt for the likewise 'variable' classical alcohol dehydrogenase, sorbitol dehydrogenase, and cytosolic aldehyde dehydrogena~ but in contrast to the 'constant' class Ill '.alcohol dehydrogenase and mitoehondrial aldehyde dehydrogenas¢, This establishes that rates of divergence in the aldehyde and alcohol dehydrog©nascs are unrelated to subunit size or quaternary structure, highlights the unique nature of class 111 alcohol dchydrogena~, and positions the stomach aldehyde dehydrogenas¢ in a group with more ordinary i'eaturcs.

The 3B-hydroxysteroid dehydrogenase of Pseudomonas testosteroni commercially available was purified by an FPLC step and submitted to sequence determination by peptide analysis. The structure obtained reveals a 253-residue polypeptide chain, with an N-terminal, free a-amino group, and a low cysteine content. Comparisons with other hydroxysteroid dehydrogenases recently characterized reveal distant similarities with prokaryotic and, to some extent, also eukaryotic forms of separate specificities. Residue identities with a Streptomyces 20g-hydroxysteroid dehydrogenase are 35% and distributed over the entire molecule, whereas residue identities with the mammalian 17P-hydroxysteroid dehydrogenase only constitute 20%, and are essentially limited to the Nterminal and central parts, Nevertheless, all these enzymes exhibit a conserved tyrosine residue (position 151 in the present enzyme) noted as possibly having a functional role in some members of this protein family. Combined, the results establish the prokaryotic 3B-hydroxysteroid dehydrogenase as belonging to the family of shortchain alcohol dehydrogenases, reveal that the hydroxysteroid dehydrogenases are no more closely related than dehydrogenases with other enzyme activities within the family (e. g. glucose, ribitol, hydroxyprostaglandin dehydrogenases), show several of the mammalian hydroxysteroid dehydrogenases to have subunits of longer size with different patterns of similarity than those of the prokaryotic family members characterized, and define important segments of the coenzyme-binding region for this enzyme group.

Isozyme patterns differ widely among the classical type (class I) of mammalian alcohol dehydrogenases. For the rabbit enzyme, the possibility of isozymes has been reported but structural evidence is lacking. This system was now studied at both the mRNA/cDNA and protein levels. Ten cDNA clones, coding for class‐I alcohol dehydrogenase, were isolated from a rabbit liver cDNA library using a human DNA fragment as probe. The cDNA spanned 1296 bp, including the entire coding region. All clones coded for the same polypeptide and Northern blots identified a single mRNA corresponding to about 1.5 kb. Comparison of two protein forms (CC and BC) by HPLC peptide fingerprinting and structural analysis revealed peptide segments identical in amino acid sequence. Consequently, direct protein analyses and Northern blots show the presence of only one primary translation product. The data suggest that lagomorphic alcohol dehydrogenase, like the rodent enzyme, is not as isozyme rich as it may appear superficially, and that secondary modifications contribute substantially to mammalian alcohol dehydrogenase multiplicity. The active center of the rabbit enzyme suggests similarities to the horse S, human γ, and rat enzyme structures, compatible with a steroid dehydrogenase activity shown experimentally. Typical class‐I properties were established by direct analysis and confirmed by structural properties (Km for cyclohexanol 0.8–1.1 mM, for ethanol 1.6–1.9 mM). The isozyme versus species differences mark the variability of class‐I alcohol dehydrogenase versus class III and suggest a parallelism between rapid mutational differences and frequent duplicatory events.