Isozyme developments in mammalian class-I alcohol dehydrogenase. cDNA cloning, functional correlations, and lack of evidence for genetic isozymes in rabbit.
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 gamma, 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.
Höög
J O
JO
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
Vagelopoulos
N
N
Yip
P K
PK
Keung
W M
WM
Jörnvall
H
H
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats
D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D000426:Alcohol Dehydrogenase