Structure of the class II enzyme of human liver alcohol dehydrogenase: combined cDNA and protein sequence determination of the pi subunit.
The class II enzyme of human liver alcohol dehydrogenase was isolated, carboxymethylated, and cleaved with CNBr and proteolytic enzymes. Sequence analysis of peptides established structures corresponding to the pi subunit. Two segments from the C-terminal region unique to pi were selected for synthesis of oligodeoxyribonucleotide probes to screen a human liver cDNA library constructed in plasmid pT4. Sequence analysis of two identical hybridization-positive clones with cDNA inserts of about 2000 nucleotides gave the entire coding region of the pi subunit, a 61-nucleotide 5' noncoding region and a 741-nucleotide 3' noncoding region containing four possible polyadenylation sites. Translation of the coding region yields a 391-residue polypeptide, which in all regions except the C-terminal segment corresponds to the protein structure as determined directly by peptide analysis. With the class I numbering system, the exception concerns a residue exchange at position 368, the actual C-terminus which is Phe-374 by peptide data but a 12-residue extension by cDNA data, and possibly two further residue exchanges at positions 303 and 312. The size difference might indicate the existence of posttranslational modifications of the mature protein or, in combination with the residue exchanges, the existence of polymorphism at the locus for class II subunits. The pi subunit analyzed directly results in a 379-residue polypeptide and is the only class II size thus far known to occur in the mature protein.(ABSTRACT TRUNCATED AT 250 WORDS)
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003488:Cyanogen Bromide; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D006801:Humans; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D009693:Nucleic Acid Hybridization; D010446:Peptide Fragments / Q000032:analysis
D046911:Macromolecular Substances; D010446:Peptide Fragments; D004247:DNA; D000426:Alcohol Dehydrogenase; D004262:DNA Restriction Enzymes; D003488:Cyanogen Bromide