Sorbitol dehydrogenase. The primary structure of the sheep-liver enzyme.
The first primary structure for a sorbitol dehydrogenase has been determined by analysis of the tetrameric enzyme from sheep liver. The [14C]carboxymethylated protein was cleaved with CNBr and proteolytic enzymes. Peptides were purified by several methods, often utilizing exclusion chromatography for pre-fractionation and reverse-phase high-performance liquid chromatography for final purification. Different methods of sequence analysis complemented each other, mainly the manual dimethylaminoazobenzene isothiocyanate method and and the use of liquid-phase sequencer degradations. All eight major CNBr fragments were purified and form the basis of the work. Three minor CNBr fragments derived from an acid cleavage and from a partly resistant Met-Thr bond were also obtained, as well as evidence for a contaminating homologous polypeptide. Most of the tryptic peptides were purified, including all with methionine residues, thus overlapping the CNBr fragments. Combined, all data permit the deduction of a 354-residue amino acid sequence for the polypeptide chain of sorbitol dehydrogenase. The N terminus is acyl-blocked, the C terminus is formed by a proline residue, tryptophan is the least common residue (two, at positions 50 and 301) and there are 10 cysteine residues, including the residue previously shown to be especially reactive (at position 43). Similarities to 'long' alcohol dehydrogenases have functional implications.
D000595:Amino Acid Sequence; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D007064:L-Iditol 2-Dehydrogenase / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D008715:Methionine; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D012756:Sheep; D013401:Sugar Alcohol Dehydrogenases / Q000302:isolation & purification*
D010446:Peptide Fragments; D008715:Methionine; D013401:Sugar Alcohol Dehydrogenases; D007064:L-Iditol 2-Dehydrogenase; D003488:Cyanogen Bromide