Purification and some properties of a hepatic NADPH-dependent reductase that specifically acts on 1,5-anhydro-D-fructose

Glycogen gives rise to 1,5-anhydro-D-fructose (AF), which is then reduced to 1,5-anhydro-D-glucitol (AG) in animal livers, An enzyme that catalyzes NADPH-dependent reduction of AF to AG was isolated and purified to homogeneity from porcine liver, Its apparent molecular mass was about 38 kDa on the basis of SDS-PAGE, and its monomeric dispersion in aqueous solution was indicated by gel filtration on a Superose 12 column, Amino acid sequences were determined for four peptides obtained from the purified enzyme, The resulting sequences covered about 50% of the whole sequence and indicated a remarkable similarity between the enzyme and aldose reductase, The purified enzyme showed molecular activity of 8.7 s(-1) on the basis of a molecular mass of 38 kDa, and a K-m value of 0.44 mM for AF at the optimum pH of 7.0, It reduced pyridine-3-aldehyde and 2,3-butanedione effectively, acetaldehyde, glucosone, and glucuronic acid poorly and showed no detectable action on glucose, mannose and fructose, It was inactivated by p-chloromercuribenzoic acid to a considerable extent, and the inactivation was partially reversed by 2-mercaptoethanol treatment, It was also sparingly inhibited by relatively high concentrations of glucose, glucose-1(6)-phosphate and 1,5-anhydroglucitol, The reverse reaction, i.e., NADP(+)-dependent AG oxidation, was not observed, The observed catalytic properties and partial amino acid sequences rule out the possibility that the isolated protein is identical with any known reductase.