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 Km 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.
Sakuma
M
M
Department of Life Sciences (Chemistry), Graduate School of Arts and Sciences, The University of Tokyo.
Kametani
S
S
Akanuma
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D004791:Enzyme Inhibitors; D005632:Fructose / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine; D013696:Temperature
D004791:Enzyme Inhibitors; D005632:Fructose; C048927:1,5-anhydrofructose; D013401:Sugar Alcohol Dehydrogenases; C110944:1,5-anhydro-fructose reductase