title: Purification and characterization of a novel cytosolic NADP(H)-dependent retinol oxidoreductase from rabbit liver.
abstract: Rabbit liver cytosol exhibits very high retinol dehydrogenase activity. At least two retinol dehydrogenases were demonstrated to exist in rabbit liver cytosol, and the major one, a cytosolic NADP(H)-dependent retinol dehydrogenase (systematic name: retinol oxidoreductase) was purified about 1795-fold to electrophoretic and column chromatographic homogeneity by a procedure involving column chromatography on AF-Red Toyopearl twice and then hydroxyapatite. Its molecular mass was estimated to be 34 kDa by SDS-PAGE, and 144 kDa by HPLC gel filtration, suggesting that it is a homo-tetramer. The enzyme uses free retinol and retinal, and their complexes with CRBP as substrates in vitro. The optimum pH values for retinol oxidation of free retinol and CRBP-retinol were 8.8-9.2 and 8.0-9.0, respectively, and those for retinal reduction of free retinal and retinal-CRBP were the same, 7.0-7.6. Km for free retinol and Vmax for retinal formation were 2.8 microM and 2893 nmol/min per mg protein at 37 degrees C (pH 9.0) and the corresponding values with retinol-CRBP as a substrate were 2.5 microM and 2428 nmol/min per mg protein at 37 degrees C (pH 8.6); Km for free retinal and Vmax for retinol formation were 6.5 microM and 4108 nmol/min per mg protein, and the corresponding values with retinal-CRBP as a substrate were 5.1 microM and 3067 nmol/min per mg protein at 37 degrees C, pH 7.4. NAD(H) was not effective as a cofactor. 4-Methylpyrazole was a weak inhibitor (IC50 = 28 mM) of the enzyme, and ethanol was neither a substrate nor an inhibitor of the enzyme. This enzyme exhibits relatively broad aldehyde reductase activity and some ketone reductase activity, the activity for aromatic substitutive aldehydes being especially high and effective. Whereas, except in the case of retinol, oxidative activity toward the corresponding alcohols was not detected. This novel cytosolic enzyme may play an important role in vivo in maintaining the homeostasis of retinal, the substrate of retinoic acid synthesis, at least in rabbit liver, since a high concentration of retinol in liver and the lower Km of the enzyme for retinol force the oxidative reaction, while higher activity of retinal reductase at physiological pH forces the reductive reaction.
authors.affiliation: Department of Biochemistry, School of Medicine, Kagawa Medical University, Japan. dhuang@kms.ac.jp
authors.forename: D Y
authors.initials: DY
authors.lastname: Huang
authors.forename: Y
authors.initials: Y
authors.lastname: Ichikawa
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D000072467:Cytochrome P450 Family 2; D003600:Cytosol / Q000201:enzymology; D017886:Durapatite; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D012172:Retinaldehyde / Q000378:metabolism; D013379:Substrate Specificity; D014801:Vitamin A / Q000378:metabolism
chemical_list: D009243:NAD; D014801:Vitamin A; D009249:NADP; D017886:Durapatite; D000429:Alcohol Oxidoreductases; C022520:retinol dehydrogenase; C504568:Cyp2d1 protein, rat; D000072467:Cytochrome P450 Family 2; D012172:Retinaldehyde