title: Biosynthesis of all-trans-retinoic acid from retinal. Recognition of retinal bound to cellular retinol binding protein (type I) as substrate by a purified cytosolic dehydrogenase.
abstract: An NAD-dependent rat liver cytosolic dehydrogenase accepted as substrate retinal generated in situ by microsomes from retinol bound to excess CRBP (cellular retinol binding protein, type I). This activity, which was not retained by anion-exchange chromatography at pH 9.15, was designated P1. P1 activity increased 2.5-fold, with no statistically significant change in its K or Hill coefficient, in liver cytosol from rats fed a retinoid-deficient diet. Orally dosed retinoic acid partially suppressed the increase. Activities chromatographically similar to hepatic P1 were observed in cytosols from rat kidney and testes. P1, purified from rat liver cytosol, had a pI of approximately 8.3, migrated as a tetramer (214 kDa) on a Sephadex G-200 column, and had a subunit molecular mass of 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With free retinal it catalyzed a maximum rate of retinoic acid synthesis of 265 nmol/min/mg of protein and exhibited allosteric kinetics with a K of 0.76 +/- 0.35 microM and a Hill coefficient of 1.5 +/- 0.13 (mean +/- S.D., n = 4). Substrate inhibition was noted with retinal concentrations greater than 6 microM. The purified enzyme not only recognized retinal generated by microsomes as substrate, but also recognized retinal bound to CRBP. The rates of retinoic acid synthesis from CRBP-retinal, with a series of increasing apoCRBP concentrations, exceeded the rates that would be supported by the free retinal present. The CRBP-retinal complex exhibited allosteric kinetics (K, 0.13 microM; Hill coefficient, 1.75; averages of duplicates) in the presence of excess apoCRBP (the ratio total CRBP/total retinal at each concentration of retinal was 2). This enzyme is likely to play a significant role in retinoic acid synthesis in vivo, because it participates in the synthesis of retinoic acid from a physiologically occurring form of retinol (holoCRBP), reflects retinoid status, and is distributed in extrahepatic tissues in addition to liver. These results also suggest a novel role for CRBP in retinoid metabolism, facilitating the conversion of retinal into retinoic acid.
authors.affiliation: Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214.
authors.forename: K C
authors.initials: KC
authors.lastname: Posch
authors.forename: R D
authors.initials: RD
authors.lastname: Burns
authors.forename: J L
authors.initials: JL
authors.lastname: Napoli
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D050697:Retinal Dehydrogenase; D012172:Retinaldehyde / Q000378:metabolism*; D012177:Retinol-Binding Proteins / Q000378:metabolism*; D054840:Retinol-Binding Proteins, Cellular; D013379:Substrate Specificity; D013997:Time Factors; D014212:Tretinoin / Q000378:metabolism*
chemical_list: D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D014212:Tretinoin; D000445:Aldehyde Oxidoreductases; D050697:Retinal Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D012172:Retinaldehyde