title: Effects of sulfhydryl reagents, retinoids, and solubilization on the activity of microsomal retinol dehydrogenase.
abstract: A microsomal retinol dehydrogenase (RoDH) that recognizes holo-cellular retinol binding protein (CRBP) as substrate is inhibited by phenylarsine oxide (IC50 = 3 microM) in the presence of 10 mM cysteine. Inhibition was reversible with dithiothreitol, indicating that two cysteine residues in close proximity are essential for RoDH activity. Bromophenylarsine oxide was an irreversible inhibitor (IC50 = 0.2 microM), suggesting that a nucleophile lies close to the two cysteine residues. N-Ethylmaleimide inhibited reactions supported by holo-CRBP, but not from free retinol, suggesting that it obstructed holo-CRBP access to RoDH without affecting the catalytic site. RoDH activity was similar in microsomes from vitamin A-sufficient or vitamin A-deficient rats and was not inhibited by relatively high concentrations (5 microM) of all-trans-retinoic acid, holo-cellular retinoic acid binding protein, apo-cellular retinoic acid binding protein, or 9-cis-retinoic acid. Triton X-100 stimulated RoDH activity eightfold at a detergent to protein ratio of 0.25 to 1 (w/w). A combination of Tween 80, Brij 92, and Triton X-100 (2:1:2) stimulated RoDH activity eightfold at a detergent to protein ratio of 2.5 to 1 (w/w). Detergent-solubilized RoDH, partially purified through a PAO-Sepharose resin, preferred NADP(H) as cofactor, had a Km for retinal synthesis from holo-CRBP of 0.6 microM (Vmax = 115 pmol/min/mg protein) and a Km for reduction of retinal bound to CRBP of 0.6 microM (Vmax = 613 pmol/min/mg protein). This work provides further insight into microsomal RoDH and strengthens the evidence of an interaction between RoDH and holo-CRBP.
authors.affiliation: Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo 14214, USA.
authors.forename: M H
authors.initials: MH
authors.lastname: Boerman
authors.forename: J L
authors.initials: JL
authors.lastname: Napoli
mesh_terms: D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D000072467:Cytochrome P450 Family 2; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D050697:Retinal Dehydrogenase; D012172:Retinaldehyde / Q000378:metabolism / Q000494:pharmacology; D012176:Retinoids / Q000494:pharmacology*; D012177:Retinol-Binding Proteins / Q000378:metabolism; D054840:Retinol-Binding Proteins, Cellular; D012995:Solubility; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014801:Vitamin A / Q000378:metabolism / Q000494:pharmacology
chemical_list: D003902:Detergents; D011994:Recombinant Proteins; D012176:Retinoids; D012177:Retinol-Binding Proteins; D054840:Retinol-Binding Proteins, Cellular; D013439:Sulfhydryl Reagents; D014801:Vitamin A; D000429:Alcohol Oxidoreductases; C022520:retinol dehydrogenase; C504568:Cyp2d1 protein, rat; D000072467:Cytochrome P450 Family 2; D000445:Aldehyde Oxidoreductases; D050697:Retinal Dehydrogenase; D012172:Retinaldehyde