title: Purification and partial characterization of a cellular carotenoid-binding protein from ferret liver.
abstract: A cellular carotenoid-binding protein was purified to homogeneity from beta-carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinity chromatography. The final purification was 607-fold. [14C]beta-Carotene co-purified with the binding protein throughout the purification procedures. SDS-PAGE of the purified protein showed a single band with an apparent molecular mass of 67 kDa. Scatchard analysis of the specific binding of the purified protein to beta-carotene showed two classes of binding sites, a high affinity site with an apparent Kd of 56 x 10(-9) M and a low affinity site with a Kd of 32 x 10(-6) M. The Bmax for beta-carotene binding to the high affinity site was 1 mol/mol, while that for the low affinity site was 145 mol/mol. The absorption spectrum of the complex showed a 32-nm bathochromic shift in lambdamax with minor peaks at 460 and 516 nm. Except for alpha-carotene and cryptoxanthin, none of the model carotenoids or retinol competed with beta-carotene binding to the protein. Thus, a specific carotenoid-binding protein of 67 kDa has been characterized in mammalian liver with a high degree of specificity for binding only carotenoids with at least one unsubstituted beta-ionone ring.
authors.affiliation: Department of Medicine, George Washington University, Washington, D. C. 20037, USA.
authors.forename: M N
authors.initials: MN
authors.lastname: Rao
authors.forename: P
authors.initials: P
authors.lastname: Ghosh
authors.forename: M R
authors.initials: MR
authors.lastname: Lakshman
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005289:Ferrets; D008024:Ligands; D008099:Liver / Q000378:metabolism*; D008297:Male; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D019207:beta Carotene / Q000008:administration & dosage / Q000378:metabolism*
chemical_list: D008024:Ligands; D011506:Proteins; D019207:beta Carotene