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.
Highlights
THE JOURNAL OF BIOLOGICAL CHEMISTRY24455–24460, 1997 Printed in U.S.A. Purification and Partial Characterization of a Cellular Carotenoid-binding Protein from Ferret Liver*
In contrast to the elution profile of the CCBP, all of the labeled -carotene was eluted from these columns in the void volume of elution buffer
Summary of Purification—Table I shows the summary of purification of CCBP
Summary
24455–24460, 1997 Printed in U.S.A. Purification and Partial Characterization of a Cellular Carotenoid-binding Protein from Ferret Liver*. A cellular carotenoid-binding protein was purified to homogeneity from -carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinity chromatography. [14C]-Carotene co-purified with the binding protein throughout the purification procedures. 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 -ionone ring. We have previously reported the isolation of a partially purified carotenoid-protein complex from rat liver [10]. In this paper we present, for the first time, the purification to homogeneity and partial characterization of a CCBP from the livers of -carotene-fed male ferrets. Characterization of a CCBP in the ferret would have relevance to humans
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.