Cholesterol interaction with recombinant human sterol carrier protein-2 |
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| Authors: | S M Colles J K Woodford D Moncecchi S C Myers-Payne L R McLean J T Billheimer F Schroeder |
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| Affiliation: | (1) Division of Pharmacology and Medicinal Chemistry, Department of Pharmacology and Cell Biophysics, University of Cincinnati Medical Center, 45267-0004 Cincinnati, Ohio;(2) Department of Physiology and Pharmacology, Texas Veterinary Medical Center, Texas A&M University, 77843-4466 College Station, Texas;(3) Department of Analytical and Structural Sciences, Marion Merrell Dow Research Institute, 45215-6300 Cincinnati, Ohio;(4) Cardiovascular Department, Du Pont Merck Pharmaceutical Company, 19898-0400 Wilmington, Delaware;(5) Present address: Cleveland Clinic Foundation, 9500 Euclid Ave., NC10, 44195 Cleveland, OH;(6) Present address: The Andrew Jergens Co., 2535 Spring Grove Ave., 44214-1773 Cincinnati, OH;(7) Present address: 6103 Browning Dr., 82007 Cheyenne, WY;(8) Present address: Laboratory of Membrane Biochemistry and Biophysics, NIAAA, National Institutes of Health, FLOW Bldg., Rm. 55A, 12501 Washington St., 20852 Rockville, MD |
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| Abstract: | The interaction of human recombinant sterol carrier protein-2 (SCP-2) with sterols was examined. Two independent ligand binding
methods, Lipidex 1000 binding of 3H]cholesterol and a fluorescent dehydroergosterol binding assay, were used to determine the affinity of SCP-2 for sterols.
Binding analysis indicated SCP-2 bound 3H]cholesterol and dehydroergosterol with aK
d of 0.3 and 1.7 μM, respectively, and suggested the presence of a single binding site. Phase fluorometry and circular dichroism
were used to characterize the SCP-2 sterol binding site. Alterations in dehydroergosterol lifetime, SCP-2 tryptophan lifetime,
and SCP-2 tryptophan quenching by acrylamide upon cholesterol binding demonstrated a shielding of the SCP-2 tryptophan from
the aqueous solvent by bound sterol. Differential polarized phase fluorometry revealed decreased SCP-2 tryptophan rotational
correlation time upon cholesterol binding. Circular dichroism of SCP-2 indicated that cholesterol elicited a small decrease
in SCP-2 alpha helical content. The data suggest that SCP-2 binds sterols with affinity consistent with a lipid transfer protein
that may act either as an aqueous carrier or at a membrane surface to enhance sterol desorption. |
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