J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.D500008-JLR200 on April 16, 2005

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Journal of Lipid Research, Vol. 46, 1561-1568, July 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

A simple and rapid method to measure cholesterol binding to P450s and other proteins

Natalia Mast and Irina A. Pikuleva1

Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston TX 77555-1031

Published, JLR Papers in Press, April 16, 2005. DOI 10.1194/jlr.D500008-JLR200

1 To whom correspondence should be addressed. e-mail: irpikule{at}utmb.edu

Cholesterol plays an important role in cellular function and membrane compartmentalization and is involved in the interaction with more than a dozen of different proteins. Using three cholesterol-metabolizing cytochrome P450s (P450s 7A1, 46A1, and 11A1), we have developed a rapid and simple assay for measurements of nanomolar to micromolar cholesterol affinities. In this assay, the P450 is incubated with a fixed amount of radiolabeled cholesterol and varying concentrations of cold cholesterol followed by separation of free and protein-bound cholesterol via filtration through a membrane. Free cholesterol is found in the flow-through fraction, whereas P450 binds to the membrane. The radioactivity of the membranes is then measured, and a saturation curve is generated after correction for nonspecific binding of cholesterol to the filter. The validity of the filter assay was confirmed by spectral assay, a traditional method to evaluate the interaction of the P450 enzymes with their substrates. Two types of membranes, one binding positively charged proteins and another binding negatively charged proteins, were identified. These membranes were also found to hold proteins through hydrophobic interactions.

Thus, the cholesterol binding properties of a wide variety of proteins could be characterized using this filter assay.

Abbreviations: CYP, cytochrome P450; HPCD, 2-hydroxypropyl-ß-cyclodextrin

Supplementary key words cytochrome P450 7A1 • cytochrome P450 46A1 • cytochrome P450 11A1 • spectral binding • alternative assay


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Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.