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Journal of Lipid Research, Vol. 46, 1779-1785, August 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Transfer of cholesteryl esters and phospholipids as well as net deposition by microsomal triglyceride transfer protein

Paul Rava, Humra Athar, Caroline Johnson and M. Mahmood Hussain¹

Department of Anatomy and Cell Biology and Department of Pediatrics, State University of New York Downstate Medical Center, Brooklyn, NY 11203

Published, JLR Papers in Press, May 16, 2005. DOI 10.1194/jlr.D400043-JLR200

¹ To whom correspondence should be addressed. e-mail: mahmood.hussain{at}downstate.edu

Microsomal triglyceride transfer protein (MTP) activity is classically measured using radioactive lipids. We described a simple fluorescence assay to measure its triacylglycerol (TAG) transfer activity. Here, we describe fluorescence-based methods to measure the transfer of phospholipids (PLs) and cholesteryl esters (CEs) by MTP. Both transfer activities increased with time and MTP amounts and were inhibited to different extents by an MTP antagonist, BMS197636. We also describe a method to measure the net deposition of fluorescent lipids in acceptor vesicles. In this procedure, negatively charged donor vesicles are incubated with MTP and acceptor vesicles, and lipids transferred to acceptors are quantified after the removal of donor vesicles and MTP by the addition of DE52. Lipid deposition in acceptor vesicles was dependent on time and MTP. Using these methods, TAG transfer activity was the most robust activity present in purified MTP; CE and PL transfer activities were 60–71% and 5–13% of the TAG transfer activity, respectively. The method to determine lipid transfer is recommended for routine MTP activity measurements for its simplicity.

These methods may help identify specific inhibitors for individual lipid transfer activities, in characterizing different domains involved in transfer, and in the isolation of mutants that bind but cannot transfer lipids.

Abbreviations: apoB, apolipoprotein B; CE, cholesteryl ester; MTP, microsomal triglyceride transfer protein; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PL, phospholipid; TAG, triacylglycerol

Supplementary key words lipoprotein assembly • phosphatidylethanolamine • triacylglycerol • fluorescent lipids • apolipoprotein B • transfer assays • lipid transfer

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