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Methods |
* Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center, Brooklyn, NY 11203
Department of Pediatrics, State University of New York Downstate Medical Center, Brooklyn, NY 11203
1 To whom correspondence should be addressed. e-mail: mahmood.hussain{at}downstate.edu
Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB) lipoproteins. Its activity is classically measured by incubating purified MTP or cellular homogenates with donor vesicles containing radiolabeled lipids, precipitating the donor vesicles, and measuring the radioactivity transferred to acceptor vesicles. Here, we describe a simple, rapid, and sensitive fluorescence assay for MTP. In this assay, purified MTP or cellular homogenates are incubated with small unilamellar donor vesicles containing quenched fluorescent lipids (triacylglycerols, cholesteryl esters, and phospholipids) and different types of acceptor vesicles made up of phosphatidylcholine or phosphatidylcholine and triacylglycerols. Increases in fluorescence attributable to MTP-mediated lipid transfer are measured after 30 min. MTP's lipid transfer activity could be assayed using apoB lipoproteins but not with high density lipoproteins as acceptors. The assay was used to measure MTP activity in cell and tissue homogenates. Furthermore, the assay was useful in studying the inhibition of the cellular as well as purified MTP by its antagonists.
This new method is amenable to automation and can be easily adopted for large-scale, high-throughput screening.
Abbreviations: apoB, apolipoprotein B; CE, cholesteryl ester; PC, phosphatidylcholine; PL, phospholipid; MTP, microsomal triglyceride transfer protein; TAG, triacylglycerol
Supplementary key words lipoprotein assembly • cholesteryl esters • phospholipids • triacylglycerol • apolipoprotein B
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