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Papers In Press, published online ahead of print February 1, 2004
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Department of Anatomy and Cell Biology, SUNY Downsate Medical Center, Brooklyn, NY 11203
Corresponding Author: mahmood.hussain{at}downstate.edu
Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of 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, cholesterol esters, and phospholipids) and different types of acceptor vesicles made up of phosphatidylcholine or phosphatidylcholine and triacylglycerols. Increases in fluorescence due 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 the 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 through put screening.
Revised on January 20, 2004
Accepted on January 21, 2004
A simple, rapid and sensitive fluorescence assay for microsomal triglyceride transfer protein
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