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Papers In Press, published online ahead of print May 16, 2005
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Anatomy and Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY 11203
Corresponding Author: mhussain{at}downstate.edu
Microsomal triglyceride transfer protein (MTP) activity is classically measured using radioactive lipids. We have previously described a simple fluorescence assay to measure triacylglycerol (TAG) transfer activity of MTP. Here, we describe fluorescence-based methods to measure the transfer of phospholipids (PL) and cholesterol esters (CE) by MTP. Both lipid transfer activities increased with time as well as the amount of MTP, 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 the time and amounts of MTP. Using these methods, we also determined relative transfer activities for TAG, CE, and PL in MTP. 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. Although both assays are sensitive, the method determining lipid transfer is recommended for routine MTP activity measurements for its simplicity. Availability of these methods may help to identify specific inhibitors for individual lipid transfer activities, in characterizing different domains involved in transferring these lipids, and isolation of mutants that bind but cannot transfer lipids.
Revised on May 9, 2005
Accepted on May 10, 2005
Measuring cholesterol ester and phospholipid transfer and net deposition by microsomal triglyceride transfer protein using fluorescent lipids
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