出版社：American Society for Biochemistry and Molecular Biology
摘要：Microsomal triglyceride transfer protein (MTP) activity is classicallymeasured using radioactive lipids. We described a simple fluorescenceassay to measure its triacylglycerol (TAG) transfer activity.Here, we describe fluorescence-based methods to measure thetransfer of phospholipids (PLs) and cholesteryl esters (CEs)by MTP. Both transfer activities increased with time and MTPamounts and were inhibited to different extents by an MTP antagonist,BMS197636. We also describe a method to measure the net depositionof fluorescent lipids in acceptor vesicles. In this procedure,negatively charged donor vesicles are incubated with MTP andacceptor vesicles, and lipids transferred to acceptors are quantifiedafter the removal of donor vesicles and MTP by the additionof DE52. Lipid deposition in acceptor vesicles was dependenton time and MTP. Using these methods, TAG transfer activitywas the most robust activity present in purified MTP; CE andPL transfer activities were 60–71% and 5–13% ofthe TAG transfer activity, respectively. The method to determinelipid transfer is recommended for routine MTP activity measurementsfor its simplicity.
These methods may help identify specific inhibitors for individuallipid transfer activities, in characterizing different domainsinvolved in transfer, and in the isolation of mutants that bindbut 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
