Revised Manuscript Received July 2, 1993* abstract: The microsomal triglyceride transfer protein (MTP) is found in the lumen of microsomes isolated from liver and intestine. This protein, which catalyzes the transport of neutral lipids between membranes, appears to play an important role in the biogenesis of plasma very low density lipoproteins and chylomicrons. Enzyme kinetic studies were used to investigate the mechanism of MTP-catalyzed lipid transport. Initial rates of [14C] triolein and [14C] cholesteryl oleate transport from donor to acceptor smallunilamellar vesicles were determined at varying donor and acceptor membrane concentrations. Results using two different kinetic analyses demonstrated lipid transport was best described by ping-pong bi-bi kinetics, indicating that MTP is a lipid binding protein which shuttles triglyceride and cholesteryl ester molecules between membranes. This model forlipid transport was supported by a fluorescent lipid binding assay in which MTP was able to extract pyrene-labeled cholesteryl ester from a vesicle. MTP-membrane interactions and lipid transport were regulated by membrane surface charge. Equilibrium gel filtration chromatography demonstrated MTP has a higher affinity for neutrally charged membranes than negatively charged membranes. In agreement with the membrane binding studies, MTP-mediated lipid transfer was inhibited by increasing the concentration of negatively charged phospholipid molecules in donor membranes.

The microsomal triglyceride transfer protein (MTP) 1 catalyzes the transport of triglyceride (TG), cholesteryl ester (CE), and phosphatidylcholine (PC) between membranes (Wetterau & Zilversmit, 1984). MTP activity is found within the lumen of microsomes isolatedfrom the liver and intestinal mucosa (Wetterau & Zilversmit, 1986). Purified MTP from bovine liver is a soluble heterodimer consisting of 88-and 58-kDa subunits noncovalently associated (Wetterau& Zilversmit, 1985; Wetterau et al., 1990, 1991a). The 58-kDa component has been identified as the multifunctional protein, protein disulfide isomerase (Wetterau et al., 1990). The dimeric structure and subcellular location of MTP make it unique among lipid transfer proteins. Most other mammalian intracellular lipid transfer proteins have been isolated from the postmicrosomal supernatant (cytosol) of various tissues [reviewed in Helmkamp (1986) and Rueckert (1990)]. All are monomeric proteins with molecular masses in the range of 8-35 kDa. MTP is also distinct from the monomeric 58-74-kDa plasma cholesterylester transfer protein (CETP)(Ihm et al., 1982a; Morton & Zilversmit, 1982; Albers et al., 1984; Hesler et al., 1987), which is a glycoprotein containing a 52-kDa polypeptide backbone (Drayna et al., 1987). CETP, like