The specific aims of this proposal are: 1) To determine the rates and mechanisms of transfer of digestive lipid products from micelles, vesicles, and mixed populations of postprandial digestive phases. These studies will use a recently developed experimental system which allows the principal investigator (P.I.) to quantitatively examine the kinetic mechanisms of luminal fatty acid (FA) and monoglyceride (MG) transfer; 2) To determine the molecular mechanisms by which the two enterocyte FABP's modulate the transfer of FA and MG to membranes. The P.I. has recently shown that IFABP transfers FA to membranes by collisional interactions with the membrane. These studies will determine the structural domains responsible for these interactions using site-directed mutagenesis combined with biochemical and biophysical analysis of protein-membrane interactions. In addition, the rate and mechanism of MG transfer from LFABP to membranes will be determined, as it has been recently demonstrated that MG is a specific ligand for LFABP; and 3) To examine the role of IFABP and LFABP in cellular FA and MG uptake and transport. The P.I. has shown previously that Caco-2 cells, unlike actual enterocytes, express LFABP but not IFABP. In these studies, the P.I. will use Caco-2 cells, unlike actual enterocytes, express only LFABP but not IFABP. In these studies, the P.I. will use Caco-2 cells transfected with wild type or specific mutant IFABP's to examine the effects of L- and IFABP on both the intracellular trafficking of FA and MG, and on enterocyte lipid metabolism. The long term goal of this research is to enable the control of lipid assimilation by modulation of specific transport processes.
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