The long-term goal of this project is to describe the biochemical mechanisms of the lipolytic enzymes responsible for dietary fat digestion. Dietary fats provide a major source of energy, essential fatty acids, a vehicle for fat-soluble vitamins, and components of cellular membranes. Fats eaten in excess adversely affect health increasing the risk of cardiovascular disease and obesity. Before fats can be absorbed, they must be digested. Lipases in the stomach and duodenum digest dietary fats. This proposal focuses on the lipase responsible for the majority of fat digestion, pancreatic triglyceride lipase, and another pancreatic protein, colipase, which is required, by pancreatic triglyceride lipase for activity. We propose that specific interactions among lipase, colipase, lipids, and bile salts, which are necessary for absorption of the fatty acids released by lipase, dictate the function of pancreatic triglyceride lipase. The first specific aim is to characterize the mechanisms that mediate specific interactions among lipase, colipase, bile salts, and lipids. We will substitute selected amino acids in lipase or colipase by site-specific mutagenesis to identify functionally important regions of both proteins. The second specific aim is to determine the mechanisms that trigger the conversion of lipase from an inactive to an active conformation. In aqueous solution lipase is inactive. When lipase encounters the lipid substrate its conformation changes and it becomes active. We will follow the conformational change by tryptophan fluorescence to determine the factors that influence this critical conformational change. The last specific aim is to develop an expression system for labeling recombinant colipase with stable isotopes. The ability to label colipase with stable isotopes will enable NMR studies of the conformation of colipase in aqueous solutions under different conditions. We will test various culture conditions to improve our current system for the production of 15N labeled colipase and we will develop a system for economically labeling colipase with 13C and 15N. These studies will determine the biochemical details of the interactions between lipase, colipase, lipids, and bile salts that impact lipase function. The results will ultimately permit us to manipulate these interactions for therapeutic purposes.
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