Vitamin A is an essential nutrient required for growth, reproduction, differentiation of epithelial tissues and vision. Vitamin A deficency is a major cause of blindness and is associated with increased mortality in children of third world countries. An understanding of the mechanisms involved in intestinal absorption of vitamin A may prove valuable in the design of oral supplementation programs. Our interest is in understanding the mechanisms of intestinal vitamin A absorption, particularly in the role of cellular retinol binding protein II (CRBP II). Rat CRBP II is present in the adult rat in high concentrations exclusively in the small intestinal absortive cells, suggesting that it is uniquely adapted to the intestinal metabolism of retinol. It is closely homologous to rat cellular retinol binding protein (CRBP), which in contrast to CRBP II, is found many epithelial tissues. Intracellular movement of retinoids involves flux between the CRBPs ard membranes. This process is affected by the ligand binding specificities of the protein, rates of partitioning of ligands between the CRBPs and membranes as well as possible direct interactions of the CRBPs with membranes. The interactions between ligands, CRBPS, phospholipid bilayers and membrane proteins make this a complex problem to study. Efforts to study CRBP II has also been hampered by difficulties in obtaining pure apoprotein from rat tissues. We propose to approach the problem by obtaining pure apoCRBP II and apoCRBP by prokaryotic expression of these two proteins. Fluorescence and NMR spectroscopy along with engineering of mutant proteins by site-directed mutagenesis, will be used to study structural aspects of ligand protein interactions. Liposomes will be used to model interactions of proteins and ligands with membranes. CRBP serves as a """"""""natural mutagenesis"""""""" experiment for identifying molecular characteristics of CRBP II that make it uniquely adapted to the enterocyte. Based on the results of these in vitro studies, we hope to develop a model for how these proteins function in vivo.
