The long term objectives of this project are to understand the molecular events involved in the absorption, storage, transport, and nonvisual functions of vitamin A and the roles that specific binding proteins play in these processes. In this project period particular attention is given to cellular retinol-binding protein, type(II) [CRBP(II)]. This protein binds both retinol and retinal and is found in the mature absorptive cells of the small intestine and in the perinatal liver of rat as well as human small intestine. CRBP(II) may play a central role in the necessary metabolism of vitamin A during the absorptive process. CRBP(II) will be investigated as the acceptor of retinal generated in vitro or in vivo by the cleavage of Beta-carotene, and as carrier of this retinal for its subsequent reduction to retinol. Retinol bound to CRBP(II) has been shown to be esterified in vitro by a microsomal retinyl ester synthase that is not acylCoA dependent. This reaction will be further examined to identify the endogenous acyl donor, to purify the enzyme and produce specific antibodies for immunolocalization. The induction of CRBP(II) in the differentiating enterocyte will be examined by in situ hybridization of its mRNA, compared to the appearance of CRBP(II) itself as revealed by immunolocalization. Both CRBP(II) and CRBP, the other intracellular retinol binding protein, will be followed (both mRNA and protein levels and by immunohistochemical localization) during the perinatal period in gut and liver for rat and man with particular attention to the development of the villi in the intestine. The level of CRBP(II) will be determined in the small intestine of the rat in pregnancy/lactation. CRBP(II) is present in the small intestine in two forms, one form being N-blocked. Using the purified proteins and antibodies specific for one form, the potential roles of these two forms will be investigated in the above systems by examining the ability of each to interact with each enzyme. The distribution of endogenous ligand between the two forms as affected by dietary intake of Beta-carotene or retinyl esters and nutritional and developmental effects on the relative level of the two forms will be examined. Finally, the possible roles of CRBP and cellular retinoic acid binding protein, CRABP, in the intercellular movements of vitamin A in the liver or the testis will be examined by immunolocalization at the electron microscope level, to determine if either protein can be found in the intercellular space.
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