CHARACTERIZATION OF RETINOIC ACID METABOLISM

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Retinoic acid is the endogenous retinoid that acts directly to support specific vitamin A-dependent processes. A long-term goal of this project is to determine whether impairment of retinoic acid biogenesis causes and/or contributes to the development of oncological and dermatological diseases that are prevented or arrested by retinoid therapy. The immediate goal is to identify and characterize the retinoid- specific oxidoreductases that catalyze the biosynthesis of retinoic acid from retinol and retinal. The hypotheses to be tested are: A) retinoic acid is generated in situ in a spectrum of tissues through the interaction of a retinol specific oxidoreductase and a low Km, low Vmax retinal dehydrogenase; B) the rate of retinoic acid synthesis is controlled by retinol availability and the activity/amount of the low Km, low Vmax retinal dehydrogenase; C) intracellular transport of retinoids occurs by direct transfer from protein to protein--e.g. retinol from cellular retinol binding protein (CRBP) to retinol dyhydrogenase, retinal from retinol dehydrogenase to retinal dehydrogenase, retinoic acid from retinal dehydrogenase to cellular retinoic acid binding protein (CRABP). The specific aims are: 1) purify retinol and retinal dehydrogenases from rat tests cytosol; 2) characterize the pure retinoid dehydrogenases; 3) determine the tissue distribution of retinoid dehydrogenases, their concentrations in vivo and their specificity for retinoids; 4) determine whether transfer of retinoids in the metabolic pathway from retinol to retinoic acid occurs thru protein-protein complexes. The enzymes will be purified by traditional, as well as newer techniques, including affinity-, fast-protein liquid-, and immunoadsorbent chromatography. Monoclonal antibodies, raised against each dehydrogenase, will be used in enzyme-linked immunoadsorbent assays to determine tissue distribution. Kinetic experiments will be done in vitro with CRBP, CRABP and the purified dehydrogenases to determine whether protein complexes contribute to the biogenesis of retinoic acid.