CHARACTERIZATION OF RETINOIC ACID METABOLISM

视黄酸是一种内源性类维生素A， 支持特定的维生素A依赖过程。 一个长期目标 这个项目的目的是确定是否损害维甲酸 酸生物成因导致和/或有助于 预防的肿瘤和皮肤病，或 被类维生素A治疗阻止了 当前的目标是识别和表征类维生素A- 催化视黄酸生物合成的特异性氧化还原酶 从视黄醇和视网膜酸。 待检验的假设为：A） 视黄酸是在一系列组织中原位产生的， 视黄醇特异性氧化还原酶和低Km的相互作用， 低Vmax视黄酸脱氢酶; B）视黄酸速率 维生素A的合成受视黄醇的可用性控制， 低Km、低Vmax的视黄醇脱氢酶的活性/量; C）类维生素A的细胞内转运通过直接转移发生 从蛋白质到蛋白质--例如从细胞视黄醇结合 蛋白质（CRBP）转化为视黄醇脱氢酶，视黄醇转化为视黄醇 脱氢酶转化为视黄酸脱氢酶， 视黄酸脱氢酶-细胞视黄酸结合蛋白 （CRABP）。 具体目的是：1）纯化视黄醇和视黄醇 从大鼠测试胞质溶胶中分离的酶; 2）表征纯的 类维生素A分解酶; 3）确定 类视色素氧化酶，它们在体内的浓度和它们的 对类维生素A的特异性; 4）确定是否转移 从视黄醇到视黄酸代谢途径中的类维生素A 通过蛋白质-蛋白质复合物发生。 这些酶将 通过传统和更新的技术进行纯化，包括 亲和、快速蛋白液体和免疫吸附剂 层析 单克隆抗体，针对每种 脱氢酶，将用于酶联免疫吸附剂 测定以确定组织分布。 动力学实验将 在体外用CRBP、CRABP和纯化的 酶来确定蛋白质复合物 有助于视黄酸的生物合成。

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.