CHARACTERIZATION OF RETINOIC ACID METABOLISM

视黄酸代谢的表征

• 批准号: 3235423
• 负责人: JOSEPH L NAPOLI
• 金额: $ 16.51万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3235423
• 关键词: affinity chromatography; alcohol dehydrogenase; aldehyde dehydrogenases; all trans retinol; antineoplastics; bioassay; cancer prevention; cell differentiation; chemical structure function; cofactor; embryo /fetus cell /tissue; enzyme linked immunosorbent assay; enzyme mechanism; enzyme substrate; enzyme substrate complex; epithelium; gel electrophoresis; germ cell neoplasms; growth /development; high performance liquid chromatography; isozymes; laboratory mouse; laboratory rat; mass spectrometry; monoclonal antibody; nutrition related tag; ornithine decarboxylase; oxidoreductase inhibitor; radiotracer; retinaldehyde; retinoate; retinoid binding proteins; retinoids; squamous cell carcinoma; stereochemistry; testis; tissue /cell culture; tritium; vitamin A deficiency; vitamin metabolism

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.

维甲酸是一种内源性类维A酸，它直接作用于 支持特定的维生素A依赖过程。长期目标 这个项目的目的是确定维甲酸是否受损 酸的生物发生引起和/或促进了 预防或预防的肿瘤学和皮肤病 因维甲酸治疗而被捕。 眼下的目标是识别和描述维甲酸- 催化维甲酸生物合成的特异性氧化还原酶 来自视黄醇和视网膜的酸。需要检验的假设是：a) 维甲酸是通过在一系列组织中原位生成的 视黄醇特异性氧化还原酶与低Km的相互作用， 低Vmax视网膜脱氢酶；B)维甲酸比率 合成受视黄醇可利用性和 低Km、低Vmax视网膜脱氢酶活性/量； C)类维甲酸的细胞内运输是通过直接转移进行的 从蛋白质到蛋白质--例如细胞内视黄醇结合产生的视黄醇 从视黄醇到视黄醇脱氢酶的蛋白质(CRBP) 脱氢酶转化为视网膜脱氢酶，维甲酸从 视黄酸脱氢酶对细胞维甲酸结合蛋白的作用 (CRABP)。 具体目标是：1)提纯视黄醇和视网膜 来自大鼠胞浆的脱氢酶；2)表征纯的 视黄酸脱氢酶；3)测定组织分布 维甲酸脱氢酶及其在体内的浓度 维甲酸的特异性；4)确定是否转移 视黄醇到维甲酸代谢途径中的维甲酸 通过蛋白质-蛋白质复合体发生。这些酶将会是 通过传统和较新的技术进行提纯，包括 亲和、快速蛋白质液体和免疫吸附剂 层析法。针对每个人产生的单抗 脱氢酶，将用于酶联免疫吸附剂 用于确定组织分布的分析。动力学实验将 用CRBP、CRABP和纯化的 脱氢酶确定蛋白质复合体是否 有助于维甲酸的生物发生。