CHARACTERIZATION OF RETINOIC ACID METABOLISM

视黄酸代谢的表征

• 批准号: 3235416
• 负责人: JOSEPH L NAPOLI
• 金额: $ 15.51万
• 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/3235416
• 关键词: affinity chromatography; alcohol dehydrogenase; aldehyde dehydrogenases; all trans retinol; antineoplastics; binding proteins; 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; 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.

视黄酸是内源性类视黄醇，直接作用于 支持特定的维生素 A 依赖性过程。 长期目标 该项目的目的是确定视黄酸是否受损 酸生物发生导致和/或促进发展 可以预防或预防的肿瘤和皮肤病 通过类维生素A治疗而停止。 近期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 来自视黄醇和视黄醛的酸。 要检验的假设是：A) 视黄酸是通过以下方式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， Vmax 视网膜脱氢酶低； B) 视黄酸率 合成由视黄醇的可用性和 低Km、低Vmax视网膜脱氢酶的活性/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 来自细胞视黄醇结合的视黄醇 蛋白质 (CRBP) 转化为视黄醇二氢酶，视黄醇转化为视黄醛 脱氢酶 至 视网膜脱氢酶，视黄酸 视网膜脱氢酶与细胞视黄酸结合蛋白 （螃蟹BP）。 具体目的是：1）纯化视黄醇和视黄醛 来自大鼠测试细胞质的脱氢酶； 2）表征纯 类维生素A脱氢酶； 3）确定组织分布 类视黄醇脱氢酶、它们的体内浓度及其 类维生素A的特异性； 4）判断是否转移 从视黄醇到视黄酸的代谢途径中的类视黄醇 通过蛋白质-蛋白质复合物发生。 酶将是 通过传统和新技术纯化，包括 亲和、快速蛋白质液体和免疫吸附剂 色谱法。 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 可以在体外用 CRBP、CRABP 和纯化的 脱氢酶以确定蛋白质是否复合 有助于视黄酸的生物发生。