STUDIES ON RETINOID-BINDING PROTEINS

由于其疏水性质，类维生素A与 体内蛋白质 这些蛋白质参与运输的 维生素从肝脏中的储存部位转移到需要维生素A的 外周组织 此外，细胞对蛋白质的摄取- 相关维生素与类维生素A的细胞内转运 是由蛋白质促成的。 目前的建议旨在 阐明了维生素E转运的各个步骤 A到其最终作用部位。 维生素A是从肝脏动员的视黄醇结合 蛋白（RBP）。 将尝试开发一种体外 一个系统，允许详细检查视黄醇是如何 通过内质网膜转移 并被新生的RBP收购。 此外，稳定转染 生产RBP的细胞系，在正常和维生素A- 缺乏培养基，将用于解开为什么视黄醇- 缺陷型RBP分子不离开内质网。 基于已知的RBP的三维结构， 前白蛋白，将尝试绘制相互作用的位点 在两种蛋白质之间连续使用抗肽 抗体、定点突变和计算机图形学 建模 与细胞表面受体相互作用的RBP位点 将通过类似的方法进行识别。 大多数细胞含有两种细胞内类维生素A结合蛋白， CRBP和CRABP。 将尝试获得cDNA克隆 并阐明了CRABP基因的结构。 的 CRBP在维生素A细胞间转运中的可能作用 在肝脏的脂肪储存细胞和肝细胞之间， 走近。 CRBP在分离的脂肪储存细胞中的表达将被检测。 检查和CRBP和RBP mRNA的分布， 正常和维生素A缺乏的大鼠组织将使用 原位杂交技术。 CRBP和CRABP将它们的配体递送到 然而，尚未发现的细胞内结构将被检查， 探索抗独特型抗体的应用。 这种方法将 通过尝试设计体外系统来补充， 允许类维生素A从细胞内结合蛋白转移 到亚细胞结构。

Due to their hydrophobic nature retinoids are associated with proteins in vivo. Such proteins are engaged in the transport of the vitamin from its storage site in the liver to vitamin A-requiring peripheral tissues. Moreover, the uptake by cells of the protein- associated vitamin and the intracellular transport of the retinoids are facilitated by proteins. The current proposal is aimed at elucidating the discrete steps involved in the transport of vitamin A to its ultimate site(s) of action. Vitamin A is mobilized from the liver by the retinol-binding protein (RBP). Attempts will be made to develop an in vitro system that allows detailed examination of how retinol is transferred across the membrane of the endoplasmic reticulum and is acquired by the nascent RBP. Moreover, stably transfected cell lines manufacturing RBP, grown in normal and vitamin A- deficient culture media, will be employed to unravel why retinol- deficient RBP molecules do not exit the endoplasmic reticulum. Based on the known three-dimensional structures of RBP and prealbumin, attempts will be made to map the sites of interaction between the two proteins using, in succession, antipeptide antibodies, site-directed mutagenesis and computer graphics modeling. The RBP-site interacting with a cell surface receptor will be identified by a similar approach. Most cells contain two intracellular-retinoid-binding proteins, CRBP and CRABP. Attempts will be made to obtain cDNA clones for CRABP and elucidate the structure of the CRABP gene. The possible role of CRBP in the intercellular transfer of vitamin A between fat storing cells of the liver and hepatocytes will be approached. CRBP-expression in isolated fat storing cells will be examined and the distribution of CRBP and RBP mRNAs in normal and vitamin A-deficient rat tissues will be studied using in situ hybridization techniques. The possibility the CRBP and CRABP deliver their ligands to as yet undiscovered intracellular structures will be examined by exploring the use of anti-idiotypic antibodies. This approach will be complimented by attempts to devise in vitro systems that allow transfer of retinoids from the intracellular binding proteins to subcellular structures.