REGENERATION OF 11-CIS-RETINAL IN THE RETINA

描述(摘自申请者的摘要)：脊椎动物的视觉周期是 包括参与处理全反式视网膜的生化反应 由11-顺式视网膜在视紫红质中的光异构化产生。视觉效果 周期对视力和视觉适应是必不可少的。这个项目的目标是 是在分子水平上表征视觉的基本成分 并了解循环是如何调节的。其中的两个关键反应 视觉周期是由视网膜色素上皮膜结合所催化的 酶，卵磷脂视黄醇酰基转移酶(LRAT)和异麦芽水解酶。LRAT 将酰基从卵磷脂转移到维生素A以产生全反式 视黄酸酯和全水解酶将酯加工成11-顺式 视黄醇。这两种酶对视觉都是必不可少的。对LRAT的理解 分子水平是这项拨款提案的主要兴趣所在。LRAT有一个独特的 不揭示其所属的机械类的序列。 生化研究，包括使用该小说进行的化学测绘研究 生物素亲和标记技术和定点突变研究 提出了既有地图元素又有LRAT活性部位结构的定义 它的分子作用机制。还提出了化学测绘研究。 开始阐明LRAT在膜上的结构并揭示 RPE膜中的最近邻蛋白质。LRAT相关蛋白之一 可能是异构体水解酶。细菌的鉴定和表征 异麦角水解酶是这一提议的另一个重要方面。实现以下目标的方法 对这种酶系统的鉴定将涉及到对 与LRAT相互作用亲和纯化或交联型异构体水解酶 以及光亲和标记方法来标记该酶。什么时候 鉴定出异构体水解酶，将对其进行锥体测序并在LRAT中表达 转染组HEK细胞。其结构、作用机制及调控 将探索异麦角水解酶与疾病的可能关系。 由酶的突变引起的视觉。

DESCRIPTION (Adapted from applicant's abstract): The vertebrate visual cycle is comprised of biochemical reactions involved in processing all-trans retinal produced by the photoisomerisation of 11-cis retinal in rhodopsin. The visual cycle is essential for vision and visual adaptation. The goal of this project is to characterize on a molecular level essential components of the visual cycle and to learn how the cycle is regulated. Two of the key reactions in the visual cycle are catalyzed by the retinal pigment epithelium membrane bound enzymes, lecithin retinol acyl transferase (LRAT)and the isomerohydrolase. LRAT transfers an acyl group from lecithin to vitamin A to generate all-trans retinyl esters and the omerohydrolase processes the esters to produce 11-cis retinol. Both enzymes are essential for vision. An understanding of LRAT at the molecular level is of major interest in this grant proposal. LRAT has a unique sequence which does not reveal the mechanistic class to which it belongs. Biochemical studies including chemical mapping studies using the novel technique of biotin affinity labeling and site-specific mutagenic studies are proposed to both map elements of the active-site structure of LRAT and define its molecular mechanism of action. Chemical mapping studies are also proposed to begin to elucidate the structure of LRAT in the membrane and to reveal nearest neighbor proteins in RPE membranes. One of the LRAT associated proteins may be the isomerohydrolase. Identification and characterization of isomerohydrolase is another important aspect of this proposal. Approaches to the identification of this enzyme system will involve both exploiting interactions with LRAT to either affinity purify or cross-link isomerohydrolase and photoaffinity labeling approaches to label the enzyme. When isomerohydrolase is identified it will be coned sequenced and expressed in LRAT transfected HEK cells. The structure, mechanism of action and regulation of the isomerohydrolase will be explored as will possible relationships to diseases of vision caused by mutations in the enzyme.