REGENERATION OF 11-CIS RETINAL IN THE RETINA

视网膜中 11-CIS 视网膜的再生

基本信息

批准号：
2888124
负责人：
ROBERT R RANDO
金额：
$ 45.89万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
1982
资助国家：
美国
起止时间：
1982-04-01 至 2000-08-31
项目状态：
已结题

项目摘要

The studies described here are concerned with elucidating the molecular and physiological basis of rhodopsin regeneration. Specifically, the studies are focused on understanding the process by which 11-cis-retinol, the ultimate precursor of the visual chromophore, is synthesized in the vertebrate pigment epithelium. We have shown that the membranes of the pigment epithelium contain a novel biosynthetic enzyme system capable of transforming free all-trans-retinol into its 11-cis congener. This thermodynamically uphill process derives its energy from the phospholipids of the membranes in a novel process which utilizes the free energy of hydrolysis of the ester bonds to drive the isomerization. The mechanism employed involves the transesterification of all-trans-retinol with a phospholipid followed by enzymatic processing of the retinyl ester directly to 11-cis-retinol. We have solubilized and partially purified the enzymes involved. In this grant request, we propose to completely purify the enzyme components of this isomerization process, namely, the retinyl ester synthetase and isomerase. We propose to study the mechanisms of action of these enzymes and prepare mechanism-based enzyme inhibitors of them to be used to study their physiological roles. Furthermore, monoclonal and monospecific polyclonal antibodies will be prepared against the enzymes. These antibodies will be used to determine the steady state levels, the turnover rates, and the possible post-translational modification of the enzymes. The mechanisms by which the enzymes might be regulated as well as the mechanism by which the enzymes interact with the membrane are of exceptional interest here. The availability of sequence information on the enzymes and the availability of specific antibodies to the enzymes should enable us to clone them and fully sequence them. This information is important for determining what these enzymes are related to, what their major structural motifs might be, and how the enzymes interact with the membrane. Sequence information will also be of interest clinically for determining whether or not these enzymes, which are the sine qua non of vertebrate vision, are altered in major diseases of vision.

此处描述的研究涉及阐明分子和视紫红质再生的生理基础。具体而言，研究专注于理解11-CIS-返回醇的过程视觉发色团的最终前体是合成的脊椎动物色素上皮。我们已经证明了颜料上皮包含一种能够的新型生物合成酶系统将免费的全反替诺醇转变为11木cis同类物。这热力学上山过程从磷脂中得出其能量在一个新型过程中使用的膜，该过程利用了酯键的水解以驱动异构化。机制使用的涉及将全反替诺醇的静止磷脂，然后直接酶促加工视网膜酯到11-CIS-RETINOL。我们已经溶解并部分纯化了酶涉及。在此赠款请求中，我们建议完全净化酶该异构化过程的组成部分，即视乙酯合成酶和异构酶。我们建议研究这些酶并准备基于机制的酶抑制剂用于研究其生理角色。此外，单克隆和将针对酶制备单特异性多克隆抗体。这些抗体将用于确定稳态水平，周转率以及可能的翻译后修改酶。可以调节酶的机制以及酶与膜相互作用的机制是这里的兴趣尤其。序列信息的可用性酶和对酶的特定抗体的可用性应使我们能够克隆它们并完全对它们进行排序。此信息是确定这些酶与什么相关的重要可能是主要的结构基序，以及酶如何与膜。序列信息也将在临床上引起人们的关注确定这些酶是否是正弦的非正弦酶脊椎动物的视力在主要视力疾病中改变了。