REGENERATION OF 11-CIS RETINAL IN THE RETINA

这里描述的研究涉及到阐明分子和 视紫红质再生的生理基础。具体地说，这些研究 都专注于了解11-顺式视黄醇， 视觉发色团的最终前体是在 脊椎动物的色素上皮。我们已经证明了细胞的膜 色素上皮含有一种新的生物合成酶系统，能够 将游离的全反式视黄醇转化为其11顺式同系物。这 热力学上坡过程的能量来源于磷脂。 在一种新的工艺中，该工艺利用了 酯键的水解以驱动异构化。这一机制 Employee涉及全反式视黄醇与 磷脂直接酶法制备视黄酯 至11-顺式视黄醇。我们已经溶解并部分提纯了这些酶 牵涉其中。 在这项拨款申请中，我们建议完全提纯这种酶 这种异构化过程的成分，即视黄酸酯 合成酶和异构酶。我们建议对其作用机制进行研究。 这些酶，并制备它们的基于机理的酶抑制剂 用来研究它们的生理作用。此外，单克隆和 将制备针对这些酶的单特异性多克隆抗体。 这些抗体将被用来确定稳态水平， 流动率，以及可能的翻译后修改 酵素。酶可能被调节的机制以及 酶与膜相互作用的机制是 对此格外感兴趣。上的序列信息的可用性 酶和酶的特异性抗体的可用性应该 使我们能够克隆它们并对它们进行全序列测定。此信息是 对于确定这些酶与什么相关，它们的 主要的结构基序可能是，以及酶如何与 薄膜。序列信息也将在临床上用于 确定这些酶是不是 脊椎动物的视力，在主要的视力疾病中是改变的。

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.