MOLECULAR MECHANISMS IN VISUAL TRANSDUCTION

视网膜和视网膜中维生素A的通量和代谢的调节 视网膜色素上皮尚未被很好地了解，它是 这项拨款申请。这个问题的核心是对 眼睛中处理维生素A的某些酶和结合蛋白，以及 这可能是监管的要点。在这项拨款申请中，我们建议 通过化学和生化手段研究视黄酸酯酶、合成酶、 视紫红质和某些视黄醇(Al)结合蛋白。两种酶 将被提纯到均一，并确定它们的作用机制。 为了研究这些机制，建立了一种新的定量检测系统 这些酶将与维甲酸的转移有关。 从小的单层囊泡(SUV)到蛋白质。这个化验结果会 允许精确计算类维甲酸浓度以及 为他们提供一个已知和稳定的环境，从而允许 热力学和动力学常数的有意义的计算。这个化验 系统通常对于涉及疏水的结合研究是有用的 配基。有了手头的酶的定量分析，我们将 确定它们在白化大鼠和青蛙体内是否受光调节。 高度特异的、不可逆转的、基于机理的抑制剂将是 为这些酶合成的，它们将在体内进行测试，以确定 它们的生理作用。 上述相同的检测系统将用于定量 测定视黄醇结合量S确定视黄醇结合量 蛋白质并确定这些蛋白质的活性是否受调控 用光。这些蛋白质的特定不可逆失活剂也会 做好准备并在体内进行研究。最后，以新的具体机制为基础 视蛋白的灭活剂已经准备好了。这些将主要用于 研究视紫红质活性是如何受光调节的。定量研究 缓蚀剂(S)-视蛋白络合物的可见吸收光谱 也将有兴趣确定视蛋白的机制(S) 扰动结合生色团的吸收光谱。

The regulation of the fluxes and metabolism of vitamin A in the retina and retinal pigment epithelium is not yet well understood and is the subject of this grant request. Central to this problem are quantitative studies on certain enzymes and binding proteins which process vitamin A in the eye and which could be points of regulation. In this grant request we propose to study by chemical and biochemical means retinal ester esterase, synthetase, rhodopsin and certain of the retinol(al) binding proteins. The two enzymes will be purified to homogeniety and their mechanisms of action determined. In order to study these mechanisms a new quantitative assay system for these enzymes will be developed involving the transfer of the retinoids from small unilamellar vesicles (SUV's) to the protein. This assay will allow for the precise calculation of retinoid concentrations as well as providing a known and stable environment for them which allows for a meaningful calculation of thermodynamic and kinetic constants. This assay system should be generally useful for binding studies involving hydrophobic ligands. Having a quantitative assay for the enzymes in hand, we will determine whether they are regulated by light in the albino rat and frog. Highly specific, irreversible, mechanism-based inhibitors will be synthesized for these enzymes and they will be tested in vivo to determine their physiological roles. The same assay system mentioned above will be used to quantitatively measure the binding of the retinol(al)s to ascertain the retinol binding proteins and determine whether the activity of these proteins is regulated by light. Specific irreversible inactivators of these proteins will also be prepared and studied in vivo. Finally, new specific mechanism-based inactivators for opsin have been prepared. These will be primarily used to study how rhodopsins activity is regulated by light. Quantitative studies on the visible absorption spectra of these inhibitor(s) - opsin complexes will also be of interest in determining the mechanism(s) by which opsin perturbs the absorption spectra of bound chromophores.