BIOCHEMICAL MECHANISMS OF TASTE FUNCTION

味觉与维持营养健康有关。 化学感官功能的妥协可能导致 营养和生活质量的下降。作为一个感觉系统， 味觉在感受器水平上的区别在于它使用了许多 转导机制这些机制的特征需要 它们的成分被分离、纯化、鉴定和重组。 我们对味觉感受器特异性的大部分认识都是通过 对癍点叉尾鮰氨基酸味觉系统的研究，一。 斑点状。这些受体通过以下方式抑制它们与刺激的相互作用： 或者打开相邻的离子通道或者启动G蛋白 依赖性第二信使级联反应。该补助金详细说明了 将允许纯化足够量的这些味道 用于抗体产生的受体和（部分）氨基酸序列 保持战略定力由于通过增溶和分馏进行纯化， 技术依赖于功能的维持，现有的用于 可溶状态的受体将被精炼， 开发重点将放在检测受体结合的分析上 并测量刺激门控离子通道活性。我们还将利用 重组DNA方法分离代表性的cDNA克隆， 鲶鱼的味觉感受器将制备代表性cDNA文库 从鲶鱼的味觉组织中分离的poly A+ RNA。探针从 从纯化的受体或产生的抗体获得的微序列数据 部分纯化的受体蛋白将用于筛选鲶鱼 味觉文库的推定的受体克隆。功能性表达 非洲爪蟾卵母细胞中的cDNA转录本将允许决定性的 cDNA克隆的鉴定。一旦确定，这些 然后将克隆的一级序列、组织 特异性，通过引物分析和用于估计基因拷贝数。 最后，cDNA克隆将被用于转染哺乳动物细胞系 用于表达味觉受体蛋白。转染研究将 允许结合实验和电生理学研究， 描述这些受体/通道的特性。很可能 味觉刺激门控离子通道受体将是多聚体的， 那些与G蛋白结合的蛋白会有亚型。我们的程序是 来检测这些复杂性这项工作将提供基础 扩大了我们对这些分子实体的了解， 在味觉的最初识别阶段。

The sense of taste is involved in maintenance of nutritional well being. Compromises in the functioning of the chemical senses can lead to compromises in nutrition and a lower quality of life. As a sensory system, taste is distinguished at the receptor level by its use of a number of transductive mechanisms. Characterization of these mechanisms requires that their components be isolated, purified, identified and reconstituted. Much of our insight into taste receptor specificity has been gamed through study of the amino acid taste system of the common channel catfish, I. punctatus. These receptors transduce their interaction with stimuli by either opening contiguous ion channels or by initiating a G-protein dependent second messenger cascade. This grant details procedures that will allow the purification of sufficient quantities of these taste receptors for antibody production and (partial) amino acid sequence determination. Since purification through solubilization and fractionation techniques is dependent upon maintenance of function, existing assays for the receptors in the soluble state will be refined, and new ones developed. Emphasis will be placed on assays that detect receptor binding and measure stimulus-gated ion channel activity. We will also utilize a recombinant DNA approach to isolate representative cDNA clones for the taste receptors of catfish. Representative cDNA libraries will be prepared from poly A+ RNA isolated from taste tissue of the catfish. Probes from microsequence data obtained from purified receptor, or antibodies raised to partially purified receptor protein will be used to screen the catfish taste library for putative clones of the receptors. Functional expression of cDNA transcripts in Xenopus oocytes will allow for the definitive identification of the cDNA clones. Once definitively identified, these clones will then be characterized for primary sequence, tissue specificity, by primer analysis and for estimation of gene copy number. Finally, the cDNA clones will be used to transfect a mammalian cell line for expression of the taste receptor proteins. Transfection studies will permit binding experiments and electrophysiological studies to characterize the properties of these receptor/channels. It is probable that the taste stimulus gated ion channel receptors will be multimeric and that those coupled to G-proteins will have subtypes. Our procedures are designed to detect these complexities. This work will provide the basis for expanding our knowledge of the molecular entities that are responsible for the initial recognition step in taste.