BIOCHEMICAL MECHANISMS OF TASTE FUNCTION

味觉与营养健康的维持有关。 化学感官功能的妥协可能会导致 在营养和较低的生活质量方面做出妥协。作为一种感觉系统， 味觉在感受器水平上的区别在于它使用了许多 传导机制。对这些机制的描述需要 对其成分进行分离、提纯、鉴定和重组。 我们对味觉感受器特异性的大部分洞察都是通过 共同斑点叉尾鱼氨基酸味觉系统的研究I。 斑点。这些受体通过以下方式传递它们与刺激的相互作用 要么打开连续的离子通道，要么启动G蛋白 依赖的第二信使级联。这项拨款详细说明了 将允许提纯足够数量的这些味道 抗体产生受体和(部分)氨基酸序列 决心。由于通过增溶和分馏提纯 技术依赖于功能的维护，现有的检测方法 处于可溶状态的受体将被提纯，而新的受体 发展起来的。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的活性。我们还将利用 利用重组DNA技术分离具有代表性的基因克隆 鲶鱼的味觉受体。将准备具有代表性的cDNA文库 从鲶鱼的味觉组织中分离出聚A+RNA。探查自 从纯化的受体或产生的抗体中获得的微序列数据 将部分提纯的受体蛋白用于叉尾鱼的筛选 可能的受体克隆的味觉文库。函数表达式 在非洲爪哇卵母细胞中的cdna转录本的研究将允许最终的 克隆的鉴定。一旦确定，这些 然后将对克隆的主要序列、组织进行表征 特异性，通过引物分析和基因拷贝数的估计。 最后，这些克隆将被用来转染哺乳动物细胞系 用于味觉受体蛋白的表达。转基因研究将 允许进行绑定实验和电生理学研究 描述这些受体/通道的特性。这是有可能的 味觉刺激门控离子通道受体将是多聚体和 那些与G蛋白偶联的基因将会有亚型。我们的程序是 旨在检测这些复杂性。这项工作将为以后的研究提供依据 以扩大我们对分子实体的了解 对于味觉的初始识别步骤。

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.