BIOCHEMICAL MECHANISMS OF TASTE FUNCTION

这项工作的总体目标是了解生物化学基础， 识别味觉刺激。 斑点叉尾鱼（Ictalurus punctatus）， 一种具有广泛的皮肤味觉系统的动物，被用作 实验模型 这种动物的行为和 在电生理学上，低浓度的氨基酸作为味觉 刺激。 使用生物化学测定，味觉刺激氨基酸已经被 显示其特异性结合于味觉的质膜部分 这只动物的上皮细胞 电生理学研究加生化 我们实验室的结果已经定义了非重叠和 重叠的受体类别。 我们实验室最近的工作表明， 至少有四种这样的受体类别，并且每种类别的特异性具有 被调查了 刺激的敏感性和特异性 氨基酸与质膜的结合使得这种制备接近于 是研究味觉生物化学的理想实验模型， available. 目的是溶解血浆中的受体蛋白 从味觉上皮细胞的膜，分离这些受体结合蛋白， 并表征它们的结合特异性和大分子 参数 将使用清洁剂进行溶解， 保持受体的识别（特异性）能力 proteins. 可溶状态下受体蛋白的结合试验 将被开发。 结合蛋白将通过亲和层析分离。 色谱法，使用氨基酸作为亲和配体，并通过 免疫亲和层析，使用开发的单克隆抗体 对抗味觉质膜结合蛋白。 这些单克隆 抗体在我们的实验室制备，并测试其特异性结合 对代表以下氨基酸的氨基酸的抑制活性 特定的受体类别。 分离后，结合蛋白 将通过色谱法、蔗糖密度离心和 蛋白质印迹电泳测定分子量，斯托克斯 半径、亚基特征和对活性单克隆抗体的特异性 抗体的 将测定每种分离蛋白的结合动力学 以确定每种蛋白质对刺激氨基酸的特异性。 的 研究将允许详细的分子描述的初始 味觉识别中的结合步骤。

The overall goal of this work is to understand the biochemical basis for recognition of taste stimuli. The channel catfish (Ictalurus punctatus), an animal that has an extensive cutaneous taste system, is used as the experimental model. This animal responds both behaviorally and electrophysiologically to low concentrations of amino acids as taste stimuli. Using a biochemical assay, taste stimulus amino acids have been shown to bind specifically to the plasma membrane fraction of taste epithelium from this animal. Electrophysiological studies plus biochemical results from our laboratory have defined both non-overlapping and overlapping receptor classes. Recent work from our laboratory suggests at least four such receptor classes, and the specificity of each class has been investigated. The sensitivity and specificity with which stimulus amino acids bind to the plasma membranes makes this preparation as close to an ideal experimental model for studying the biochemistry of taste as is available. The aims are to solubilize the receptor proteins of the plasma membranes from taste epithelium, isolate these receptor binding proteins, and characterize them as to their binding specificity and macromolecular parameters. Solubilization will be performed with detergents that allow retention of the recognition (specificity) ability of the receptor proteins. Binding assays for the receptor proteins in the soluble state will be developed. The binding proteins will be isolated via affinity chromatography, using amino acids as affinity ligands, and via immunoaffinity chromatography, using monoclonal antibodies developed against the taste plasma membrane binding proteins. These monoclonal antibodies are prepared in our laboratory and tested for specific binding inhibitory activity against amino acids that are representatives of particular receptor classes. Following isolation, the binding proteins will be characterized by chromatography, sucrose density centrifugation and electrophoresis with Western blots to determine molecular weight, Stokes radius, subunit characteristics and specificity against active monoclonal antibodies. Binding kinetics will be determined for each isolated protein to define the specificity of each protein for stimulus amino acids. The research will permit a detailed molecular description of the initial binding step in taste recognition.