BIOCHEMICAL MECHANISMS OF TASTE FUNCTION

味觉功能的生化机制

• 批准号: 3407341
• 负责人: JOSEPH G BRAND
• 金额: $ 12.58万
• 依托单位: MONELL CHEMICAL SENSES CENTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-01 至 1989-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3407341
• 关键词: affinity chromatography; alanine; arginine; chemical binding; chemoreceptors; density gradient ultracentrifugation; fresh water environment; gel electrophoresis; immunochemistry; monoclonal antibody; solubility; stereoisomer; taste

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.

这项工作的总体目标是了解生化基础 识别口味刺激。 通道cat鱼（Ictalurus punctatus）， 具有广泛皮肤味道系统的动物被用作 实验模型。 这只动物在行为和 从电生理到低浓度的氨基酸作为味道 刺激。 使用生化测定，味道刺激氨基酸已经 显示与质膜的质膜分数特别结合 该动物的上皮。 电生理研究以及生化 我们实验室的结果已定义了非重叠和 重叠的受体类。 我们实验室的最新工作建议 至少四个这样的受体类，每个类的特异性具有 被调查。 刺激的灵敏度和特异性 氨基酸与质膜结合，使得这种制剂与接近 一个理想的实验模型，用于研究味道的生物化学 可用的。 目的是溶解血浆的受体蛋白 来自味道上皮的膜，分离这些受体结合蛋白， 并将其描述为它们的结合特异性和大分子 参数。 溶解度将使用允许的洗涤剂进行 保留受体的识别（特异性）能力 蛋白质。 可溶性受体蛋白的结合测定 将开发。 结合蛋白将通过亲和力隔离 色谱法，使用氨基酸作为亲和力配体，并通过 使用单克隆抗体开发的免疫亲和力色谱法 针对质量质膜结合蛋白。 这些单克隆 抗体是在我们的实验室中制备的，并测试了特定的结合 对代表的氨基酸的抑制活性 特定的受体类。 分离后，结合蛋白 以色谱法，蔗糖密度离心和 带有蛋白质印迹的电泳，以确定分子量，斯托克斯 半径，亚基特性和针对活性单克隆的特异性 抗体。 将确定每种分离蛋白的结合动力学 定义每种蛋白质对刺激氨基酸的特异性。 这 研究将允许对初始的详细分子描述 味觉识别的绑定步骤。