BIOCHEMICAL MECHANISMS OF TASTE FUNCTION

味觉功能的生化机制

• 批准号: 3564488
• 负责人: JOSEPH G BRAND
• 金额: $ 14.53万
• 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/3564488
• 关键词: affinity chromatography; alanine; arginine; binding proteins; catfish; chemical binding; chemoreceptors; density gradient ultracentrifugation; fresh water environment; gel electrophoresis; immunochemistry; laboratory mouse; membrane proteins; monoclonal antibody; receptor; 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 味觉刺激的识别。 斑点叉尾鮰（Ictalurus punctatus）， 具有广泛皮肤味觉系统的动物被用作 实验模型。 这种动物在行为和 电生理学上以低浓度氨基酸作为味道 刺激。 使用生化测定，味觉刺激氨基酸已被 显示与味道的质膜部分特异性结合 来自该动物的上皮。 电生理学研究加上生化 我们实验室的结果定义了非重叠和 重叠的受体类别。 我们实验室最近的工作表明 至少有四个这样的受体类别，并且每个类别的特异性都有 被调查。 刺激的敏感性和特异性 氨基酸与质膜结合使得该制剂尽可能接近 研究味觉生物化学的理想实验模型 可用的。 目的是溶解血浆的受体蛋白 从味觉上皮细胞膜上分离出这些受体结合蛋白， 并表征它们的结合特异性和大分子 参数。 将使用允许的去垢剂进行溶解 保留受体的识别（特异性）能力 蛋白质。 可溶态受体蛋白的结合测定 将被开发。 结合蛋白将通过亲和力分离 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 对抗味道质膜结合蛋白。 这些单克隆 抗体在我们的实验室制备并测试特异性结合 对代表氨基酸的抑制活性 特定的受体类别。 分离后，结合蛋白 将通过色谱法、蔗糖密度离心法和 蛋白质印迹电泳测定分子量，Stokes 半径、亚基特征和针对活性单克隆抗体的特异性 抗体。 将确定每种分离蛋白质的结合动力学 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 味觉识别中的结合步骤。