BIOCHEMICAL MECHANISMS OF TASTE FUNCTION

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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.