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.
| Smutzer, G; Bayley, D; Kalinoski, D L et al. (1991) A simplified method for drying agarose gels. Biotechniques 11:468, 470 |
