categoría: feed additives
autores: roura, e., tedo, g., holt, r. and klasing, k.c.
libro/revista:journal of animal science, 81 (e-suppl.2): 46
humans and mice perceive the umami taste via a trans-membrane heterodimeric receptor consisting of t1r1 and t1r3 proteins that are g-protein coupled to intracellular calcium release. to identify the porcine umami taste receptor, vallate papilla tissue samples were obtained from a 6-month-old male pig and total rna was extracted, purified, and reverse transcribed. a porcine expressed sequence tag (est) with high homology to human t1r3 was located in a public domain library (pig ests database from iowa state university). the race pcr technique was used to obtain cdna for adjacent 3′ and 5′ regions of the t1r3 est sequence. candidate pcr products were sequenced and the race process was repeated until the full length 2568 nucleotide sequence was determined. to obtain the pig t1r1 receptor sequence, degenerate pcr primers were designed that covered areas of high homology to the mouse, human, and cat genes. primer sets were found that amplified a portion of the t1r1 and the complete 2535 nucleotide t1r1 sequence was obtained using the race pcr technique. full length products of t1r1 and t1r3 were amplified by pcr, sequenced, and found to have high (>80%) homology to respective genes from other mammals. the open reading frame of t1r1 and t1r3 was recombined into pcdna6.2/ v5-dest gateway vectors for expression in mammalian cells. cho-k1 cells were transfected with both full-length pig taste receptors constructs and a mouse g alpha 15 g protein sub-unit construct. transfected cells were seeded into 96-well-plates, loaded with a calcium detection die cocktail and then exposed to individual amino acids. ligand binding was determined by fluorescence using an elisa plate reader. the porcine umami taste receptor was most sensitive to non-essential amino acids, with glutamic acid and alanine giving strong responses. in general, essential amino acids gave lower responses. these results indicate that the porcine umami receptor is tuned to detect amino acids.