| Abstract: | The taste receptor membrane fraction (Fraction P2) was prepared from a homogenate of the taste tissue of the channel catfish Ictalurus punctatus. This included the rostral, dorsal, and dorsolateral surfaces of the catfish in addition to those of the barbels. The yield of Fraction P2 is 4-7 mg protein from an individual fish, with a purification averaging 8- to 15-fold over that of the crude whole homogenate and essentially quantitative recovery of binding activity in Fraction P2. Treatment of Fraction P2 in vitro with a high concentration of the taste stimulus molecule L-alanine led to a several-fold enhancement of binding activity. Enhancement of the binding of 3H L-alanine was observed after treatment with unlabeled 10 mM L-alanine and removal of the L-alanine by washing. Enhancement occurred whether the preparation was stored frozen (-65 degrees C) for an extended period in the presence of the L-alanine, or merely exposed to it in the cold without freezing. D-Alanine enhanced the binding activity of 3H L-alanine to about 60% of the level induced by L-alanine. Nonspecific binding of 3H L-alanine was unaffected by the treatment. Scatchard analyses of saturation curves for binding of 3H L-alanine to freshly prepared Fraction P2 and to L-alanine-treated Fraction P2 revealed no change in the KD value, but a several-fold increase occurred in the amount bound. Binding activity is operationally defined. Because the enhancement observed here is reminiscent of an increase in transport due to a countertransport effect, further studies were carried out to examine whether the phenomenon reflects transport or true binding. The measured binding was not increased in the presence of Na+, indicating that it is not due to an Na+-coupled transport of L-alanine. When Fraction P2 was preloaded with L-alanine (10(-6)--10(-2) M) prior to assay, no stimulation of binding was observed; instead, binding decreased. This result is consistent with a true binding phenomenon but not with a carrier-mediated transport process to explain the enhancement phenomenon. Binding assays carried out over a range of osmolarities revealed decreased binding at high osmotic strengths, suggesting that a significant portion of the ligand might be contained in vesicles. It is postulated that "hidden" or "buried" receptor sites exist in the Fraction P2 as isolated, and that these are exposed upon perturbation of the membrane structure by a high ligand concentration. |
