Biochemical Studies of Taste Sensation

Large numbers of taste buds are distributed over the body surface of the channel catfish Zctalurus punctatus, with the barbels having an especially high density. L-Alanine, as well as certain other amino acids, are taste stimuli in this animal. Epithelial tissue obtained by gentle scraping of the barbel surface was fractionated by differential centrifugation. A sedimentable fraction (P2) was prepared that was enriched in I,-[BH]alanine binding activity, the plasma membrane marker enzyme 5’-nucleotidase, and the mitochondrial marker succinate cytochrome c reductase, but not the microsomal marker NADH cytochrome c reductase. Binding of I,-[aH]alanine was measured using a Millipore filter method in which correction for nonspecific binding was also determined. Time, temperature, and pH for measuring binding activity were established. At the optimal pH of 7.8, the K, for L-alanine is 4.8 x lOma M. The first order dissociation rate constant at 6’ is 3.8 x lo-’ s-’ and at 24” it is 12.1 x lo-’ s-l. The second order rate constant for association is between lo2 and lOa M-I s-l. Reversibility of the binding interaction was also demonstrated by the rapid displacement of bound L[‘Hlalanine by a large excess of unlabeled L-alanine. That the binding does not represent incorporation into protein was confirmed by the lack of effect of puromycin. The amounts bound of several other chemostimulatory amino acids were also determined. When fraction P2 was stored at -65’, the specific binding activity for L.-[‘Hlalanine declined by about one-half; the remainder of the activity was stable for at least 1 month, suggesting that more than one type of binding site for L-alanine may be present. Storage in glycerol or in various other media did not protect against this loss of activity. Treatment of Fraction P2 with either Triton X-100 (0.1, 0.5, and 2%, v/v) or Lubrol PX (20 mM) completely abolished L[‘Hlalanine binding activity. Following denervation of the barbel, which is known to lead to degeneration of the taste buds, the sedimentable Fraction P2 showed decreased specific binding activity for L[‘Hlalanine after 2, 5, and 10 days of denervation. Furthermore, preparations of fraction P2 from ventral skin, which has a lower density of taste buds than does barbel skin, showed less binding activity than preparations from barbel. These observations indicate that the binding is a physiologically relevant measure of an early event in taste sensation.