Biochemical studies of taste sensation. XI. Isolation, characterization and taste ligand binding activity of plasma membranes from catfish taste tissue

Variants of creatine kinase-MM (variant of ATP:creatine N-phosphotransferase, EC 2.7.3.2), present in human heart and skeletal muscle, have been purified to homogeneity using DEAE-Sepharose column chromatography and column chromatofocusing techniques. Creatine kinase-MM I-IV were present in both heart and skeletal muscle, while MM-V was found only in heart. The number, ratio and elution profile of the variants during chromatofocusing remained identical even when they were purified in the presence of proteinase inhibitors. MM-I-V, on chromatofocusing, were eluted at pH 8.3, 7.9, 7.6, 7.2 and 6.8, respectively. Isoelectric focusing revealed the pI of MM-I-V to be 7.2, 6.9, 6.7, 6.4 and 6.2. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a doublet pattern for creatine kinase-MM variants III-V. However, polyacrylamide gel electrophoresis without SDS indicated homogeneity because each variant showed a single band. The doublet pattern observed in the presence of SDS may reflect the presence of two subunits of slightly different mass.     

Physicochemical studies of taste reception. III. Interpretation of the water response in taste reception

The model membrane composed of a Millipore filter paper and the total lipids from bovine tongue epithelium or phosphatidylcholine from egg yolk simulated well the water response of a living taste cell. The water response observed with the model membrane adapted to various salt solutions was interpreted in terms of changes in electric potential at the membrane-solution interface, i.e. the water response was attributed to the e.m.f. change produced by diffusion of the electrolytes dissolved in (or adsorbed on) the membrane surface into the bulk solution.The water response of the frog tongue was also investigated by measuring the neural response of the glossopharyngeal nerve. The results obtained were consistent with the mechanism proposed in the present paper. The response of the frog to Ca²⁺ was examined under the condition where the water response was suppressed, and it was concluded that the water response of the frog is different from the response to Ca²⁺.     

Biochemical studies of taste sensation. VII. Enhancement of taste stimulus binding to a catfish taste receptor preparation by prior exposure to the stimulus.

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.     

Biochemical studies of taste sensation--XII. Specificity of binding of taste ligands to a sedimentable fraction from catfish taste tissue

The specificity of amino acid binding sites in a sedimentable fraction prepared from catfish taste epithelium was examined. Using seven 3H-labeled amino acids as ligands and the unlabeled amino acids in binding competition assays, the presence of possibly three classes of amino acid binding sites was deduced. Site 1 binds L-THR, L-SER, L-ALA and possibly D-ALA and beta-ALA, Site 2 binds L-SER, L-ALA, GLY, D-ALA, and beta-ALA and Site 3 binds L-ARG and L-LYS. Additional evidence supporting the specificity of Site 2 was obtained from the specificity of enhancement of L-ALA binding. The results demonstrate the presence of some major classes of taste receptor sites, and provide a basis for understanding taste receptor specificity at the biochemical level.     

Location of taste buds in intact taste papillae by a selective staining method.

Taste buds were found to stain strongly and selectively in intact papillae with highly acidic dyes such as ponceau S. In intact tongues the taste buds in the fungiform, circumvallate and foliate papillae of the cynomolgus monkey and in the fungiform papillae of the rat as well as the taste discs in the fungiform papillae of the frog could be visualized. This method enables a rapid location and counting of taste buds in taste papillae without preparing histological sections. In cynomolgus tongue material fixed in formalin, the dyes penetrate into the buds. In fresh tongues only the taste pore region of the buds stains, which suggests that in vivo taste buds are impenetrable underneath the pore.     

Biochemical studies of taste sensation. VII. Enhancement of taste stimulus binding to a catfish taste receptor preparation by prior exposure to the stimulus

The technical assistance of Ardithanne Boyle, Linda Graham and Pamela Burke is appreciated. I thank Dr. H. Ronald Kaback and Dr. Leonard D. Kohn for their suggestions of experiments relevant to transport questions. This research was supported in part by NIH research grant No. NS-08775 from NINCDS. 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 10mM L-alanine and removal of the L-alanine by washing. Enhancement occurred whether the preparation was stored frozen (?65°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 K_D 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 ^?2M) 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 expiain 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 ahigh ligand concenration.     

Bitter taste of saccharin and acesulfame-K.

The relationships among suprathreshold taste responses to acesulfame-K, Na-saccharin and 6-n-propylthiouracil (PROP) were examined in two studies. In the first study, the labeled magnitude scale was used with the high anchor labeled as 'strongest imaginable oral sensation' and in the second study, it was labeled as 'strongest imaginable sensation of any kind'. Results from the two procedures were similar. Individual differences among 65 subjects were seen in bitter responses to acesulfame-K and saccharin. Bitter responses to acesulfame-K ands accharin were positively correlated, but showed no significant relationship with responses to PROP bitterness or with PROP taster groups. Saccharin and acesulfame-K may share a common mechanism for bitter taste reception and transduction, one that varies across individuals and is different from mechanisms mediating bitter responses to PROP. Changing the instructions of the labeled magnitude scale induced a context effect. Ratings of sweetness referenced to the 'strongest imaginable sensationof any kind' were lower than ratings referenced to just oral sensations.     

Biochemical studies of taste sensation XI. Isolation,characterization and taste ligand binding activity of plasma membranes from catfish taste tissue

Plasma membranes were isolated from taste receptor-containing epithelium of the channel catfish, Ictalurus punctatus. The membranes were prepared by ultracentrifugation of a sedimentable fraction in sucrose, using either a discontinous density gradient or a continous linear density gradient. The plasma membranes were characterized by their increased content of 5′-nucleotidase and by electron microscopy, as well as by a greatly diminished content of NADH-cytochrome c reductase and succinate-cytochrome c reductase. The recovery of binding activity for taste ligands was low, because of the long time-period required for ultracentrifugation, but of the recovered activity 80% occurred in the plasma-membrane preparation. Binding of seven chemostimulatory amino acids was demonstrated and found to correspond reasonably well with earlier binding data obtained using a less pure sedimentable fraction. The data provide direct evidence supporting the long-standing hypothesis that taste receptor sites are localized to the plasma membranes.