Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.
The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding. (+info)
On the sweetness of N-(trifluoroacetyl)aspartame.
A panel of tasters has found that the N-trifluoroacetyl derivative of aspartame is five times less sweet than the parent compound, contrary to the tenet in the literature, but consistent with sweet receptor models which require this nitrogen to exist in protonated form. (+info)
Genetic taste responses to 6-n-propylthiouracil among adults: a screening tool for epidemiological studies.
Genetically mediated taste responsiveness to 6-n-propylthiouracil (PROP) has been linked to reduced acceptance of some bitter foods. In this community-based study male (n = 364) and female (n = 378) adults enrolled in a self-help dietary intervention trial were screened for PROP taster status. Respondents, aged 18--70 years, were mailed filter papers impregnated with PROP or with aspartame solutions. They received instructions to rate taste intensity and hedonic preference using nine point category scales. Women rated PROP as more bitter than did men. Both sweetness and bitterness ratings were lower for older adults. Taste responsiveness to PROP was unrelated to body mass index in women or men. Higher bitterness ratings for PROP were weakly associated with higher sweetness ratings for aspartame, but were unrelated to sweet taste preferences. Successful administration of PROP filter papers by mail suggests new avenues for the screening of taste phenotypes in epidemiological studies. (+info)
Whole nerve chorda tympani responses to sweeteners in C57BL/6ByJ and 129P3/J mice.
The C57BL/6ByJ (B6) strain of mice exhibits higher preferences than does the 129P3/J (129) strain for a variety of sweet tasting compounds. We measured gustatory afferent responses of the whole chorda tympani nerve in these two strains using a broad array of sweeteners and other taste stimuli. Neural responses were greater in B6 than in 129 mice to the sugars sucrose and maltose, the polyol D-sorbitol and the non-caloric sweeteners Na saccharin, acesulfame-K, SC-45647 and sucralose. Lower neural response thresholds were also observed in the B6 strain for most of these stimuli. The strains did not differ in their neural responses to amino acids that are thought to taste sweet to mice, with the exception of L-proline, which evoked larger responses in the B6 strain. Aspartame and thaumatin, which taste sweet to humans but are not strongly preferred by B6 or 129 mice, did not evoke neural responses that exceeded threshold in either strain. The strains generally did not differ in their neural responses to NaCl, quinine and HCl. Thus, variation between the B6 and 129 strains in the peripheral gustatory system may contribute to differences in their consumption of many sweeteners. (+info)
Application of the U and gamma' models in binary sweet taste mixtures.
The U and Gamma' models of sensory interactions, successfully applied in olfaction for several years, are tested here using data from published studies on sweetness. The models are subsequently tested on new data obtained in studies of binary mixtures of four sodium sulfamates. The U model allows for the estimation of a global interaction, whereas the Gamma' model allows for the distinction between that which is due to an intrinsic interaction in the mixture itself and that which may be due to the power function exponents in the mixture. The models give satisfactory predictions for observed phenomena of sweet taste suppression, synergism or pure additivity. Additionally, they appear to be more suitable than other models recently applied in taste, particularly the equiratio model. Application of the models to the sulfamate mixtures, reveals additivity for sodium cyclohexylsulfamate (cyclamate)/potassium cyclohexylsulfamate and sodium cyclohexylsulfamate/sodium exo-2-norbornylsulfamate, respectively; whereas for sodium cyclohexylsulfamate/sodium 3-bromophenylsulfamate, the models revealed a slight hypo addition which is simply due to the dissimilarity values of the power function exponents of the components. (+info)
The effect of various substances on the suppression of the bitterness of quinine-human gustatory sensation, binding, and taste sensor studies.
The purpose of this study was to quantify the degree of suppression of the perceived bitterness of quinine by various substances and to examine the mechanism of bitterness suppression. The following compounds were tested for their ability to suppress bitterness: sucrose, a natural sweetener; aspartame, a noncaloric sweetener; sodium chloride (NaCl) as the electrolyte; phosphatidic acid, a commercial bitterness suppression agent; and tannic acid, a component of green tea. These substances were examined in a gustatory sensation test in human volunteers, a binding study, and using an artificial taste sensor. Sucrose, aspartame, and NaCl were effective in suppressing bitterness, although at comparatively high concentrations. An almost 80% inhibition of bitterness (calculated as concentration %) of a 0.1 mM quinine hydrochloride solution required 800 mM of sucrose, 8 mM of aspartame, and 300 mM NaCl. Similar levels of bitterness inhibition by phosphatidic acid and tannic acid (81.7, 61.0%, respectively) were obtained at much lower concentrations (1.0 (w/v)% for phosphatidic acid and 0.05 (w/v)% for tannic acid). The mechanism of the bitterness-depressing effect of phosphatidic acid and tannic acid was investigated in terms of adsorption and masking at the receptor site. With phosphatidic acid, 36.1% of the bitterness-depressing effect was found to be due to adsorption, while 45.6% was due to suppression at the receptor site. In the case of 0.05 (w/v)% tannic acid, the total bitterness-masking effect was 61.0%. The contribution of the adsorption effect was about 27.5% while the residual masking effect at the receptor site was almost 33%. Further addition of tannic acid (0.15 (w/v)%), however, increased the bitterness score of quinine, which probably represents an effect of the astringency of tannic acid itself. Finally, an artificial taste sensor was used to evaluate or predict the bitterness-depressing effect. The sensor output profile was shown to reflect the depressant effect at the receptor site rather well. Therefore, the taste sensor is potentially useful for predicting the effectiveness of bitterness-depressant substances. (+info)
Formaldehyde-induced shrinkage of rat thymocytes.
To test the possibility that micromolar formaldehyde, a metabolite of methanol derived from aspartame, exerts cytotoxicity, its effect on rat thymocytes was examined under the in vitro condition using a flow cytometer. Incubation of thymocytes with formaldehyde at 100 micro M or more for 24 h significantly increased the populations of shrunken cells and cells with hypodiploid DNA. The peak blood concentration of methanol in human subjects administered abuse doses of aspartame has been reported to exceed 2 mg/dL (625 micro M). It would increase the population of thymocytes undergoing apoptosis if formaldehyde at 100 micro M or more appears in the blood after administration of aspartame. (+info)
Modified DNA aptamers against sweet agent aspartame.
We obtained a modified DNA aptamer against sweetener, aspartame, by in vitro selection method. The modified DNA was prepared from dATP, dGTP, dCTP and a modified dTTP bearing a terminal amino group at C-5 position in place of thymidine by PCR using a hyper thermophilic DNA polymerase, KOD Dash DNA polymerase. The synthetic 102-mer DNA with a 60-mer random region was used as an initial template for the PCR. The PCR-amplified modified DNA library was applied to an aspartame-agarose column, and then the bound modified DNA was eluted from the column for the affinity chromatography selection. Repeating the procedure, we selected the modified DNA aptamer against aspartame. (+info)