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T Imoto

Publications and source records attributed to T Imoto.

At least 109 records · Page 6Linked to original sources
PubMed1994

Histological localization of the sweet taste receptor in rat taste buds by the use of gurmarin, a sweet taste-suppressing peptide.

The binding site of gurmarin, a peptide inhibiting the sweet-taste sensation, was studied in taste buds in rat circumvallate papillae by means of a histochemical technique. Frozen sections of tongues were incubated with gurmarin conjugated with biotin and thereafter examined with a light microscope. Positive reactivity for the peptide was localized to the taste hairs, the apical projections of taste bud cells. The reaction appeared in about 10% of the circumvallate taste buds examined. As electrophysiological studies indicate that gurmarin suppresses the sweet-taste sensation at the level of reception, the present study suggests that the receptor for sweet taste is located on the taste hairs, and, furthermore, is present only in a certain, limited number of the taste buds.

Animals↗
PubMed1993

The primary structures and properties of non-stomach lysozymes of sheep and cow, and implication for functional divergence of lysozyme.

Lysozymes were purified from the homogenate of cow and sheep kidneys, and their amino-acid sequences as well as some enzymic properties were determined. Like most mammalian lysozymes both sheep and cow kidney lysozymes are composed of 130 amino acids. The sequences of these two lysozymes are the most similar to each other (95% identity), the second most similar to the conventional mammalian lysozymes like human, rat and rabbit lysozymes (74-85% identity), and much less similar to their own stomach lysozymes (65-70% identity). Cow kidney lysozyme is also different from cow milk lysozyme (partial sequence), indicating that cow contains at least three kinds of chicken type lysozymes, that is kidney, milk and stomach lysozymes. The activities of cow and sheep kidney lysozymes were 3% and 29% against Micrococcus luteus at pH 7.0, ionic strength of 0.1 and 30 degrees C, and 57% and 84% against glycol chitin at pH 5.5 and 40 degrees C, which were expressed as percentages relative to hen lysozyme. The net charges of cow and sheep lysozymes at pH 7 were less positive (+1.5 and +2.5, respectively) than human and hen lysozymes (both +8.0) and rather close to the stomach ones (-2 to 0). The decreased net positive charge observed in cow and sheep kidney lysozymes may suggest that the ruminant kidney lysozyme had functioned once as a digestive enzyme in the stomach of an ancestral ruminant.

Amino Acid Sequence↗
PubMed1993

Detection of subtle differences in the surface structure of lysozymes by use of an immobilized Fab fragment.

A method was developed to evaluate the association constant at physiological pH (pH 7.5) between a lysozyme and the Fab fragment derived from anti-lysozyme monoclonal antibody 37-7, which was immobilized to the adsorbent for HPLC. Comparison of the association constants between lysozymes and the immobilized Fab fragment indicated that mAb 37-7 recognized the prominently exposed regions (hills and ridges) around His15 of hen lysozyme, but His15 itself was not directly involved in the binding with mAb 37-7. Moreover, the epitope was confirmed by the reactivity of His15 with monoiodoacetic acid in the presence of mAb 37-7. The association constant of 15-carboxymethylated histidine lysozyme (15CM lysozyme) with the immobilized Fab fragment was smaller by one-seventh than that of 15-carboxamidated histidine lysozyme, though the side chains introduced were almost identical in size. From the pH titration of 15CM lysozyme with 13C-enriched carboxyl group by use of 13C-NMR, the pKa of the introduced carboxyl group was evaluated to be 5.06. Since the carboxyl group was fully ionized under the conditions of measurement (pH 7.5), electrostatic repulsion was found to disturb severely the association between mAb 37-7 and hen lysozyme. Moreover, it was demonstrated that, because of the high reproducibility of measurement, the immobilized Fab fragment could detect subtle differences in the surface structure of lysozymes.

Animals↗
PubMed1993

Endopeptidase activity of cathepsin C, dipeptidyl aminopeptidase I, from bovine spleen.

By employing various synthetic substrates, as well as soluble denatured protein substrate (TAP-lysozyme) and its derivatives, endopeptidase activity of cathepsin C, dipeptidyl aminopeptidase I [EC 3.4.14.1], from bovine spleen was investigated. Cathepsin C efficiently degraded Z-Phe-Arg-MCA, Pro-Phe-Arg-MCA, and Suc-Leu-Leu-Val-Tyr-MCA. This endopeptidase activity required sulfhydryl reagents and halide ions, as in the case of the dipeptidyl aminopeptidase (DAP) activity. We confirmed that this endopeptidase activity is due to cathepsin C itself based on the results on gel-filtration and anion-exchange chromatographies, comparative studies of the inhibitory effects of leupeptin and E-64 on this activity and those of cathepsins B and L, and further the competitive inhibitions by mutual substrates for the DAP and endopeptidase activities of cathepsin C. We also found that cathepsin C endopeptidase activity towards TAP-lysozyme and its N-alpha-acetylated tryptic peptides showed marked dependence on sulfhydryl reagents and chloride ion. Thus, we concluded that cathepsin C has endopeptidase activity as well as DAP activity. The binding energy between the enzyme and the amino acid side chains of the substrate may be as important for the endopeptidase activity as is the electrostatic interaction between the enzyme and the free alpha-amino group of the substrate for the DAP activity.

Animals↗
PubMed1993

Thermodynamic and kinetic stabilities of hen-egg lysozyme and its chemically modified derivatives: analysis of the transition state of the protein unfolding.

For the stabilization of a protein against irreversible denaturation caused by rapid reaction of the unfolded form of the protein (kinetic stabilization), the free energy change of activation for unfolding should be increased. First, we demonstrated that this strategy was effective to stabilize a protein against protease digestion. For kinetic stabilization, it is important to stabilize a protein at a site where the local structures are largely unfolded in the transition state for unfolding. We developed a method to find such sites by comparison of the thermodynamic stabilities and the unfolding rate constants between unmodified and modified proteins. Application of this method to analyze the transition state of hen-egg lysozyme using some chemically modified derivatives is also described. Moreover, it was confirmed that the protease digestion method is superior to the relaxation method for estimation of the unfolding rate constant. Namely, the protease digestion method may be useful in analyzing the transition state of protein unfolding.

Animals↗
PubMed1993

Stabilization of lysozyme by the introduction of Gly-Pro sequence.

Three mutant lysozymes where the Asp101-Gly102 sequence of lysozyme was converted to Asp101-Pro102, Gly101-Pro102 and Pro101-Gly102 were prepared to investigate the effect of proline residues on the stabilization of proteins. The free energy changes of lysozymes for the unfolding in aqueous solution at pH 5.5 and 35 degrees C were 10.0, 10.1, 11.0 and 7.7 kcal/mol for wild type, Asp101Pro102, Gly101Pro102 and Pro101Gly102 lysozyme respectively. When the energy level in the unfolded state of wild type lysozyme was fixed at a standard level, the energy levels in the folded state of Asp101Pro102 and Pro101Gly102 lysozymes were found to be higher than that of wild type lysozyme on the basis of delta GD(H2O) and entropy losses of their polypeptide chains in the unfolded state. The presence of some strain in the folded state of these lysozymes was supported by both the calculation of conformational energy for a trans-L-prolyl residue [Schimmel, P.R. and Flory, P.J. (1968) J. Mol. Biol., 34, 105-120] and the analysis of structures of energy-minimized mutant lysozymes. Therefore, it is concluded that the formation of the Gly-Pro sequence is effective in avoiding possible strain in the folded state of a protein caused by the introduction of proline residue(s).

Amino Acid Sequence↗
PubMed1992

Left-sided substrate binding of lysozyme: evidence for the involvement of asparagine-46 in the initial binding of substrate to chicken lysozyme.

The "right-sided" and "left-sided" substrate binding modes at the lower saccharide binding subsites (D-F sites) of chicken lysozyme were investigated by utilizing mutant lysozymes secreted from yeast. We constructed the following mutant lysozymes; "left-sided" substitution of Asn46 to Asp, deletion of Thr47, and insertion of Gly between Thr47 and Asp48 and "right-sided" substitution of Asn37 to Gly. Analyses of their activities and substrate binding abilities showed that Asn46 and Thr47 are involved in the initial enzyme-substrate complex and Asn37 is involved in the transition state. These results support an earlier proposal that interactions between substrate and residues at the left side of lysozyme stabilize a catalytically inactive enzyme-substrate complex, while interactions between substrate and residues at the right side stabilize the catalytically active complex [Pincus, M. R., & Scheraga, H. A. (1979) Macromolecules 12, 633-644]. These results are also consistent with the proposed kinetic mechanism for lysozyme reaction that the rearrangement of an initial enzyme-substrate complex (beta-complex) to another complex (gamma-complex) is required for catalytic hydrolysis [Banerjee S. K., Holler, E., Hess, G. P., & Rupley, J. A. (1975) J. Biol. Chem. 250, 4355-4367].

Amino Acid Sequence↗
PubMed1992

Stabilization of a protein by removal of unfavorable abnormal pKa: substitution of undissociable residue for glutamic acid-35 in chicken lysozyme.

Glu35 in chicken lysozyme has an abnormally high pKa (6.1) partly due to the hydrophobic environment provided by Trp108. The relationship between protein stability and abnormal pKa was investigated in detail by using mutant lysozymes in which Glu35 was replaced by undissociable residues and an oppositely ionizable residue. It was found that lysozyme was stabilized at alkaline pH range by the replacement of Glu35 with an undissociable residue, Gln (E35Q lysozyme) or Al (E35A lysozyme). On the other hand, when Glu35 was replaced by His (E35H lysozyme), which could have an opposite charge to Glu by ionization, the introduced His35 was found to have an abnormally low pKa (3.6), leading to the destabilization of lysozyme at acidic pH. These observations are completely consistent with the situation that the environment around Glu35 is highly hydrophobic and therefore the placement of either a positive or negative charge in such an environment leads to destabilization of lysozyme. These observations also indicate that the replacement of an acidic residue having abnormally high pKa or a basic residue having abnormally low pKa by an undissociable residue is a very efficient and general method for stabilization of a protein.

Animals↗
PubMed1992

Multiple role of hydrophobicity of tryptophan-108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35.

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively. The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

An extract of Gymnema sylvestre leaves and purified gymnemic acid inhibits glucose-stimulated gastric inhibitory peptide secretion in rats.

Gastric inhibitory peptide release into the portal vein in response to duodenal infusion of D-glucose was studied in the presence of a leaf extract of Gymnema sylvestre, purified gymnemic acid and inhibitors of some putative glucose sensors and carriers in the intestinal lumen. Intraduodenal infusion of D-glucose significantly increased the portal immunoreactive gastric inhibitory peptide concentration in a dose-dependent manner. The increase in the portal immunoreactive gastric inhibitory peptide induced by glucose was significantly depressed by concomitantly infused leaf extract of Gymnema sylvestre, purified gymnemic acid and phlorizin but not by cytochalasin B. Mannoheptulose, which inhibits glycolysis, and procaine and lidocaine, which inhibit the vagal glucoreceptor in the lumen, did not affect portal immunoreactive gastric inhibitory peptide concentrations. These results suggest that a glucose receptor, which interacts with the leaf extract of Gymnema sylvestre, purified gymnemic acid and phlorizin, exists for the release of immunoreactive gastric inhibitory peptide and that the glucose receptor for gastric inhibitory peptide release is not likely to be identical with a glucose transporter or a vagal glucoreceptor in the lumen.

3-O-Methylglucose↗
PubMed1992

Amino acid sequence of sweet-taste-suppressing peptide (gurmarin) from the leaves of Gymnema sylvestre.

The complete amino acid sequence of a sweet-taste-suppressing peptide, gurmarin, from the leaves of Gymnema sylvestre was determined by the Edman analysis of peptides derived from digests obtained with Staphylococcus aureus V8 protease, pyroglutamyl aminopeptidase, and lysyl endopeptidase. Gurmarin consists of 35 amino acid residues with an amino-terminal pyroglutamyl residue and has the molecular weight of 4,209. Gurmarin has no significant homology with other known proteins.

Amino Acid Sequence↗
PubMed1992

Colorimetric assay for lysozyme using Micrococcus luteus labeled with a blue dye, Remazol brilliant blue R, as a substrate.

Micrococcus luteus (M. lysodeikticus) labeled with Remazol brilliant blue R (blue ML) was prepared as a novel substrate for the colorimetric assay of lysozyme. The treatment of the labeled substrate with lysozyme resulted in the release of soluble blue products which can be easily measured spectrophotometrically at 600 nm. The blue color was most efficiently released at pH 7 and ionic strength of 0.2 on incubation with hen lysozyme at 40 degrees C. A new colorimetric method for the assay of lysozyme using this substrate was developed. The assay system gave a linear dose-response curve, and as little as 0.1 microgram of human lysozyme (1 microgram/ml, 100 microliters) can be detected. The present method is more convenient and reproducible than the conventional lysozyme assay with bacterial cells. Application of the system to the determination of lysozyme in human serum is described.

Adult↗
PubMed1992

Endo- and aminopeptidase activities of rat cathepsin H.

Employing soluble denatured protein substrates and their derivatives, the proteolytic activity of rat cathepsin H was investigated. The enzyme showed aminopeptidase activity which sequentially released amino acid from the N-terminal of the substrate. The aminopeptidase activity did not act on N alpha-acetylated peptides and showed moderate ionic-strength dependence when methionyl-methylcoumarylamide was employed as a substrate. These results indicate that the activity essentially requires an N-terminal free amino group of the substrate and recognizes it electrostatically to some extent. On the other hand, the enzyme was also indicated to exhibit endopeptidase activity by employing appropriate N alpha-acetylated peptide substrates. In contrast to the aminopeptidase activity, the endopeptidase activity showed rather strict specificity, preferring hydrophobic residues at P2 and P3 sites. Because of the broad specificity and high efficiency of the aminopeptidase activity, it was difficult to directly observe endopeptidase activity in the digestion of large peptide substrates with a free alpha-amino terminal. Thus, this is the first experimental evidence that indicates endopeptidase activity by assigning internal peptide bonds cleaved by this activity. From this data, we proposed a model of the binding site of this enzyme.

Amino Acid Sequence↗
PubMed1991

Mechanisms of a conversion from membrane associated lysosomal acid phosphatase to content forms.

We reported that membrane-associated APase (M-APase) is anchored in the lipid bilayer through its hydrophobic sequence close to the COOH-terminus [Biochem. Biophys. Res. Commun. (1989) 162, 1044-1053] and is released from lysosomal membranes into the lysosomal contents by limited proteolysis with cathepsin D [J. Biochem. (1990) 108, 287-291]. We here report the conversion process of M-APase to three forms of the content enzyme (C-APase I, II, and III) by assigning the COOH-terminus of each APase in lysosomes. The purified M-APase (67 kDa) was subjected to COOH-terminal determination after digestion with cathepsin D. The COOH-terminus of cathepsin D-digested M-APase (65 kDa) ended at the position of the 382nd leucine residue. The COOH-termini of C-APase I (48 kDa) and III (64 kDa) were also determined. Since the two enzymes ended at the same position of the 373rd alanine residue, this COOH-terminal is 9 amino acid residues shorter than that of cathepsin D-digested M-APase. Then, we compared NH2-terminal sequences of the three enzymes, and found that those of three enzymes are exactly the same. Therefore, protein portions of C-APase I and III proved to be identical. The above results indicate that in lysosomes M-APase is first hydrolyzed between amino acid residues 382 and 383 by cathepsin D, and after solubilization, the enzyme is converted to C-APase III by losing 9 amino acid residues by lysosomal carboxypeptidase(s). Molecular weight differences among three C-APases (III, 64 kDa; II, 55 kDa; I, 48 kDa) probably are due to different degrees of carbohydrate chain degradations as reported previously [J. Biochem. (1989) 105, 449-456].

Acid Phosphatase↗
PubMed1991

Electrophysiological and behavioral studies on the taste of umami substances in the rat.

Electrophysiological and behavioral experiments were performed to reveal taste properties of "umami" substances such as monosodium glutamate (MSG) and disodium inosine monophosphate (IMP) in rats. To eliminate the taste effects of Na ions contained in these umami substances, we dissolved them in 0.01 mM amiloride, which is known to block sodium responses. In the electrophysiological study, taste responses of the whole chorda tympani nerve were recorded. The magnitude of responses to MSG (or IMP) at concentrations below 0.1 M (or 0.01 M) was less than 10% of that to 0.1 M NaCl. On the other hand, the mixtures of MSG and IMP showed responses 2-7 times larger than the arithmetric sum of the responses to each component of the mixtures. A new sweet taste inhibitor (Gymnema sylvestre extract) strongly suppressed neural responses to mixtures of MSG and IMP as well as sucrose, but only weakly or negligibly to individual solutions of these umami substances. In the behavioral study, the brief exposure two-bottle preference test and conditioned taste aversion paradigm were used. MSG was most preferred at 0.3 M (preference ratio = 57%), IMP, at 0.01 M (61%), and both were less preferred or rejected at higher concentrations. In contrast, mixtures of MSG and IMP were more preferred at a broad concentration range (e.g., 82% for 0.1 M MSG + 0.01 M IMP). Aversive conditioning to umami substances was generalized to sucrose, and vice versa, but not to 0.1 M NaCl, 0.01 M HCl, and 0.1 mM quinine hydrochloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

A novel peptide isolated from the leaves of Gymnema sylvestre--I. Characterization and its suppressive effect on the neural responses to sweet taste stimuli in the rat.

1. A new substance that suppressed selectively the neural responses of the rat to sweet taste stimuli was isolated from the leaves of Gymnema sylvestre. 2. The substance was proved to be a peptide consisting of 35 amino acids and having a molecular weight of about 4,000. 3. The inhibitory effect on the sweet responses appeared after treating the tongue surface with the peptide at a concentration of more than 1 x 10(-6) M.

Animals↗