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T Imoto

Publications and source records attributed to T Imoto.

At least 91 records · Page 5Linked to original sources
PubMed1995

Three-dimensional structure of gurmarin, a sweet taste-suppressing polypeptide.

The solution structure of gurmarin was studied by two-dimensional proton NMR spectroscopy at 600 MHz. Gurmarin, a 35-amino acid residue polypeptide recently discovered in an Indian-originated tree Gymnema sylvestre, selectively suppresses the neural responses of rat to sweet taste stimuli. Sequence-specific resonance assignments were obtained for all backbone protons and for most of the side-chain protons. The three-dimensional solution structure was determined by simulated-annealing calculations on the basis of 135 interproton distance constraints derived from NOEs, six distance constraints for three hydrogen bonds and 16 dihedral angle constraints derived from coupling constants. A total of 10 structures folded into a well-defined structure with a triple-stranded antiparallel beta-sheet. The average rmsd values between any two structures were 1.65 +/- 0.39 A for the backbone atoms (N, C alpha, C) and 2.95 +/- 0.27 A for all heavy atoms. The positions of the three disulfide bridges, which could not be determined chemically, were estimated to be Cys3-Cys18, Cys10-Cys23 and Cys17-Cys33 on the basis of the NMR distance constraints. This disulfide bridge pattern in gurmarin turned out to be analogous to that in omega-conotoxin and Momordica charantia trypsin inhibitor-II, and the topology of folding was the same as that in omega-conotoxin.

Amino Acid Sequence↗
PubMed1995

Effects of additives on irreversible inactivation of lysozyme at neutral pH and 100 degrees C.

The mechanism of irreversible inactivation of lysozyme at neutral pH at 100 degrees C, and effects of additives on the inactivation were investigated. The thermoinactivation of lysozyme at neutral pH was caused by intra- and intermolecular disulfide exchange and the production of irreversibly denatured lysozyme, which was destabilized by multiple chemical reactions other than disulfide exchange. In addition, independently, deamidation slightly affected the inactivation by causing a decrease of electrostatic interaction between positive charges of lysozyme and negative charges of the bacterial cell wall. As for the effects of additives on the inactivation, a small amount of copper ion suppressed intra- and intermolecular disulfide exchange by catalyzing air oxidation of heat-induced trace amounts of free thiols, and organic reagents (acetamide, ethanol, and glycerol) changed the mechanism of the inactivation to that under acidic conditions by shifting the pKa values of dissociable residues and also suppressed intermolecular disulfide exchange by decreasing hydrophobic interactions.

Acetamides↗
PubMed1995

Effect of salt concentration on the pKa of acidic residues in lysozyme.

We determined the pKa values of acidic residues in hen lysozyme by comparing the pH dependency of stability between wild type and mutant lysozymes in which a negative charge is eliminated. In the comparison of the stability between wild type and a mutant lysozyme, the difference in pH titration curve between them could be expressed as a two-state process involving protonation of a single acidic residue. The results strongly indicated that the Aune and Tanford theory of protein denaturation [Aune, K.C. and Tanford, C. (1969) Biochemistry 8, 4579-4585] is applicable to protein stability in solution. On the other hand, the pKa values of acidic residues in the presence of low (5 mM) or high (400 mM) salt concentration were determined by means of two-dimensional NMR. We found that the pKa values obtained from the pH dependency of stability were close to those from the NMR experiment under the high salt condition. Moreover, by comparing pKa values at high salt and low salt concentrations, we could evaluate the dependency of two electrostatic interactions (salt bridge and charge-helix dipole interaction) on salt concentration.

Amino Acids↗
PubMed1995

Stabilization of lysozyme against irreversible inactivation by suppression of chemical reactions.

The effects of additives on the nonenzymatic deamidation of an Asn residue in a peptide and racemization of Asp and/or Asn in lysozyme were investigated at pH 6 and 100 degrees C. These chemical reactions were accelerated by the addition of phosphate ions. Several salts suppressed the deamidation in the presence of phosphate ions, while the salts did not affect the deamidation in the absence of phosphate ion at pH 6 and 100 degrees C. The results indicated that the effect of the salts was due to the suppression of phosphate catalysis. On the other hand, trifluoroethanol (TFE), which induces the conversion of random coiled polypeptides to secondary structured ones, dramatically suppressed the deamidation of an Asn residue in a peptide. The rate of deamidation in the presence of TFE was comparable to that of asparagine (free amino acid), which was very slowly deamidated. Because TFE could not suppress the deamidation of free asparagine, the suppression of the deamidation of an Asn residue in a peptide was attributed to suppression of the catalysis by the peptide bond in the carboxyl terminus. Since the inactivation of lysozyme was caused by multiple chemical reactions such as the deamidation and racemization, it was expected that the inactivation of lysozyme could be prevented by the addition of salts or TFE. Thus, it was confirmed that salts and TFE suppressed the lysozyme inactivation at pH 6 and 100 degrees C.

Amides↗
PubMed1995

Insect lysozyme from house fly (Musca domestica) larvae: possible digestive function based on sequence and enzymatic properties.

Lysozyme was purified from the homogenate of the whole body of house fly (Musca domestica) larvae by standard chromatographic techniques. The purified lysozyme was sequenced and its enzymatic properties were examined. This lysozyme was a chicken-type lysozyme composed of 122 amino acids, showing about 75% identity with fruit fly lysozymes and 38% with human lysozyme. This enzyme was inactive towards Micrococcus luteus and under the physiological conditions of PH 7.0 and ionic strength 0.1, but was as active toward glycol chitin as was hen lysozyme. The pH-dependent profile of lytic activity towards M. luteus showed that house fly lysozyme has an acidic pH optimum and shows no enzymatic activity above Ph 7. These features are analogous with those of ruminant stomach lysozymes which have evolved for the digestive function, suggesting that this lysozyme does not function as a self-defense protein, like hen and human lysozyme, but as a digestive enzyme, probably in the gut of the insect body. Although a similar functional conversion to digestive enzyme was reported in fruit fly, phylogenetic tree analysis indicates that the evolutionary change of lysozyme to a digestive enzyme occurred similarly in fruit fly and house fly, but the events are not related and occurred independently in each strain. This observation is in contrast with the case of ruminant stomach lysozymes, which were recruited before the divergence of each species of ruminants.

Amino Acid Sequence↗
PubMed1995

Correlation between the differences in the free energy change and conformational energy in the folded state of hen lysozymes with Gly-Pro and Pro-Gly sequences introduced to the same site.

We suggested for the introduction of a prolyl residue into a protein that if the N-terminus residue is glycine, an unfavorable interaction in the folded state caused by the introduction of the prolyl residue can be substantially avoided by use of mutant lysozymes in which Gly-Pro and Pro-Gly sequences are introduced to positions 101-102 in the loop region of the lysozymes [Ueda, T., Tamura, T., Maeda, Y., Hashimoto, Y., Miki, T., Yamada, H., and Imoto, T. (1993) Protein Eng. 6, 183-187]. In order to determine whether or not the information obtained is applicable to other regions, we prepared mutant lysozymes with Gly-Pro and Pro-Gly sequences at position 47, which is located in the beta-sheet, positions 70-71, which are located in the loop, positions 117-118, which are located in the beta-turn, and positions 121-122, which are located in the 3(10)-helix. The free energy changes of the native and mutant lysozymes for unfolding were determined at pH 5.5 and 35 degrees C. However, a mutant lysozyme with the Gly-Pro sequence was not always stabler than that with the Pro-Gly sequence at the same site. On the other hand, in order to determine whether or not strain caused by these sequences exists in the folded or unfolded state, the structures of these mutant lysozymes were determined by use of energy minimization. On comparison of the differences in the free energy change between the mutant lysozymes with Gly-Pro and Pro-Gly sequences at the same site with those in their total local conformational energies, it was found there is a good correlation between them. Therefore, it was suggested that the difference in total local conformational energy caused by the introduction of a Gly-Pro or Pro-Gly sequence could be estimated by use of the energy minimized structure. Moreover, the correlation indicated that the differences in the free energy change between Gly-Pro and Pro-Gly lysozymes may be reflected by the differences in the total local conformational energies in their folded state. It was suggested that the energy levels in the unfolded states of mutant lysozymes with Gly-Pro and Pro-Gly sequences at the same site in a Gdn-HCl solution were almost identical.

Amino Acid Sequence↗
PubMed1995

Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences.

Site-directed mutagenesis was performed at Asp-Gly (48-49, 66-67, 101-102) and Asn-Gly (103-104) sequences of hen egg-white lysozyme to protect the enzyme against irreversible thermoinactivation. Because the lysozyme inactivation was caused by the accumulation of multiple chemical reactions, including the isomerization of the Asp-Gly sequence and the deamidation of Asn [Tomizawa et al. (1994) Biochemistry, 33, 13032-13037], the suppression of these reactions by the substitution of Gly to Ala, or the introduction of a sequence of human-type lysozyme, was attempted and the mutants (where each or all labile sequences were replaced) were prepared. The substitution resulted in the reversible destabilization from 1 to 2 kcal/mol per substitution. The destabilization was caused by the introduction of beta-carbon to the constrained position that had conformational angles within the allowed range for the Gly residue. Despite the decrease in the reversible conformational stability, the mutants had more resistance to irreversible inactivation at pH 4 and 100 degrees C. In particular, the rate of irreversible inactivation of the mutant, which was replaced at four chemically labile sequences, was the latest and corresponded to approximately 18 kcal/mol of the reversible conformational stability. Therefore, replacement of the chemically labile sequence was found to be more effective at protecting enzymes against irreversible thermoinactivation than at strengthening reversible conformational stability.

Amino Acid Sequence↗
PubMed1995

Effective renaturation of reduced lysozyme by gentle removal of urea.

To increase the folding yield of concentrated reduced lysozyme, we developed a renaturation method by means of dialysis from concentrated urea with redox agents. After lysozyme was incubated in the reducing buffer (8 M urea solution) with oxidized glutathione, renaturation of reduced lysozyme was started by dialysis against the dialyzing buffer containing 8 M urea with redox agents. The urea concentration of the dialyzing bottle was gradually diluted with dialyzing buffer without urea at a flow rate of 0.1 ml/min by high pressure pump. Using this systematic dialysis, a concentration as high as 5 mg/ml of reduced lysozyme could be renaturated in 80% yield, while the folding yield was < 5% even at a concentration of 1 mg/ml using a conventional rapid dilution method [Goldberg et al. (1991) Biochemistry, 30, 2790-2797]. Therefore, it was concluded that gentle removal of urea from denatured proteins, dissolved in concentrated urea solution, by means of dialysis should be useful to renature denatured proteins effectively.

Buffers↗
PubMed1995

Gurmarin inhibition of sweet taste responses in mice.

The inhibitory effects of gurmarin (a peptide isolated from the leaves of Gymnema sylvestre) on sweet taste responses were studied by examining the chorda tympani nerve responses to various taste substances before and after lingual treatment with gurmarin in C57BL and BALB mice. Treatment with gurmarin at 3 micrograms/ml or more selectively suppressed responses to sucrose without affecting responses to NaCl, HCl, and quinine in C57BL mice, whereas gurmarin at 100 micrograms/ml did not significantly suppress sucrose responses in BALB mice. Responses to various sweet substances in C57BL mice decreased to approximately 45-75% of control after gurmarin, and the suppressive effect of gurmarin was reversible. The profile of the residual responses of C57BL mice to various sweeteners after gurmarin was almost identical to that of BALB mice, which was hardly affected by gurmarin. These results strongly suggest that there are at least two types of sweet taste receptors in mice, gurmarin-sensitive and -insensitive. Probably, C57BL and BALB mice share an identical gurmarin-insensitive receptor, and C57BL mice also have a gurmarin-sensitive receptor.

Amino Acids↗
PubMed1994

The mechanism of irreversible inactivation of lysozyme at pH 4 and 100 degrees C.

The mechanism of irreversible inactivation of lysozyme at pH 4, 100 degrees C, was investigated. It was elucidated that the inactivation was caused by production of molecules in an irreversibly denatured state. From analyses of the mechanism of production of the inactive enzyme, the inactivation was not evoked by a single chemical reaction. The free energy change between the folded and unfolded states decreased by the accumulation of chemical reactions (isomerization of Asp-Gly, deamidation of Asn, racemization of Asp and Asn, and cleavage of the Asp-X-peptide bond) induced at high temperature. Thus, certain molecules were ultimately in the unfolded state even at low temperature and lost activity. Moreover, a good correlation between the stability (free energy change) and the averaged number of chemical reactions that leads to the inactivation was obtained on the basis of some assumptions.

Acetylcysteine↗
PubMed1994

Isolation and characterization of 101-succinimide lysozyme that possesses the cyclic imide at Asp101-Gly102.

Lytic activity of lysozyme solution gradually increased on incubation at pH 4, 40 degrees C. When the solution was analyzed by use of cation-exchange HPLC at pH 5, a new peak appeared with increased incubation time. The derivative in the new peak was identified to be 101-succinimide lysozyme in which cyclic imide formed at Asp101-Gly102. The formation of 101-succinimide lysozyme increased with increases in concentration of acetate buffer. Kinetic analysis of the formation of 101-succinimide lysozyme indicated that the cyclic imide was stable below pH 5 due to suppression of the hydrolysis of cyclic imide. Its lytic activity against M. luteus, which has a negative charge, was 165% at pH 7, whereas its activity against glycol chitin, which has no charge, was 90%. Since the lytic activity of Asn101 lysozyme, where one negative charge is eliminated, reached a maximum of 125%, it was suggested that the increase of lytic activity against bacterial cells in 101-succinimide lysozyme was due not only to the disappearance of the negative charge at Asp101 but also to the removal of steric hindrance at the upper part of the active site cleft.

Acetates↗
PubMed1994

Formation of alpha-helix 88-98 is essential in the establishment of higher-order structure from reduced lysozyme.

Lys96 and Lys97 in lysozyme are located at the C terminus of alpha-helix 88-98. The positive charges of these residues are supposed to stabilize the helical structure, and these residues are conserved as the basic amino acids among c-type lysozymes. The renaturation rate of reduced mutant lysozyme, where both Lys96 and Lys97 were mutated together to Ala, was slower than that of native lysozyme at pH 8.0 and 37 degrees C by SH-SS interchange reactions. In order to investigate the reason, the peptide fragment 36-105 (where we can obtain information of the interaction between helix 88-98 and Trp62 and Trp63 residues) was prepared. CD spectra were compared between peptide fragment 36-105 and its acetylated form, where the positive charges of Lys96 and Lys97 were eliminated, and it was elucidated that the displacement of positive charges at the C terminus of the helix caused the shift of the advantageous structure of the fragment from alpha-helix to coil. Moreover, we obtained evidence that there was interaction of the helix with Trp62 and/or Trp63, which maintained a thermodynamically stable higher-order structure. Therefore, these results suggest that the formation of alpha-helical structure in 88-98 is a significant factor in the establishment of native structure from reduced lysozyme.

Amino Acid Sequence↗
PubMed1994

Reduction of disulfide bonds in proteins by 2-aminothiophenol under weakly acidic conditions.

We developed a method for reducing disulfide bonds in proteins under weakly acidic conditions by use of 2-aminothiophenol. The disulfide bonds in hen egg-white lysozyme, ribonuclease A, and soybean trypsin inhibitor were quantitatively reduced by 2-aminothiophenol in phosphate buffer, pH6, containing 8 M Gdn HCl, 1 mM EDTA, and 20% ethanol, for 60 min at 40 degrees C. On analysis of the RP-HPLC patterns of tryptic peptides, which were derived from reduced and S-alkylated lysozyme and ribonuclease A at pH 6, it was confirmed that no side reaction occurred. Moreover, the reduction under weakly acidic conditions was demonstrated to be applicable for the location of such a labile residue as O-acetylated tyrosine.

Aniline Compounds↗
PubMed1994

An S-alkylating reagent with positive charges as an efficient solubilizer of denatured disulfide-containing proteins.

A novel S-alkylating reagent, N-(3-bromopropyl)-N,N,N',N',N'-pentamethyl-1,3-propanedi(ammonium bromide) (TAP2-Br) which carries two positive charges in the molecule, was prepared to increase the solubility or to decrease the hydrophobicity of cysteine-containing denatured proteins (or peptides). S-Alkylation with TAP2-Br introduces two positive charges per cysteine residue, which will effectively shift the net charge of a protein in the positive direction. Disulfide-containing proteins, such as hen egg-white lysozyme, RNase A, BSA, and soybean trypsin inhibitor (Kunitz type), were reduced and S-alkylated with TAP2-Br to evaluate the potential of this reagent compared with other S-alkylating reagents such as monoiodoacetic acid, bromosuccinic acid and (3-bromopropyl)trimethylammonium bromide. The solubilities of these denatured proteins in the pH range of 2-10 indicated that S-alkylation with TAP2-Br effectively solubilized not only basic proteins (lysozyme and RNase) but also an acidic protein containing a fairly large number of cysteine residues (BSA). Moreover, the retentions of cysteine-containing tryptic peptides derived from lysozyme on reversed-phase HPLC were greatly reduced by S-alkylation with TAP2-Br. These results indicate that TAP2-Br is very useful to increase the solubility of some cysteine-containing denatured proteins and to decrease the hydrophobicity of peptides containing cysteine residue(s).

Alkylating Agents↗
PubMed1994

Colonic lysozymes of rabbit (Japanese white): recent divergence and functional conversion.

Lysozyme was extracted from the feces of rabbit (Japanese White) with 2.5% acetic acid and purified by ion-exchange chromatography. Subsequent ion-exchange HPLC at pH 4.0 revealed the presence of two isozymes, namely rabbit colonic lysozymes 1 and 2. The amino acid sequences of these lysozymes were determined. The colonic lysozymes 1 and 2 showed 98% identity with each other and 94 and 95% identities with rabbit kidney lysozyme, respectively. The very high identities between kidney and colonic lysozymes indicate that the colonic isozymes diverged from the conventional kidney lysozyme very recently, probably after the divergence of rabbit from other rodents, accompanying the gene duplication. Despite the small changes in the sequences, the enzymatic properties of colonic lysozyme differ from those of the kidney lysozyme. The activity of the colonic lysozyme against Micrococcus luteus cells showed a narrow and acidic pH optimum, in contrast to the wide and high pH optimum of the kidney lysozyme. Changes in the enzymatic properties are analogous to those of the ruminant stomach lysozymes and may implicate adaptive evolution in the functional conversion of rabbit colonic lysozymes in gut.

Adaptation, Physiological↗
PubMed1994

The role of net charge on the renaturation of reduced lysozyme by the sulfhydryl-disulfide interchange reaction.

Reduced and acetylated lysozymes are basic proteins. When their amino groups were variously acetylated and then renatured by sulfhydryl-disulfide (SH-SS) interchange reaction at pH 8.0, the final folding yield decreased as the number of positive charges decreased. The final folding yield of native and Ac1 lysozyme, with one positive charge eliminated, was less sensitive to increasing protein concentration than that of Ac2 lysozyme, where two positive charges had been eliminated. The final folding yield of reduced Ac2 lysozyme increased in the presence of 1 M urea, which reduced the aggregation of unfolded lysozyme. Thus, the aggregation of unfolded lysozymes, which leads to a decrease in the final folding yield, was found to be heavily dependent on their net charges. Moreover, the final folding yield of reduced lysozyme was shown to be increased by use of cystamine as an oxidizing reagent in comparison with 2-hydroxyethyl disulfide or dithiodiglycolic acid. This may support the idea that the final folding yield is influenced by electrostatic interaction between unfolded lysozymes in the early stage of renaturation. In contrast, the concentration dependency of the final folding yield of Ac1 lysozyme was different from those of carboxymethylated His15 and Asp106 lysozymes whose positive net charges were similar to that of Ac1 lysozyme. On the basis of the observations, it is suggested that the formation of the aggregates in the renaturation process might also be affected by the structure of the unfolded state of lysozyme in solution.

Acetylation↗
PubMed1994

Lysozyme requires fluctuation of the active site for the manifestation of activity.

Mutations around His15 which lie far away from the active site, stimulated glycol chitin activity of lysozyme at physiological temperature. Del-Arg14His15 lysozyme, a mutant lysozyme whose Arg14 and His15 were deleted together, and has the highest activity among these mutant lysozymes, had a similar binding ability to a trimer of N-acetyl-glucosamine, a substrate analogue, relative to native lysozyme. This suggests that the increased activity was due to an increased kcat in the catalysis reaction. The H-D exchange rate of the N-1 proton in the Trp63 which is located in the active site cleft, was enhanced in the Del-Arg14His15 lysozyme, while 2-D proton NMR analysis revealed no conformational change around Trp63. We conclude that some sort of fluctuation at the active site might be required for the manifestation of activity. This theory is supported by the finding that the Del-Arg14His15 lysozyme showed a shift in temperature dependency of activity to lower temperatures compared with that of native lysozyme.

Acetylglucosamine↗