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T Imoto

Publications and source records attributed to T Imoto.

At least 145 records · Page 8Linked to original sources
PubMed1986

Dinitrophenylation as a probe for the determination of environments of amino groups in protein. Reactivities of individual amino groups in lysozyme.

Dinitrophenylation of hen egg white lysozyme with 2,4-dinitrofluorobenzene (DNFB) was carried out at pH 7-11 and room temperature in order to examine whether dinitrophenylation could be applied to determine the environments of individual amino groups in lysozyme or not. Lightly dinitrophenylated lysozyme was reduced, S-carboxymethylated and then subjected to reversed-phase high-performance liquid chromatography (RP-HPLC). All tryptic peptides, which contained dinitrophenylated amino groups (one alpha-amino group, Lys 1(alpha), and six epsilon-amino groups, Lys 1(epsilon), Lys 13, Lys 33, Lys 96, Lys 97, and Lys 116), could be separated and monitored by absorbance measurement at 360 nm on RP-HPLC. The relative reactivities of individual amino groups, determined from the relative peak areas of dinitrophenylated tryptic peptides at 360 nm, were found to be sensitive to the reaction pH and to the presence of the trimer of N-acetyl-D-glucosamine or NaCl. It was concluded that dinitrophenylation of a protein with DNFB followed by peptide analysis by RP-HPLC with detection at 360 nm is a good method for probing the environments of individual amino groups in the protein.

Amines↗
PubMed1986

Specific carbodiimide-binding mechanism for the selective modification of the aspartic acid-101 residue of lysozyme in the carbodiimide-amine reaction.

A mechanism for the selective modification of Asp-101 in hen egg-white lysozyme with an amine nucleophile catalyzed by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was investigated using ethanolamine as a nucleophile at pH 5.0 and room temperature. In the presence of N-acetyl-D-glucosamine (NAG) and its oligomers [(NAG)n, n = 2 and 3] under the conditions with which about 90% of lysozyme was calculated to form complexes, the formation of Asp-101 modified lysozyme decreased markedly but to different degrees, that is (NAG)3 was the most and NAG the least effective. When the lysozyme derivative, in which Trp-62 in the active site cleft was oxidized to oxindolealanine (Ox-62 lysozyme), was used in place of native lysozyme, the formation of Asp-101 modified derivative decreased to about half, which was similar to the decrease in the presence of (NAG)2. In the presence of 0.5 M NaCl, on the other hand, the formation of Asp-101 modified lysozyme was considerably enhanced. From these observations, it is concluded that EDC binds to the active site cleft of lysozyme to specifically activate Asp-101. The affinity of EDC to the active site of lysozyme is partly due to the hydrophobic interaction of EDC with the Trp-62 residue at sub-site B of lysozyme. EDC is an activating reagent for carboxyl groups unlike most active site-directed reagents which produce final products directly. Therefore, the active site-directed nature of EDC was very useful because it made it possible to selectively introduce various amines as needed at a particular carboxyl group of lysozyme.

Acetylglucosamine↗
PubMed1986

Age-related changes in optokinetic and rotational tests.

Optokinetic nystagmus (OKN) and nystagmus induced by the rotational test were analyzed in sixty normal subjects, 13 to 68 years old. Reactivity was measured by the maximum slow phase velocity. A slight linear correlation to age was found in both tests. In the OKN test the eye speed increased with advancing age, and in the rotational test the eye speed decreased with advancing age. This pattern was found statistically significant at a target speed of 40 degrees/s in the OKN test and at 6 degrees/s2 (stimulation) in the rotational test.

Adolescent↗
PubMed1986

Blood levels and distribution of talampicillin and bacampicillin in NZW rabbits.

Parameters for blood concentrations of the peroral antibiotics talampicil in and bacampicillin, in NZW rabbits were determined using a two compartment model. On the basis of these pharmacokinetic parameters, a three compartment model was prepared for the concentrations of these drugs in the tongue, gingiva, submandibular gland, parotid gland, cervical lymph-node and mandibular bone. Simulation curves based on the parameters of tissue concentrations revealed visual conformity to the measured values.

Ampicillin↗
PubMed1985

Isolation and characterization of 101-beta-lysozyme that possesses the beta-aspartyl sequence at aspartic acid-101.

In the reaction of the intramolecular cross-linking between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride [Yamada, H., Kuroki, R., Hirata, M., & Imoto, T. (1983) Biochemistry 22, 4551-4556], it was found that two-thirds of the protein (both the recovered and cross-linked lysozymes) showed a lower affinity than the rest against chitin-coated Celite, an affinity adsorbent for lysozyme. The protein with the reduced affinity was separated on chitin-coated Celite affinity chromatography and found to be slightly different from native lysozyme in the elution position of the tryptic peptide of Ile-98-Arg-112 on reversed-phase high-performance liquid chromatography. In contrast with native lysozyme, the limited hydrolysis of this abnormal tryptic peptide of Ile-98-Arg-112 in 6 N HCl at 110 degrees C gave a considerable amount of beta-aspartylglycine. Therefore, it was concluded that two-thirds of the protein obtained from this reaction possessed the beta-aspartylglycyl sequence at Asp-101-Gly-102. As a result, we obtained four lysozymes from this reaction, the derivative with the beta-aspartyl sequence at Asp-101 (101-beta-lysozyme), the cross-linked derivative between Lys-13 and Leu-129 (CL-lysozyme), the CL-lysozyme derivative with the beta-aspartyl sequence at Asp-101 (101-beta-CL-lysozyme), and native lysozyme. In the ethyl esterification of Asp-52 in lysozyme with triethyloxonium fluoroborate [Parsons, S. M., Jao, L., Dahlquist, F. W., Borders, C. L., Jr., Groff, T., Racs, J., & Raftery, M. A. (1969) Biochemistry 8, 700-712; Parsons, S. M., & Raftery, M. A. (1969) Biochemistry 8, 4199-4205], the same bond rearrangement was detected in the same ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

An intramolecular cross-linkage of lysozyme. Formation of cross-links between lysine-1 and histidine-15 with bis(bromoacetamide) derivatives by a two-stage reaction procedure and properties of the resulting derivatives.

Hen egg white lysozyme was treated at pH 5.5 with four bifunctional reagents, bis(bromoacetamide) derivatives [BrCH2CONH(CH2)nNHCOCH2Br, 1-n, n = 0, 2, 4, and 6], to alkylate His-15 monofunctionally. The excess bifunctional reagent was then removed, and the pH was raised to 9.0 to allow the other end of the reagent molecule to react. The shortest reagent (1-0) gave no intramolecularly cross-linked lysozyme derivative but only histidine-15-modified lysozyme monomer and intermolecularly cross-linked lysozyme dimer. However, the reagents with longer arms (1-2, 1-4, and 1-6) gave lysozyme derivatives cross-linked intramolecularly between the nitrogen at epsilon 2 of His-15 and the epsilon-amino group of Lys-1 without formation of any other intramolecularly cross-linked lysozyme derivative. These results are consistent with our previous proposal that lysozyme has a small hydrophobic pocket that binds small molecules in the direction from His-15 to Lys-1 [Yamada, H., Uozumi, F., Ishikawa, A., & Imoto, T. (1984) J. Biochem. (Tokyo) 95, 503-510]. The thermal stabilities of three cross-linked lysozymes thus obtained were investigated in 0.1 M acetate buffer containing 3 M guanidine hydrochloride at pH 5.5. All derivatives were stabilized but to different degrees. The derivative cross-linked with 1-4 was most stabilized (2.3 kcal/mol), but the derivatives cross-linked with the reagents both shorter (1-2) and longer (1-6) than 1-4 were less stabilized (both 1.6 kcal/mol).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetamides↗
PubMed1985

Ciliary activity in patients with nasal allergies.

Nasal allergy is a disease ensuing from type-I allergic reactions in the nasal cavity. A correct understanding of this process requires further investigation of the non-allergic pathology present. In the allergic state, the function of the nasal cilia is considered to be involved in the invasion of allergens into the nasal mucosa, and may also be involved with the manifestation and progression of the disease produced. In the present study, we examined the ciliary activity of the nasal mucosa of 35 patients with nasal allergies by using the photo-electric method of Ohashi and Nakai to better understand the non-allergic pathologies present. The nasal cilia of four healthy volunteers served as controls. In addition, a multivalent analysis was made according to quantifying theory I in order to define the effects of various factors on ciliary activity in the nasal mucosa during the allergic state. The following results were found in our present study. Ciliary activity was observed in 32 of 35 patients with nasal allergy. The mean value of the ciliary activity in these patients was 498 +/- 195 beats/min, while that of the normal controls was 753 +/- 46 beats/min. The ciliary activity in the nasal mucosa in the allergic state declined as the morbid period was prolonged; this decline in ciliary activity was relatively great in cases of perennial allergies aggravated by season-specific allergens.

Adolescent↗
PubMed1985

Chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozyme.

Preparation of chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozymes and its application to separation of N-bromosuccinimide-oxidized lysozymes are described. By pH gradient elution, two diastereomers of oxindolealanine-62-lysozyme, delta 1-acetoxytryptophan-62-lysozyme (intermediate product in the reaction in acetate buffer), and native lysozyme were all separated within 40 min.

Animals↗
PubMed1985

A convenient S-2-aminoethylation of cysteinyl residues in reduced proteins.

The cysteinyl residues in proteins had been S-2-aminoethylated with ethylenimine quantitatively. However, ethylenimine is now listed as a carcinogen and is no longer commercially available. A method of converting cysteinyl residues to S-2-aminoethyl derivatives quantitatively using 2-bromoethylamine under mild conditions was developed here.

Chromatography, High Pressure Liquid↗
PubMed1984

1H NMR studies of aspartate aminotransferase. Histidyl residues of cytosolic and mitochondrial isoenzymes.

200 MHz proton nuclear magnetic resonance spectra were compared between the cytosolic (cAAT) and mitochondrial (mAAT) isoenzymes of aspartate aminotransferase (EC 2.6.1.1) from pig heart. The pattern of signal distribution in the whole spectral region differed considerably between the two isoenzymes, reflecting the difference in their amino acid sequences. A group of distinct signals were resolved at elevated temperatures (50 to 70 degrees C) in the low field region (9.0 to 7.5 ppm) of the spectra of both isoenzymes in the pyridoxal form. Most of these signals were also observable at 28 degrees C although some showed considerable line broadening. Among resonance lines in this spectral region, cAAT in the pyridoxal form showed four pH-titratable resonances with pKa of 9.54, 6.72, 5.69, and 4.87 at 28 degrees C. Variation in pK and line width of these signals indicated differences in the microenvironment of histidyl residues. On the other hand, mAAT showed six pH-titratable resonances with pKa of 6.73 (peak 2), 6.77 (peak 3), 6.07 (peak 4), 4.71 (peak 5), 4.54 (peak 6), and 4.33 (peak 7). Peaks 2, 3, and 4 were narrow and others were considerably broad. Thus, only part of the histidyl residues present in each isoenzyme (8 and 10 His/monomeric unit of cAAT and mAAT, respectively) appeared on the spectra as pH-titratable resonances. With both isoenzymes, chemical shift and pKa values of these signals obtained for the pyridoxal form were indistinguishable from those for the pyridoxamine form and the borohydride-reduced form. None of the observable signals were affected upon the interaction of cAAT with glutarate. By contrast, peaks 2 and 4 in mAAT showed subtle but distinct chemical shift changes upon complex formation with succinate, suggesting that these two resonances are due to histidyl residues located at the part of the enzyme molecule which undergoes a conformational change upon the interaction with the dicarboxylate.

Animals↗
PubMed1984

Responsiveness of solitario-parabrachial relay neurons to taste and mechanical stimulation applied to the oral cavity in rats.

A total of 311 units, responsive to natural stimulation of the oral cavity, were isolated from the rostral part of the solitary tract nucleus (NTS) of rats. Of these, 169 "taste neurons", activated by taste stimulation, and 142 mechanoreceptive units, exclusively sensitive to mechanical stimulation of the oral cavity, were found. Most taste units (62.3%) were also excited by mechanical stimulation. Forty-three (34.1%) of the 126 taste units examined were identified as solitario -parabrachial relay (SP) neurons by antidromic stimulation from the ipsilateral dorsal pons, while only eleven (12.6%) of the 87 mechano-receptive units were SP neurons. Taste SP neurons could be divided into two subgroups according to their antidromic latency; the fast SP units with an antidromic latency shorter than 9 ms and slow SP units with a longer antidromic latency. These two subgroups were not differentiated in any physiological properties except that the fast SP neurons were frequently excited by sucrose. Taste neurons were classified according to the best stimulus of the four basic taste solutions to produce the largest number of discharges in each neuron. All types of taste neurons were found among the SP and non-SP neurons, but only a small number of quinine-best neurons (n = 2) were found in the SP neuron group compared to the number of quinine-best neurons in the non-SP neuron group (n = 10). A histological examination of the recording sites revealed that taste relay neurons were found at the central or dorsal part of the nucleus but mechanoreceptive relay neurons were found at the peripheral part, although relay and non-relay neurons of either class were intermingled in the nucleus.

Animals↗
PubMed1984

Discrimination of peripheral and central vestibular disorders by means of a quantitative statistical method.

This paper presents an approach to the prediction of peripheral and central vestibular disorders from a mathematical statistical point of view. An analysis was performed by the quantification method for multi-dimensional qualitative data. The data analysed were from 713 patients with a complaint of dizziness or vertigo who were subjected mainly to neurootological tests on 14 items. The categorical discriminant analysis of peripheral and central vestibular disorders revealed a discriminant success rate of 82%. We used the following five tests in discrimination: optokinetic test (OKN), caloric test, eye-tracking test (ETT), hearing test, and standing test. A system for the screening of patients with vertigo was assessed by use of a microcomputer and is in daily use in our department.

Central Nervous System Diseases↗
PubMed1984

Nature of the binding site around and reactivity of histidine-15 in lysozyme.

Bromoacetamide derivatives having n-alkyl substituents (BrCH2CONH(CH2)nH, 1-n) and carboxyalkyl substituents (BrCH2CONH(CH2)nCOOH, 2-n) react with His-15 in lysozyme exclusively at N epsilon 2 at pH 5.5 and 40 degree C. Kinetic studies suggest that lysozyme has a small hydrophobic pocket that binds these reagents in the vicinity of His-15.

Alkylation↗
PubMed1984

Otoconia degradation.

During certain stages of mammalian inner ear development, small crystallized bodies which resemble otoconia may be found in the endolymphatic sac. In order to examine whether the endolymphatic sac plays any part in the process of degradation and dissolution of otoconia, we made an electron-microscopic examination on the endolymphatic sac of fetuses and adult guinea pigs injected with streptomycin sulfate (SM). In 30-day-old fetal guinea pigs we found miniature otoconium-like bodies (OLBs) in the endolymphatic sac and a giant OLB in the endolymphatic duct. In adult animals we found no otoconia in the endolymphatic sac following SM intoxication. However, the results suggested that both the dark cells of the utricle, as well as the non-sensory epithelium of the saccule, may be engaged in the absorption and dissolution of otoconia.

Animals↗