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N Tamiya

Publications and source records attributed to N Tamiya.

At least 73 records · Page 4Linked to original sources
PubMed1982

Isoelectric points of erabutoxins and monoacyl derivatives of erabutoxin b. Estimation of the pK values of amino groups in erabutoxins by using isoelectric-focusing data.

The isoelectric points of erabutoxins a, b and c, neurotoxic proteins of a sea snake, Laticauda semifasciata, were determined by density-gradient isoelectric focusing. The same measurement was also made with monoacyl derivatives of erabutoxin b, in which each one of all amino groups had been either acetylated or propionylated. Erabutoxins a and b showed the same isoelectric point at pH 9.68. The values for ]1-N alpha-acetyl-arginine]-, [15-N6-acetyl-lysine]-, [27-N6-acetyl-lysine]-, [47-N6-propionyl-lysine]- and [51-N6-acetyl-lysine]-erabutoxin b were at pH 9.52, 9.31, 9.45, 9.22 and 9.09 respectively, being definitely different from each other and lower than the value for the unmodified molecule. The isoelectric point of erabutoxin c, which is [51-asparagine]-erabutoxin b, was the same as that of [51-N6-acetyl-lysine]erabutoxin b. Assuming that no change in pK occurs on monoacylation, the pK values of amino groups in erabutoxin b were calculated from the isoelectric-point data. It is indicated that the pK values of zeta-amino groups differ markedly from each other and that the value of alpha-amino group is anomalously high.

Amino Acids↗
PubMed1982

Isolation and properties of lysophospholipases from the venom of an Australian elapid snake, Pseudechis australis.

Two lysophospholipases were isolated from the venom of an Australian elapid snake (subfamily Acanthophiinae), Pseudechis australis, by sequential chromatography on CM-52 cellulose, Sephadex G-75 and DE-52 cellulose columns. They were very similar to each other. One of them, lysophospholipase I, was obtained as a homodimer, the monomer of which consisted of 123 amino acid residues with seven disulphide bridges. The amino acid composition and the N-terminal amino acid sequence of the enzyme were similar to those of phospholipase A2, Ca2+ was required for its activity and the maximum activity was attained at 2 mM-CaCl2 in the presence of 1 mM-EDTA. The optimum pH was 7.5. Lysophospholipase I hydrolysed lysophosphatidylcholine more rapidly than lysophosphatidylethanolamine. It did not hydrolyse, however, phosphatidylcholine, 1-palmitoylglycerol, tripalmitoylglycerol or p-nitrophenyl acetate. Modification of the enzyme with p-bromophenacyl bromide or 2-nitrophenylsulphenyl chloride suppressed the activity. A strong direct haemolytic activity was exhibited when the lysophospholipase was present together with phospholipase A2.

Acetophenones↗
PubMed1982

Structural dynamics of erabutoxin b. A 13C nuclear magnetic resonance relaxation study of methyl groups.

A detailed examination has been made of the 13C NMR relaxation times of the assigned methyl groups of erabutoxin b. Anisotropic rotation was analysed using a restricted diffusion model. The results are compared with a previous study of the 1H NMR relation times. There is good agreement on the segments of the molecule which show restricted motion. Our results are compared with those of previous studies on proteins in solution and in crystals and again the general agreement is good. We have attempted to interpret the value of the limited motion seen in the protein to the reactions of the neurotoxin.

Elapid Venoms↗
PubMed1982

Dynamics of erabutoxin b as studied by nuclear magnetic resonance. Relaxation studies of methyl proton resonances.

Longitudinal and transverse relaxation times were measured for well-resolved and assigned methyl proton resonances of erabutoxin b at 270 MHz, 300 MHz and 500 MHz. Both longitudinal and transverse magnetization decay curves are non-exponential due to cross-relaxation and cross-correlation effects. The longitudinal and transverse relaxation rates were obtained from the initial slope of both magnetization decay curves. The correlation times for the isotropic tumbling motion of the protein were determined to be 2.82 ns at 300 K and 1.62 ns at 330 K from the analysis of the relaxation data of some alpha protons. Using these values, the relaxation data of methyl protons were fitted to various theoretical models. Most of the methyl resonances could be fitted well to a model which allowed methyl rotation (in the range 0.01-0.05 ns) and an external contribution from protons assumed to be in positions derived from X-ray coordinates. The data for a few methyl groups, however, could not be fitted in this way. For these a smaller number of external protons than predicted by the X-ray coordinates was assumed. Additionally, a larger amplitude motion had to be introduced into the model for particular residues. This additional motion requires concerted protein motion close to these residues, since the X-ray structure suggests that steric hindrance would prevent local motion. These results are consistent with the idea of a flexible and dynamic structure for proteins.

Elapid Venoms↗
PubMed1982

Individual assignments of the amide proton resonances involved in the triple-stranded antiparallel pleated beta-sheet structure of a long neurotoxin, Laticauda semifasciata III from Laticauda semifasciata.

THe characteristic feature of the crystal structure of erabutoxin b, a short neurotoxin from Laticauda semifasciata, and alpha-cobratoxin, a long neurotoxin from Naja naja siamensis, is the presence of a triple-stranded antiparallel pleated beta-sheet structure formed by the central and the third peptide loops. In the present study, we have studied the assignment of slowly exchangeable amide protons of Laticauda semifasciata III from L. semifasciata, using nuclear Overhauser effects (NOE) and spin-decoupling methods. The results show that nearly all of the slowly exchangeable amide protons are to be assigned to the back-bone amide protons, involved in the triple-stranded antiparallel pleated beta-sheet structure, indicating that this sheet is stable in 2H2O solution. In contrast, the amide protons in short neurotoxins are readily exchangeable under the same experimental condition, suggesting that long neurotoxins have a more rigid sheet structure than short ones. This rigidity may come from the hydrophobic and hydrogen bond interaction between the central loop and the tail, which is not present in short neurotoxins. Since the functionally important residues are located on this beta-sheet, the different kinetic properties of the neurotoxins are well correlated with the difference in the rigidity of the beta-sheet.

Amines↗
PubMed1982

Non-destructive detection of methionine sulfoxide in the resilium of a surf clam by solid-state 13C-NMR spectroscopy.

Methionine sulfoxide was detected in the resilium (internal hinge ligament) of a surf clam by high-resolution solid-state 13C-NMR spectroscopy involving cross-polarization and magic angle spinning, using no chemical procedure. The results support the previous report [Kikuchi, Y. and Tamiya, N. (1981) J. Biochem. (Tokyo) 89, 1975-1976] on a high content of methionine sulfoxide observed by chemical methods in the resilium protein of surf clam species.

Amino Acids↗
PubMed1981

The amino acid sequence and position of the free thiol group of a short-chain neurotoxin from common-death-adder (Acanthophis antarcticus) venom.

The amino acid sequence of a short-chain neurotoxin Acanthophis antarcticus c (toxin Aa c) from the venom of an Australian elapid snake, the common death adder (Acanthophis antarcticus, subfamily Acanthophiinae) was elucidated. Toxin Aa c is composed of 62 amino acid residues, including eight half-cystine residues and a cysteine residue. The amino acid sequence of toxin Aa c is homologous with those of other short-chain neurotoxins found in snakes of the family Elapidae, especially with those from snakes of the subfamily Hydrophiinae. The single cysteine residue was located in position 4. Toxin Aa c has a lethal dose (LD50) of 0.08 micrograms/g body weight of mouse on intramuscular injection.

Amino Acid Sequence↗
PubMed1981

Structural differences between erabutoxins in aqueous solution and in crystalline states.

The snake venom toxins erabutoxin a and b, have been studied in detail in aqueous solution over a wide pH range by proton NMR spectroscopy. Great care has been taken particularly to assign and locate in the structure residue histidine-7 with reference to other amino-acid sidechains. It lies in a pocket away from the surface in agreement with its lack of chemical reactivity. The data are not consistent with two structure determinations in crystals by X-ray diffraction. The aqueous solution conformation explains in greater depth the observed essentially of amino acids to the activity of the toxin.

Crystallization↗
PubMed1981

Isolation, properties and amino acid sequence of a long-chain neurotoxin, Acanthophis antarcticus b, from the venom of an Australian snake (the common death adder, Acanthophis antarcticus).

The venom of an Australian elapid snake, the common death adder (Acanthophis antarcticus), was chromatographed on a CM-cellulose CM52 column. One of the neurotoxic components, Acanthophis antarcticus b (toxin Aa b) was isolated in about 9.4% (A280) yield. The complete amino acid sequence of toxin Aa b was elucidated. Toxin Aa b is composed of 73 amino acid residues, with ten half-cystine residues, and has a formula weight of 8135. Toxin Aa b has no histidine or methionine residue in its sequence. The amino acid sequence of toxin Aa b is homologous with those of other neurotoxins with known sequences, although it is novel in having a valine residue at its N-terminus and an arginine residue at position-23, where a lysine residue is found in almost all the so-far-known neurotoxins. Irrespective of the latter replacement, the toxin Aa b is fully active, with an LD50 value (in mice) of 0.13 microgram/g body weight on intramuscular injection.

Amino Acid Sequence↗
PubMed1981

Methionine sulfoxide in the resilium protein of surf clams.

A high content of methionine sulfoxide was observed in the resiliums (internal hingeligaments) of surf clams. As no isolation procedure which might cause the oxidation of methionine to methionine sulfoxide was involved and the hydrolysis was carried out in vacuo, it is the first solid evidence for the presence of methionine sulfoxide as a constituent of natural protein.

Amino Acids↗
PubMed1981

A proton-magnetic-resonance study on the molecular conformation and structure-function relationship of a long neurotoxin, laticauda semifasciata III from Laticauda semifasciata.

The 300-MHz and 500-MHz NMR spectra of a long neurotoxin laticauda semifasciata III (LS III) from Laticauda semifasciata have ben analysed. Comparison with the NMR spectra of alpha-cobratoxin from Naja naja siamensis, a homologous long neurotoxin to laticauda semifasciata III, allowed the assignment of all the aromatic protein resonances to specific amino acid residues. All the methyl proton resonances have been assigned to specific types of amino acid residues. The pH dependences of the aromatic and methyl proton chemical shifts were analyzed by the non-linear least-square method to give the pKa values and protonation shifts. The interproton nuclear Overhauser effect enhancements were measured in order to elucidate the spatial proximity of the methyl-bearing residues and aromatic residues. On the basis of these NMR data and using the crystal structure of alpha-cobratoxin by Walkinshaw et al., more than half of the methyl proton resonances have been assigned to specific amino acid residues. A hydrophobic core comprising the first loop, the central loop and the tail part of the molecule has been defined. This hydrophobic core may be common to all long neurotoxins and may protect the three-stranded antiparallel pleated beta-sheet structure, thus making the backbone structure of long neurotoxins more rigid than that of short neurotoxins. The positively charged surface of laticauda semifasciata III, which is responsible for binding to the acetylcholine receptor protein, is confirmed as the concave surface formed by the central and the third loop. The arrangement of the amino acid residues on this surface is similar to that of all other neurotoxins. Accordingly, the slow on-off rates of association of long neurotoxins with receptor is considered to arise from the rigid backbone structure. A small conformation change is thought to be associated with binding to the receptor protein.

Amino Acid Sequence↗
PubMed1980

Molecular dynamics of two homologous neurotoxins revealed by 1H-2H exchange: an infrared spectrometry study.

Temperature effects on the hydrogen exchange kinetics and the infrared spectra of two homologous snake neurotoxins (Laticauda semifasciata erabutoxin b and Naja nigricollis toxin alpha) were investigated between 10 and 40 degrees C, at their isoionic pH. (1) Erabutoxin b is more accessible to the solvent than toxin alpha. (2) With increasing temperature, both toxin molecules undergo a global transition affecting the most accessible as well as the most buried hydrogens: the overall accessibility changes are more important for erabutoxin b than for toxin alpha. The different conformational stabilities of the toxins are also qualitatively supported by the temperature-induced shifts which affect the infrared amide I band of toxin alpha only. The existence of two conformer families could be responsible for the different conformational stability of these proteins.

Animals↗
PubMed1980

Electronic spectroscopy and deuteration kinetics of tyrosine and tryptophan residues: an application to the study of erabutoxin b.

The fluorescence increase on the deuterium oxide addition to the solvent medium was studied in various tryptophan- (or indole-) and/or tyrosine-containing model compounds. It was shown how the rates of the deuteration at the indole NH group of tryptophan and at the OH group of tyrosine could be followed independently of each other. The method was applied to a study of erabutoxin b molecule, a neurotoxic protein from a sea snake, to analyze the microenvironments of its single tryptophan and tyrosine residues. It was shown that the "functionally invariant" single tryptophan residue was exposed to the solvent in the surface of the molecule and that the "structurally conserved" single tyrosine residue was buried in the molecule. The rate of deuteration of the tyrosine residue (80 s(-1) at pH 6.3 and 33 degrees C) was 1/20 of that of an exposed tyrosine. It was also found that the amino group of Lys-27 quenched the fluorescence of Trp-29 but its deuteration had no effect on the fluorescence.

Deuterium↗
PubMed1980

The acetylation of the amino groups of Laticauda semifasciata III, a sea snake venom component.

The amino groups of Laticauda semifasciata III (LsIII) purified from the venom of a sea snake, Laticauda semifasciata, were acetylated with acetic anhydride. Three monoacetyl derivatives of LsIII, namely, [1-N2-acetyl-arginine]-LsIII, [23-N6-acetyl-lysine]-LsIII and [35-N6-acetyl-lysine]-LsIII, were obtained. These monoacetyl derivatives of LsIII showed the same CD spectra as that of native LsIII. [1-N2-Acetyl-arginine]-LsIII was half as active as the original toxin suggesting the importance of the net positive charge of the toxin molecule for the toxicity. [23-N6-Acetyl-lysine]-LsIII and [35-N6-acetyl-lysine]-LsIII showed no toxicity. Lysine-23 is one of the residues common to all neurotoxins, whereas lysine-35 is one of the residues found specifically among long-chain neurotoxins, although there are some exceptional toxins without lysine-23 or lysine-35.

Acetylation↗