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N Tamiya

Publications and source records attributed to N Tamiya.

At least 91 records · Page 5Linked to original sources
PubMed1980

Molecular conformation and function of erabutoxins as studied by nuclear magnetic resonance.

The 270-MHz proton NMR spectra of erabutoxins a, b and c from Laticauda semifasciata in 2H2O solution were observed together with [15-N6-acetyllysine]erabutoxin b, [27-N6-acetyllysine]-erabutoxin b and [47-N6-acetyllysine]erabutoxin b. The lysine epsilon-methylene proton resonances of erabutoxin b are assigned to individual residues. The epsilon-methylene proton resonance of Lys-27 is significantly broad, indicating that the mobility of this residue is restricted. Upon acetylation of Lys-27 of erabutoxin b, the pKa values of three other lysine residues are lowered by about 0.2, indicating long-range interactions among lysine residues. All the methyl proton resonances are assigned to amino acid types, primarily by the spin-echo double-resonance method. The pH dependences of proton chemical shifts were analyzed by the nonlinear least-square method, for obtaining pKa values and protonation shifts. The interproton nuclear Overhauser effect enhancements were measured for elucidating the spatial proximity of methyl-bearing residues and aromatic residues. On the basis of these NMR data and with the crystal structures by Low et al. and by Petsko et al., the methyl proton resonances of all the valine, leucine, and isoleucine residues and Thr-45 have been identified. The microenvironments of Tyr-25, His-26, Trp-29, four lysines and eight methyl-bearing residues have been elucidated. The addition of the paramagnetic hexacyanochromate ion causes broadening of the proton resonances of Thr-45, Lys-47, Ile-50, Trp-29 and Ile-36 residues located on one end of the molecule of erabutoxin b. The positively charged invariant residues of Lys-47 and Arg-33 at this part of the molecule are probably involved in the binding to the receptor protein.

Chromates↗
PubMed1979

Phospholipase A of sea snake Laticauda semifasciata venom. Isolation and properties of novel forms lacking tryptophan.

The venom gland extracts of the sea snake Laticauda semifasciata contained at least four forms of phospholipase A separable on a CM-cellulose column. They were designated as phospholipases A I-IV in the order of elution from the column. Phospholipases A I, III, and IV were isolated in a homogeneous state. They were similar to one another in amino acid composition and molecular weight (14,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phospholipase A I contained one tryptophan residue. whereas III and IV did not. Although all these forms had the same A2-type positional specificity, they were classified into two groups (I, and III and IV) on the basis of enzymic properties. Phospholipase A I had a higher specific activity and showed normal kinetics, whereas III and IV had approximately one-tenth of the specific activity of I and showed biphasic kinetics due to their activation by the reaction products. Phospholipase A I, the major form, seems to be identical with phospholipase A reported previously (Tu, A.T., Passey, R.B., & Toom, P.M. (1970) Arch. Biochem. Biophys. 140, 96-106), whereas the other two, III and IV, are new. Phospholipase A I became more like III and IV in enzymic properties on modification with N-bromosuccinimide.

Amino Acids↗
PubMed1978

Three neurotoxins from the venom of a sea snake Astrotia stokesii, including two long-chain neurotoxic proteins with amidated C-termini.

From the venom of a sea snake Astrotia stokesii three neurotoxic components, toxins Astrotia stokesii a, b and c were isolated in 40, 15 and 5% yield by weight respectively of the whole venom. Their LD50 values for 20g mice were 0.13, 0.096 and 0.098 microgram/g body wt. respectively and accounted for almost all the lethal activity of the venom. Their amino acid sequences were determined. Astrotia stokesii a was composed of 60 amino acid residues with nine half-cystine residues and was quite homologous to other sea-snake short-chain neurotoxins in its amino acid sequence. Toxins Astrotia stokesii b and c were composed of 70 and 72 amino acid residues respectively with 10 half-cystine residues. They are the first long-chain neurotoxins with high activity isolated from sea-snake venoms. The C-terminal carboxy groups of toxins b and c were found to be amidated; the amidation is known for some polypeptides, but is novel for a protein. The amide group may make a hydrogen-bond with glutamic acid-39, which replaces a lysine that has so far been found invariably in long-chain neutrotoxins. Astrotia stokesii b and c are also novel in having phenylalanine-25 and isoleucine- or valine-42. The ordinary Tyr-Glu pair, which is observed in X-ray structure [Low, Preston, Sato, Rosen, Searl, Rudko & Richardson (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2991-2994] and n.m.r.study [Inagaki, Tatsumi, Miyazawa, Hori & Tamiya (1977) Abstr. Int. Congr. Pure Appl. Chem. 26th, p. 336] on erabutoxins may be replaced by a hydrophobic pair. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 5009o (30 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7B1, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

Amides↗
PubMed1978

Conformation of snake toxic polypeptides studied by a method of prediction and circular dichroism.

Short and long neurotoxins as well as cardiotoxins belong to three distinct families of homologous toxic polypeptides extracted from cobra venoms. A study of their conformation was undertaken by using the method of Chou and Fasman for prediction of secondary structures of proteins. To improve the reliability of this method, an averaging scheme was developed. The data obtained showed that all toxins have a predominant trend for beta-sheet nucleation. Moreover, predicted beta-sheet strands fitted well those actually observed from X-ray data. Thus, it seems that all toxins share similarities in their secondary structure. This proposition was supported by a comparative study of the CD spectra of a set of toxins. Nevertheless, the present data suggest also that each type of toxins possesses localized structural individualities which might be responsible for the biological and/or immunological specificities.

Amino Acid Sequence↗
PubMed1978

Conformation of erabutoxins a and b in aqueous solution as studied by nuclear magnetic resonance and circular dichroism.

The 270-MHZ nuclear magnetic resonance (NMR) spectra of erabutoxins a and b have been observed in 2H2O solution. By the use of convolution difference and double resonance techniques, proton signals in the aromatic and methyl regions have been assigned. From the pH dependence of NMR chemical shifts, the pKa value of His-26 of erabutoxin b is found to be 5.8, whereas His-7 of erabutoxins a and b is not protonated at pH above 3. The imidazole ring of His-7 is protonated upon the denaturation at pH 2.85. The acid denaturation process has been followed by the His-26 and methyl proton signals and is found to be reversible but is slow as compared with NMR chemical shift time scale. The circular dichroism (CD) of erabutoxin b has also been observed. The denaturation is found to involve a major change from the beta-rich conformation to a disordered one. The NMR and CD changes upon acid denaturation are satisfactorily explained by the two-state process. The deuterium exchange rates of the C-2 protons of His-26 and His-7 of erabutoxin b indicate that His-26 is exposed to the solvent whereas His-7 is tightly buried in the interior of the protein globule. The pKa value of Tyr-25 is as high as about 12.0, possibly due to the hydrogen bond formation between the hydroxyl group of Tyr-25 and a carboxylate group. The hydroxyl group of Tyr-25 is reversibly titrated so that this group is not buried tightly in the interior of the protein globule. The line width of the aromatic proton signals of Tyr-25 is significantly broad at room temperature, suggesting a restricted rotation of the aromatic ring. The aromatic proton signals of Trp-29 are fairly sharp; this aromatic ring is exposed and mobile. Except for His-7, the micro-environments of Tyr-25, His-26, and Trp-29 residues and methyl proton signals of valine and isoleucine are consistent with the locations of alpha carbon atoms as elucidated by X-ray crystal analyses.

Amino Acid Sequence↗
PubMed1976

Conformational changes in two neurotoxic proteins from snake venoms.

alpha-Neurotoxin from Naja nigricollis and erabutoxin b from Laticauda semifasciata, two homologous neurotoxic proteins, are studied by circular dichroism, ultraviolet spectroscopy and fluorescence in various water/trifluoroethanol mixtures. The data obtained show that the beta structure of alpha-neurotoxin is conserved in water as well as in the organic solvent. By contrast, erabutoxin b changes from the beta-structure in water to the helix type in trifluoroethanol. The latter induces similarly for both toxins a structural modification around tryptophan 29, a residue common to all neurotoxins known to date. The vicinity of tyrosine 25, another common amino acid, is also altered by the presence of the organic solvent as demonstrated by the sudden increase of reactivity of the phenolic ring towards iodine. The present work affords some evidence for the presence of a particular structure located around the two aromatic residues, which is common to all neurotoxins and able to rearrange independently from the rest of the molecule. Biological importance of this peculiar region is highly probable.

Animals↗
PubMed1976

Preparation and activity of guanidinated or acetylated erabutoxins.

1. Erabutoxins, a, b and c, neurotoxic proteins of a sea snake Lacticauda semifasciata, were guanidinated with O-methylisourea. The amino groups of all the lysine residues and those at the N-termini of the toxins were modified. The lethal activity of the toxins decreased to 50% (erabutoxins a and b) or 17% (erabutoxin c) of the original value on the modification. The c.d. (circular dichroism) maximum at 227 nm of the modified toxins became lower, whereas the whole profile of the c.d. curve remained unchanged. 2. The amino groups of erabutoxin b were acetylated with acetic anhydride. All the five monoacetyl derivatives were isolated from the reaction products by CM-cellulose and Bio-Rex 70 column chromatography. [1-Nalpha-acetylarginine]-, [15-N6-acetyl-lysine]- and [51-N6-acetyl-lysine]-erabutoxin b retained the toxicity of the native toxin, whereas [27-N6-acetyl-lysine] and [47-N6-acetyl-lysine]-erabutoxin b were 17 and 8% active respectively. The overall profile of c.d. spectrum of erabutoxin b remained unchanged on the monoacetylation.

Acetylation↗
PubMed1976

Isolation, properties and amino acid sequences of three neurotoxins from the venom of a sea snake, Aipysurus laevis.

Aipysurus laevis venom was chromatographed on CM-cellulose and Bio-Rex 70 columns. Three neurotoxic components, toxins Aipysurus laevis a, b and c, were isolated. The toxins a, b and c corresponded to 22, 33 and 21% respectively of the proteins in the original venom, and accounted for almost all the lethal activity of the venom. The three toxins a, b and c were monodisperse on disc electrophoresis at pH4; toxins a and b moved at the same velocity and c a little faster. They were monodisperse also on sodium dodecyl sulphate-polyacrylamide-disc-gel electrophoresis, giving a molecular weight of 7600. The molecular weight of toxin b estimated by gel filtration was 7000. The amino acid sequence analyses of these toxins revealed that they consisted of 60 amino acid residues and that Aipysurus laevis b was [25-methionine, 28-arginine] Aipysurus laevis a. Aipysurus laevis c was [28-lysine] Aipysurus laevis a, the tryptic peptide sequence relying on homology. The LD50 values of these toxins for 20g mice were 0.076 mug/g body wt. They inhibited the acetylcholine-induced contracture but did not affect the CKl-induced contracture of the isolated muscle.

Amino Acid Sequence↗