Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.69 (botulinum neurotoxin)
 1,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Torpedo electric organ has been used to study the binding of botulinum neurotoxin type A to pure cholinergic synaptosomes and presynaptic plasma membrane. 125I-labeled botulinum neurotoxin type A exhibits specific binding to cholinergic fractions. Two binding sites have been determined according to data analysis: a high affinity binding site (synaptosomes: Kd = 0.11 +/- 0.03 nM, Bmax = 50 +/- 10 fmol.mg prot-1; presynaptic plasma membrane: Kd = 0.2 +/- 0.05 nM, Bmax = 150 +/- 15 fmol.mg prot-1) and a low affinity binding site (synaptosomes: Kd approximately 26 nM, Bmax approximately 7.5 pmol.mg prot-1; presynaptic plasma membrane: Kd approximately 30 nM, Bmax approximately 52 pmol.mg prot-1). The binding of 125I-botulinum neurotoxin type A is decreased by previous treatment of synaptosomes by neuraminidase and trypsin, and by a preincubation with bovine brain gangliosides or antiserum raised against Torpedo presynaptic plasma membrane. When presynaptic plasma membranes are blotted to nitrocellulose sheet, either 125I-botulinum neurotoxin or botulinum toxin-gold complexes bind to a M(r) approximately 140,000 protein. Botulinum toxin-gold complexes have also been used to study the toxin internalization process into Torpedo synaptosomes. The images fit the three step sequence model in the pathway of botulinum neurotoxin poisoning.
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PMID:Binding of botulinum neurotoxin to pure cholinergic nerve terminals isolated from the electric organ of Torpedo. 133 17

Production of botulinum-like neurotoxin by a non-Clostridium botulinum organism has profound implications in the epidemiology of the disease botulism. Molecular topography of the approximately 150 kDa neurotoxic protein produced by Clostridium butyricum (strain 5839) and its activation kinetics were examined and compared with a serologically related botulinum neurotoxin produced by C. botulinum type E to further characterize the butyricum neurotoxin. Botulinum neurotoxin was fully activated within 30 min of incubation with trypsin, whereas butyricum neurotoxin achieved maximum activation within 5 min of incubation. Molecular topography of the two neurotoxins was analyzed in terms of secondary structures and the surface accessibilities of the polypeptide domains containing aromatic amino acids. The secondary structure parameters of the butyricum neurotoxin (alpha-helix 22%, beta-sheet 41% and random coil 37%), as estimated from the far ultraviolet circular dichroic spectra, appeared similar to that of botulinum neurotoxin. (Singh, B.R. and DasGupta, B.R., (1989) Mol. Cell. Biochem. 86, 87). Second derivative ultraviolet spectral analysis revealed 37 and 41 Tyr residues exposed on the surface of butyricum and botulinum neurotoxins, respectively, suggesting a differential surface accessibility of polypeptide segments containing Tyr residues. Fluorescent Trp residues in both the botulinum type E and butyricum neurotoxins were in a relatively hydrophobic environment as indicated by the blue-shifted emission maxima (334 nm). About half of the fluorescent Trp residues of both proteins were accessible to acrylamide, a neutral fluorescence quencher, and appeared to be in a similar molecular environment. The ionic surface probe, I-, quenched the Trp fluorescence of botulinum significantly, but not that of butyricum neurotoxin. Thus, a considerable number of fluorescent Trp residues were apparently located on the surface of the botulinum, but not on that of the butyricum neurotoxin. Botulinum and butyricum neurotoxins, indistinguishable by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, migrated differently in the absence of sodium dodecyl sulfate suggesting difference(s) in their surface charge distribution. These results provide the first report of the secondary and tertiary structure parameters of the neurotoxin produced by a non-botulinum species and comparison of the molecular topography of the neurotoxin with the antigenically related botulinum neurotoxin type E.
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PMID:Comparative molecular topography of botulinum neurotoxins from Clostridium butyricum and Clostridium botulinum type E. 190 Dec 21

Clostridium botulinum synthesizes the type A botulinum neurotoxin (NT) as a approximately 150 kDa single chain protein. Post-translational proteolytic processing yields a approximately 150 kDa dichain protein composed of a approximately 50 kDa light and approximately 100 kDa heavy chain, which has higher toxicity. Trypsin's action mimics the endogenous proteolytic processing. The proteolytic cleavages could occur at 4 sites. We have examined 2 such sites and defined the peptide sequences before and after proteolytic processing. The N-terminal residues of the newly synthesized approximately 150 kDa single chain NT, Pro-Phe-Val-Asn-Lys-, remain intact at the N-terminus of the approximately 50 kDa light chain generated either in the clostridial culture or in vitro with trypsin or with a protease purified from the homologous bacterial culture. The clostridial protease cleaves the single chain NT in vitro, at 1/3 the distance from its N-terminus, on the amino side of Gly of the sequence -Gly-Tyr-Asn-Lys-Ala-Leu-Asn-Asp-Leu- before cleaving the bond Lys-Ala at a slower rate. The data indicate that the dichain NT is formed in the bacterial culture in at least 2 steps. Cleavage at X-Gly produces a approximately 100 kDa heavy chain-like fragment which is then truncated; cleavage 4 residues downstream at Lys-Ala, and excision of the tetrapeptide Gly-Tyr-Asn-Lys, generates the mature heavy chain with Ala as its N-terminal residue. The approximately 100 kDa heavy chain generated in vitro, by nicking the single chain NT with trypsin, also has Ala-Leu-Asn- as the N-terminal residues.
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PMID:Botulinum neurotoxin type A: sequence of amino acids at the N-terminus and around the nicking site. 212 6

Response of the chick ciliary ganglion-iris muscle neuromuscular junction (NMJ) preparation to the botulinum neurotoxin (NT) was investigated. The 150 kDa serotypes A and E NTs inhibited muscle contraction in a dose dependent fashion. Neurotoxicity of type E NT increased 20-40 fold after mild digestion with trypsin. The 50 kDA light and 100 kDa heavy chains of type A NT, following separation, applied individually, did not paralyze the tissues. Preincubation of the NMJ preparations with the isolated type A heavy chain delayed (antagonized) the paralytic action of the 150 kDa dichain type A NT. Sequential administration of type A heavy chain, followed by type A light chain mimicked the action of the parent NT. The chick ciliary preparation therefore is a useful NMJ preparation to study neurotoxicity of botulinum neurotoxins.
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PMID:Response of the chick ciliary ganglion-iris neuromuscular preparation to botulinum neurotoxin. 216 81

1. A large-scale purification procedure has been developed for Clostridium botulinum type F neurotoxin. Commencing with 160 litres of bacterial culture, 101 mg of purified type F neurotoxin with a specific toxicity of 2 x 10(7) mouse LD50 (median lethal dose).mg-1 were obtained. 2. Purified type F neurotoxin was labelled to high specific radioactivity (900-1360 Ci/mmol) without loss of biological activity using a chloramine-T procedure. Of the two neurotoxin subunits, the heavy chain was preferentially radiolabelled. 3. Radiolabelled type F neurotoxin displayed specific saturable binding to rat synaptosomes. At least two pools of acceptors were evident: a low content of high-affinity acceptors sites [KD approximately 0.15 nM; Bmax (maximal binding) 20 fmol/mg] and a larger pool of lower-affinity sites (KD greater than 20 nM; Bmax greater than 700 fmol/mg). Both pools of acceptors were sensitive to trypsin and neuraminidase treatment, which suggests that protein and sialic acid residues are components of the synaptosomal acceptors. 4. Experiments investigating competition among botulinum neurotoxin types A, B, E and F for acceptors on rat brain synaptosomes showed that type F neurotoxin binds to acceptor molecules which are completely distinct from those of the other three neurotoxins.
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PMID:Botulinum type F neurotoxin. Large-scale purification and characterization of its binding to rat cerebrocortical synaptosomes. 218 47

The alkaline pH induced difference spectra (270-310 nm) of three antigenically distinct forms of the botulinum neurotoxin (NT) types A, B and E were examined. When isolated from the cultures of Clostridium botulinum, type A NT is a fully toxic dichain (nicked) protein, type E is a mildly toxic single chain (unnicked) protein, and type B NT is a mixture of single and dichain proteins and near fully toxic. Trypsin nicks the single chain protein to the dichain and increases its toxicity (up to about 100 fold in type E). A strong difference spectrum peak at approximately 296 nm was found when types A, B or E NT were in the alkaline pH region. This peak was not observed at pH 4.0. For types A and B NT plots of difference absorptivity vs. pH were simple sigmoidal curves. The pK of phenolic moieties of tyrosine residues in both proteins were 10.9. Nearly all tyrosine residues in both proteins were ionized. The single chain type E, unlike type A and B NT, yielded a two step titration curve and pK values 11.3 and less than 7.5; about 60% of the total tyrosine residues present were ionized. The two step titration curve was not observed when the single chain protein was nicked with trypsin to the dichain type E NT. The titration curve of dichain type E NT, although complex, was more like those of type A and B NT.
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PMID:Botulinum neurotoxin types A, B & E: pH induced difference spectra. 305 Apr 52

The 145-kDa type A botulinum neurotoxin (NT) is produced by the bacteria Clostridium botulinum (strain, Hall). The heavy (H) and light (L) chains (97- and 53-kDa, respectively) of this protein are linked by at least one disulfide bond. The N- and C-terminal halves of the H chain appear to have different functions in the mechanism of action of the NT [1987) FEBS Lett. 226, 115-120). Well-characterized and highly purified preparations of the two halves of the H chain are needed for such studies. Two different approaches were taken to cut the H chain with trypsin and isolate the fragments. In one method the cleavage products were: (i) 94-kDa fragment made of the L chain linked to the N-terminal half of the H chain (49 kDa) by a disulfide bond(s), and (ii) the C-terminal 44-kDa fragment. The N-terminal half of H chain was separated from the L chain by reducing the disulfide bond(s) linking them and then purified by ion-exchange chromatography. The 1-27 residues of 49-kDa N-terminal half of the H chain were Ala-Leu-Asn-Asp-Leu-Cys-Ile-Lys-Val-Asn-Asn-Trp-Asp-Leu-Phe-Phe-Ser-Pro- Ser-Glu - Asp-Asn-Phe-Thr-Asn-Asp-Leu-. The sequence of the other half of the H chain (44 kDa) was X-Ile-Ile-Asn-Leu-X-Ile-Leu-Asn-Leu-Arg-Tyr-Glu-X-Asn-His-Leu-Ile-Asp-Le u-Lys- X-Tyr-Ala-Ser-. In the second method, the H chain was first separated from the L chain, purified, and then cleaved. One product of cleavage, the 44-kDa fragment, was partially sequenced; the first 25 residues were identical to the sequence of the 44-kDa fragment generated by the first method. The present work also demonstrated that (i) The cysteine residue(s) located on the N-terminal half of the H chain form the -S-S- link(s) with the L chain. (ii) The other half of the H chain (44-kDa fragment, apparently the C-terminal half) is not linked via -S-S- to the L-chain or to the N-terminal half (49-kDa fragment) of the H chain.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Botulinum neurotoxin type A: cleavage of the heavy chain into two halves and their partial sequences. 317 18

The binding ability of Cl. botulinum neurotoxin to synaptosomes upon treatment with various enzymes (neuraminidase, trypsin, and beta-bungarotoxin containing phospholipase A2 activity) was studied. When synaptosomes were treated with neuraminidase, their ability to bind toxin decreased; trypsin and beta-bungarotoxin had slightly week or no effect. The decrease in toxin-binding ability of synaptosomes was paralleled by a release of sialic acid from the synaptosomes by the neuraminidase treatment. The toxin-binding ability of synaptosomes treated with neuraminidase was lower than untreated ones at a high concentration of sodium chloride. The binding of the toxin to synaptosomes occurred at least at the two types of structural sites, one site which contained sialic acid, and other site which was sensitive to high ionic strength. It may be possible that another binding state except these is present at the synapse.
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PMID:Binding ability of Clostridium botulinum neurotoxin to the synaptosome upon treatment of various kinds of the enzymes. 356 63

Neurotoxin from Clostridium botulinum type B was purified to homogeneity by by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (Mr of approximately equal to 155 000) following trypsinisation attained a level of 2 X 10(8) mouse LD50 units/mg protein. 125I-iodination of the toxin to high specific radioactivities (19-63 TBq/mmol) yielded typically greater than 65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (Mr of approximately equal to 101 000) was labelled preferentially. Saturable binding of the 125I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (Kd = 0.3-0.5 nM;Bmax approximately equal to 30-60 fmol/mg protein, together with a much larger population of weak-affinity sites. No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinized) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction. Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 muM), and other neurotoxins were without effect, emphasising the acceptor selectivity. Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively). These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors. Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM 125I-iodinated toxin. Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity. These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.
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PMID:Botulinum neurotoxin type B. Its purification, radioiodination and interaction with rat-brain synaptosomal membranes. 375 81

The dichain type E botulinum neurotoxin, a product of nicking the single chain protein by trypsin, is composed of a heavy and light chains. Sequence of the first 13 and 20 N-terminal residues of these two chains were determined. Also, proof is provided here that (i) the light chain of the nicked (dichain) is derived from the N-terminal one-third of the parent single chain neurotoxin, and (ii) molecular events leading to the activation, of the single chain neurotoxin cannot involve tryptic cleavage at or very close to the N-terminal of the single chain protein. The partial amino acid sequence of the light chain of botulinum type E and tetanus neurotoxins show significant similarity between the two clostridial neurotoxins.
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PMID:Partial amino acid sequences of the heavy and light chains of botulinum neurotoxin type E. 398 55


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