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)

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

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

A novel assay method based on the endopeptidase activities of the botulinum neurotoxins has been developed and applied to the detection of botulinum type A and B toxins. An assay system developed for the detection of botulinum type B neurotoxin (BoNT/B) is based on the cleavage of a synthetic peptide substrate representing amino acid residues 60 to 94 of the intracellular target protein for the toxin, VAMP (vesicle-associated membrane protein, or synaptobrevin). In this assay system, immobilized VAMP (60-94) peptide substrate is cleaved by BoNT/B at the Gln-76-Phe-77 bond, leaving the C-terminal cleavage fragment on the solid phase. This fragment is then detected by the addition of an antibody-enzyme reagent which specifically recognizes the newly exposed N terminus of the cleavage product. The developed assay was specific to BoNT/B, showing no cross-reactivity with other clostridial neurotoxins, and had a sensitivity for BoNT/B of 0.6 to 4.5 ng/ml, which could be increased to 0.1 to 0.2 ng/ml by using an assay amplification system based on catalyzed reporter deposition. Trypsin treatment of BoNT/B samples, which converts the single-chain toxin to the active di-chain form, was found to increase the sensitivity of the endopeptidase assay from 5- to 10-fold. An endopeptidase assay for BoNT/A, based on the cleavage of a peptide substrate derived from the protein SNAP-25 (synaptosome-associated protein), was also developed and characterized.
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PMID:Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities. 881 85