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)

Recombinant DNA techniques were used to develop an expression system for a 51-amino acid peptide fragment that encompasses residues 44-94 of human synaptobrevin 2. This protein is associated with secretory vesicles of nerve terminals and is a substrate for four of the seven serotypes of botulinum neurotoxin (BoNT). The DNA for the recombinant peptide was amplified by the polymerase chain reaction and cloned into the pTrxFus vector. The resulting synaptobrevin peptide was expressed as a thioredoxin fusion protein in E. coli and released into the medium by osmotic lysis. The 18.7-kDa thioredoxin-synaptobrevin protein, designated as TSB-51, is intended for use in a cell-free assay to test potential inhibitors of BoNT/B-mediated proteolysis of synaptobrevin with the ultimate aim of developing clinically effective therapeutic agents to counteract botulism. Incubation of TSB-51 with the purified light chain of BoNT/B resulted in proteolysis which was evident within 30 min and increased with time until completion (approximately 4 hr). Cleavage of TSB-51 appeared to be at the appropriate BoNT/B cleavage site as indicated by a reduced intensity of the 18.7-kDa band and the appearance of a band at 16.4 kDa on Tris-tricene polyacrylamide gradient gels. The concentration of free Zn2+ had a significant effect on the cleavage rate; low Zn2+ concentrations stimulated substrate cleavage, whereas high concentrations were inhibitory. Cleavage was not significantly depressed by the naturally occurring metalloprotease inhibitor phosphoramidon when tested at concentrations up to 5 mM. TSB-51 appears to be a useful substrate for studying BoNT/B and is expected to aid in the discovery of effective BoNT inhibitors.
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PMID:Production of an expression system for a synaptobrevin fragment to monitor cleavage by botulinum neurotoxin B. 971 40

In this work, the kinetics of the binding of botulinum neurotoxin A (BoNT/A) to ganglioside GT1b were studied using surface plasmon resonance (SPR). The neurotoxin bound polysialylated gangliosides, and that binding was affected by the ionic strength of the buffer. Although the level of binding was decreased at higher ionic strengths, it could be easily observed in Tris buffer, containing 150 mM NaCl. Data analysis revealed that the binding of BoNT/A to a GT1b-containing phospholipid monolayer did not fit a traditional 1:1 model. Subsequent studies, in which the time of contact between BoNT/A and GT1b was varied, indicated that the BoNT/A-GT1b complex became more stable over time, as evidenced by its reduced rate of dissociation. Circular dichroism indicated that when BoNT/A was incubated with GT1b, it underwent a conformational change that resulted in an increase in alpha-helix content and a decrease in beta-sheet content. Therefore, the SPR kinetic data were fit to a conformational change model and kinetic rate constants determined. The apparent K(D) values obtained for the binding of BoNT/A to ganglioside GT1b ranged from 2.83 x 10(-7) to 1.86 x 10(-7) M, depending on the ionic strength of the buffer.
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PMID:Botulinum neurotoxin A changes conformation upon binding to ganglioside GT1b. 1527 27

Fourteen monoclonal antibodies (mAbs) against Clostridium botulinum type A neurotoxin were raised in mice using a carboxy terminal fragment of the toxin for immunization. A two-site immunometric assay was developed which allowed detection of 8 LD(50)/ml (40 pg/ml) botulinum neurotoxin A and an accurate quantification close to 25 LD(50)/ml (150 pg/ml). No cross-reactivity was observed with other toxinotypes. During the development of this assay, interference induced by associated protein was observed. By comparing the effect of different buffers, a buffer composed with Tris-HCl and NaCl salts was demonstrated to dissociate protein complexed with the neurotoxin A. Applied to the measurement of the toxin in different matrices, this dissociating buffer ensures the correct quantification.
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PMID:A sensitive sandwich enzyme immunoassay for free or complexed Clostridium botulinum neurotoxin type A. 1809 12