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)

1. A 50-kDa fragment representing the NH2-terminus of the heavy subunit of botulinum type A neurotoxin was found, at low pH, to evoke the release of K+ from lipid vesicles loaded with potassium phosphate. Similar K+ release was also observed with the intact neurotoxin, its heavy chain and a fragment consisting of the light subunit linked the 50-kDa NH2-terminal heavy chain fragment. The light subunit alone, however, was inactive. 2. In addition to K+, the channels formed in lipid bilayers by botulinum neurotoxin type A or the NH2-terminal heavy chain fragment were found to be large enough to permit the release of NAD (Mr 665). 3. The optimum pH for the release of K+ was found to be 4.5. Above this value K+ release rapidly decreased and was undetectable above pH 6.0. 4. The binding of radiolabelled botulinum toxin to a variety of phospholipids was assessed. High levels of toxin binding were only observed to lipid vesicles with an overall negative charge; much weaker binding occurred to lipid vesicles composed of electrically neutral phospholipids. 5. A positive correlation between the efficiency of toxin-binding and the efficiency of K+ release from lipid vesicles was not observed. Whereas lipid vesicles containing the lipids cardiolipin or dicetyl phosphate bound the highest levels of neurotoxin, the toxin-evoked release of K+ was low compared to vesicles containing either phosphatidyl glycerol, phosphatidyl serine or phosphatidyl inositol. 6. The implications of these observations to the mechanism by which the toxin molecule is translocated into the nerve ending are discussed.
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PMID:A 50-kDa fragment from the NH2-terminus of the heavy subunit of Clostridium botulinum type A neurotoxin forms channels in lipid vesicles. 244 87

The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated. Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation. At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained. Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins. The biological activity of BNTA was more sensitive to irradiation than the immunological activity. Staphylococcal enterotoxin was more resistant to irradiation than BNTA. Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation.
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PMID:Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus. 323 88

The receptor structure of Clostridium botulinum neurotoxin type A was analysed by TLC immunostaining. GQ1b was found to be the most potent receptor, and the neurotoxin also bound to GT1b and GD1a, but not to GM3, GM2, GM1, GD3, GD1b and GT1a. Optimum binding of neurotoxin to the ganglioside appeared in 0.01 M phosphate buffer (pH 7.2) containing 0.2% NaCl. Higher and lower NaCl concentrations diminished neurotoxin binding to the ganglioside. In addition, the neurotoxin was able to bind to free fatty acids. Maximum binding was observed on stearic acid and neurotoxin binding to free fatty acids was not affected by NaCl concentration.
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PMID:TLC immunostaining characterization of Clostridium botulinum type A neurotoxin binding to gangliosides and free fatty acids. 370 10

The purpose of these experiments was to compare the spread of three formulations of botulinum neurotoxin A. A gelatin/phosphate buffer (C), DysportR (D {0.5 U}, BotoxR (B {0.167 U}), or a purified preparation of botulinum neurotoxin A [Bont A] (BA {0.5 U}) was injected into the tibialis anterior of male, Wistar rats. After 4 days, the adjacent extensor digitorum longus muscle was isolated in situ and the nerve was maximally stimulated to determine contractile properties and the rate of fatigue. There were no differences in body or muscle weights between any of the groups after 4 days of treatment. Maximal twitch and tetanic tensions were decreased reverse similar 25% (p < 0.05) in all treatment groups compared to C. In addition, rate of tension development was significantly less in all treatment groups compared to C but one-half relaxation time and time to peak tension were not different between any groups. Fatigue of the muscle was significantly faster in all groups compared to C but there was no difference between treatment groups. These data indicated that botulinum toxin A injected intramuscularly was likely to spread to adjacent muscles but that the spread was not different between the three formulations tested. The effect of the spread ranged from a slight to a severe reduction in maximal tension but this did not occur in all animals studied. Copyright Rapid Science Ltd
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PMID:A comparison of the spread of three formulations of botulinum neurotoxin A as determined by effects on muscle function. 1021 Aug 30

1. The aim of the present study is to characterize the role of the P2X receptor in spinal nociceptive processing in vivo. We investigated the mechanisms of the P2X receptor agonist alpha,beta-methylene ATP (alpha,betameATP)-induced modulation of acute nociceptive signalling in mouse spinal cord. 2. Intrathecal administration of alpha,betameATP produced a significant and dose-dependent thermal hyperalgesic response. This response was completely blocked by intrathecal pretreatment with the non-selective P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and the selective P2X1, P2X3 and P2X2-3 receptor antagonist, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). Pretreatment with alpha,betameATP 15, 30 and 60 min prior to administration of a second dose of alpha,betameATP diminished the alpha,betameATP-induced thermal hyperalgesia. 3. A potent agonist for the P2X1 receptor, beta,gamma-methylene-L-ATP, did not show the hyperalgesic response, indicating that the P2X1 receptor is not involved in the spinal nociceptive pathway. 4. In fura-2 experiments using mouse dorsal root ganglion (DRG) neurons, alpha,betameATP (100 microM) increased intracellular Ca2+ ([Ca2+]i). This was not produced by a second application of alpha,betameATP. The same DRG neurons also showed a marked [Ca2+]i increase in response to capsaicin (3 microM). 5. Intrathecal pretreatment with the Ca2+-dependent exocytosis inhibitor, botulinum neurotoxin B, abolished the thermal hyperalgesia by alpha,betameATP. Furthermore, thermal hyperalgesia was significantly inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonists, 2-amino-5-phosphonopentanoate (APV), dizocilpine and ifenprodil. 6. These findings suggest that alpha,betameATP-induced thermal hyperalgesia may be mediated by the spinal P2X3 receptor subtype that causes unresponsiveness by repetitive agonist applications, and that alpha,betameATP (perhaps through P2X3 receptors) may evoke spinal glutamate release which, in turn, leads to the generation of thermal hyperalgesia via activation of NMDA receptors.
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PMID:In vivo pathway of thermal hyperalgesia by intrathecal administration of alpha,beta-methylene ATP in mouse spinal cord: involvement of the glutamate-NMDA receptor system. 1038 45

The catalytic domain of clostridial neurotoxins is a substrate of tyrosine-specific protein kinases. The functional role of tyrosine phosphorylation and also the number and location of its (their) phosphorylation site(s) are yet elusive. We have used the recombinant catalytic domain of botulinum neurotoxin E (BoNT E) to examine these issues. Bacterially expressed and purified BoNT E catalytic domain was fully active, and was phosphorylated in vitro by the tyrosine-specific kinase Src. Tyrosine phosphorylation of the catalytic domain increased the protein thermal stability without affecting its proteolytic activity. Covalent modification of the endopeptidase promoted a disorder-to-order transition, as evidenced by the 35% increment of the alpha-helical content, which resulted in a 4 degrees C increase of its denaturation temperature. Site-directed replacement of tyrosine at position 67 completely abolished phosphate incorporation by Src. Constitutively unphosphorylated endopeptidase mutants exhibited functional properties virtually identical to those displayed by the nonphosphorylated wild-type catalytic domain. These findings indicate the presence of a single phosphorylation site in the catalytic domain of clostridial neurotoxins, and that its covalent modification primarily modulates the protein thermostability.
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PMID:Thermal stabilization of the catalytic domain of botulinum neurotoxin E by phosphorylation of a single tyrosine residue. 1132 92

The light chain of botulinum neurotoxin serotype A undergoes autocatalytic fragmentation into two major peptides during purification and storage (Ahmed S. A. et al. 2001, J. Protein Chem. 20:221-231) by both intermolecular and intramolecular mechanisms (Ahmed S. A. et al. 2003, Biochemistry 42:12539 12549). In this study, we investigated the effects of buffers and salts on this autocatalytic reaction in the presence and absence of zinc chloride. In the presence of zinc chloride, the fragmentation reaction was enhanced in each of acetate, MES, HEPES and phosphate buffers with maximum occurring in acetate when compared to those in the absence of zinc chloride. Adding sodium chloride in phosphate buffer in the presence of zinc chloride increased the extent of proteolysis. Irrespective of the presence of zinc chloride, adding sodium chloride or potassium chloride in phosphate buffer elicited an additional proteolytic reaction. Higher concentrations of sodium phosphate buffer enhanced the autocatalytic reaction in the absence of zinc chloride. In contrast, in the presence of zinc chloride, higher concentrations of sodium phosphate decreased the autocatalytic reaction. Optimum pH of autocatalysis was not affected significantly by the absence or presence of zinc chloride. Like zinc chloride, other chlorides of divalent metals, such as magnesium, cobalt, iron and calcium also enhanced the autocatalytic reaction. Polyols such as ethylene glycol protected the light chain from fragmentation. Exposure of light chain to UV radiation led to enhanced fragmentation. In order to avoid fragmentation, the protein should be stored frozen in a low concentration buffer of neutral or higher pH devoid of any metal. Our results provide a choice of buffers and salts for isolation, purification and storage of intact botulinum neurotoxin serotype A light chain.
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PMID:Factors affecting autocatalysis of botulinum A neurotoxin light chain. 1563 36

Heavy chain fragments of botulinum neurotoxin serotypes A and B are being developed as a bivalent vaccine for botulism. To potentiate the immune response, an aluminum containing adjuvant will be formulated with the two antigens. The adsorption mechanisms of each antigen to aluminum phosphate and aluminum hydroxide adjuvants were studied. The adsorption of the serotype A antigen to each adjuvant, and the serotype B antigen to aluminum phosphate adjuvant, is dependent on electrostatic attractive forces. The serotype A antigen is basic, and pretreatment with phosphate anions is required for favorable adsorption conditions to aluminum hydroxide adjuvant. In contrast, the serotype B antigen displays a high affinity to aluminum hydroxide adjuvant even when the two species possess the same charge. It is proposed that the serotype B antigen is adsorbed to aluminum hydroxide adjuvant by a ligand exchange mechanism.
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PMID:Formulation of botulinum neurotoxin heavy chain fragments for vaccine development: mechanisms of adsorption to an aluminum-containing adjuvant. 1596 60

To ensure safety and predictable clinical efficacy, the biological activity of type A botulinum toxin (BoNT-A) preparations must remain consistent. Several methods have been employed to assess consistency but lack clinical applicability and/or are associated with animal welfare concerns. Here, we describe a novel in vivo rat muscle force model for evaluating the biological activity of formulated BoNT-A product (Dysport) prepared from bulk toxin batches manufactured at different facilities. Toxin activity was assessed by measuring muscle force generation over time in the triceps surae muscles in the rat hind leg. Animals received 0.1 ml gelatine phosphate buffer (negative vehicle control) or 0.1 or 1.0 LD50 units of BoNT-A in phosphate buffer. Batch equivalence and consistency were confirmed by the lack of significant differences in muscle force generation and duration of effect between each test batch and the reference preparation tested in the same series of experiments. The reduction in muscle force generation was dose-related and reproducible for all active treatment groups. At appropriate dose levels, the rat muscle force model is a reliable tool for measuring biological activity in bulk toxin batches used to formulate clinical product and demonstrates the consistency of batches manufactured over many years.
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PMID:The in vivo rat muscle force model is a reliable and clinically relevant test of consistency among botulinum toxin preparations. 1863 70

The adsorption of recombinant botulinum neurotoxin (BoNT) protein-derived vaccine antigens to aluminum salt adjuvants has been previously studied for the development of a trivalent vaccine against the neurotoxins (Vessely et al., in press, J Pharm Sci). The current paper describes an investigation of the stability of recombinant BoNT antigens adsorbed to aluminum salt adjuvants during storage in aqueous solution. Both chemical and physical changes occurred during storage. Phosphate groups in the buffer exchanged with hydroxyl groups on the adjuvant surface. The resulting changes in solution pH and adjuvant surface chemistry promoted more favorable electrostatic interaction between the BoNT proteins and the surface, possibly increasing the affinity of the proteins for the surface during storage. Fluorescence and UV spectroscopy suggested changes to protein structure during storage, whereas differential scanning calorimetry showed changes to thermal processes related to protein conformation and/or surface adsorption. The consequence of the chemical and physical changes to the proteins was a decrease in the ability to desorb protein from the adjuvant surface during storage. Overall, the results of this study emphasize the utility of a thorough characterization of the interactions between protein antigens and aluminum salt adjuvants.
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PMID:Stability of a trivalent recombinant protein vaccine formulation against botulinum neurotoxin during storage in aqueous solution. 1868 Jan 75


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