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)

Botulism is widely known to result from ingestion of food containing botulinum neurotoxin produced in situ by certain strains of Clostridium botulinum. Infant botulism caused by C. botulinum, unlike the food-borne intoxication, is the toxicoinfectious form of botulism (S. S. Arnon, p. 331-345, in G. E. Lewis, ed., Biomedical Aspects of Botulism, 1981). The strain of Clostridium baratii implicated in infant botulism produced a neurotoxin that was neutralized with antiserum for botulinum neurotoxin serotype F (J. D. Hall, L. M. McCroskey, B. J. Pincomb, and C. L. Hatheway, J. Clin. Microbiol. 21:654-655, 1985). We developed a procedure to culture the toxigenic C. baratii (strain 6341) in dialysis bags and a simple purification scheme (precipitation of 900-ml culture supernatant with ammonium sulfate and two anion-exchange chromatographic steps at pH 5.5 and 8.0) that yielded up to 150 micrograms of purified neurotoxin. It is an approximately 140-kDa single-chain protein and has the following sequence of amino acid residues at the N terminus: Pro-Val-Asn-Ile-Asn-Asn-Phe-Asn-Tyr-Asn-Asp-Pro-Ile-Asn-Asn-Thr-Thr-Ile- Leu. Comparison of this amino acid sequence with those of the botulinum neurotoxin serotypes A, B, and E showed 40 to 50% identical residues in comparable positions. The specific toxicity of the neurotoxin, approximately 2 x 10(6) 50% lethal doses for mice per mg of protein injected, was not enhanced significantly by mild trypsinization, although the protease cleaved the neurotoxin within a disulfide loop that generated at least two primary fragments, approximately 47 and approximately 86 kDa, that remained linked by an interchain disulfide. These two fragments resembled the light and heavy chains of the well-characterized neurotoxin serotypes A, B, C, D, E, and F produced by C. botulinum.
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PMID:Characterization of the neurotoxin isolated from a Clostridium baratii strain implicated in infant botulism. 173 Apr 84

Secondary structures of botulinum neurotoxin type A have been determined using Fourier transform infrared spectroscopy in the amide I and amide III frequency regions. Using Fourier self-deconvolution, second derivatization, and curve-fit analysis, the amide I frequency contour was resolved into Gaussian bands at 1678, 1654, 1644, and 1634 cm-1. In the amide III frequency region, several small bands were resolved between 1320 and 1225 cm-1. Assignments of the bands in both amide I and amide III frequency regions to various types of secondary structures and the estimation of spectral band strengths by integrating areas under each band suggested that the neurotoxin contains 29% alpha-helix, 45-49% beta-sheets and 22-26% random coils. These values agreed very well with those determined earlier from CD spectra. The neurotoxin was treated with a micellar concentration of sodium dodecyl sulfate to simulate interaction between the protein and the amphipathic molecules. Sodium dodecyl sulfate micelles induced significant alterations both in the spectral band positions, and their strengths suggest refolding of the neurotoxin polypeptides. However, these changes were not entirely reversible, which could implicate the role of the altered structures in the function of the neurotoxin.
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PMID:Botulinum neurotoxin type A: structure and interaction with the micellar concentration of SDS determined by FT-IR spectroscopy. 181 89

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

A protease that nicks the approximately 150-kilodalton (kDa) single-chain type A botulinum neurotoxin into the approximately 150-kDa di-chain form in vitro was isolated from Clostridium botulinum type A (Hall strain) cultures. The di-chain neurotoxin generated in vitro is composed of an approximately 50-kDa light chain and an approximately 100-kDa heavy chain which are disulfide linked and is indistinguishable from the di-chain neurotoxin that forms in vivo and is routinely isolated (M.L. Dekleva and B.R. DasGupta, Biochem. Biophys. Res. Commun. 162:767-772, 1989). This enzyme was purified greater than 1,000-fold by ammonium sulfate precipitation, QAE-Sephadex Q-50, Sephadex G-100, and CM-Sephadex C-50 chromatography steps with the synthetic substrate N-benzoyl-DL-arginine-p-nitroanilide. The approximately 62-kDa amidase (protease) is a complex of 15.5- and 48-kDa polypeptides (determined by polyacrylamide gel electrophoresis) that could not be separated without sodium dodecyl sulfate. The enzyme has an isoelectric point of pH 5.73, a pH optimum of 6.2 to 6.4, an absolute requirement for a thiol-reducing agent as well as a divalent metallic cation (probably Ca2+) for activity, and a temperature optimum of 70 degrees C. Tests with several synthetic substrates indicated the high specificity of the enzyme for arginyl amide bonds.
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PMID:Purification and characterization of a protease from Clostridium botulinum type A that nicks single-chain type A botulinum neurotoxin into the di-chain form. 218 24

A purification procedure for type E botulinum neurotoxin has been developed, based solely on high-performance ion exchange chromatography. The method exploits the differential chromatographic behavior of the free neurotoxin versus the neurotoxin-protein 12S complex. The high purity of the product was demonstrated with sodium dodecyl sulfate-gel electrophoresis and amino acid sequencing. Beginning with dialyzed crude extract, at least 4 mg of pure neurotoxin could be obtained in two working days. The method has been adapted to user-prepared columns for processing large volumes of crude neurotoxin in one batch.
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PMID:Purification of type E botulinum neurotoxin by high-performance ion exchange chromatography. 374 Apr 11

To characterize type B botulinum neurotoxin based on reliable data on the amino acid composition, three batches of the neurotoxin were analyzed. Each batch was isolated from a separate neurotoxin producing bacterial culture (strain Okra). Two batches were purified by the same method and one was purified by a different method. The toxin preparations were comparable in purity (judged by polyacrylamide gel--sodium dodecyl sulfate electrophoresis) and similar in amino acid composition. The best estimate of the number of amino acid residues per toxin molecule (mol. wt 152,000) was: Asp212,Thr54,Ser83,Glu130,Pro46,Gly61++ +,Ala44,Val54,CyS11,Met23,Ile144,Leu107 , Tyr81,Phe77,Lys118,His7,Arg39,Trp18.
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PMID:Amino acid composition of Clostridium botulinum type B neurotoxin. 672 47

Preincubation of botulinum neurotoxin serotype A, B, or E with ganglioside GT1b was previously found to enhance adherence of botulinum neurotoxin to synapsin I and an approximately 116-kDa bovine brain synaptosomal protein; in contrast, adherence to these two proteins by tetanus neurotoxin required preincubation with GT1b. We have now found that preincubation of the neurotoxins with ganglioside GD3 enhances their adherence to the approximately 116-kDa protein more than that with GT1b. A purified preparation of the water-soluble approximately 116-kDa protein was obtained from bovine brain synaptosomes by preparative column sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. N-Terminal amino acid sequences were obtained for two tryptic fragments of the approximately 116-kDa protein. These sequences matched with the data bank sequences for beta-adducin, a cytoskeletal protein. The carboxy-terminal tail region of adducin, but not the head region, was adhered to by the neurotoxins. Adherence of the neurotoxin to adducin and synapsin I may facilitate presentation of the neurotoxin to its specific substrate(s).
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PMID:Ganglioside-induced adherence of botulinum and tetanus neurotoxins to adducin. 863 82

Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamate is involved in the negative regulation of norepinephrine-stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCI or A23187 (a Ca2+ ionophore) in the presence of Ca2+ and was proportional to the amount of L-glutamate released. Furthermore, the release of aspartate was inhibited by both botulinum neurotoxin type E and L- or N-type voltage-gated Ca2+ channel blockers. Bay K 8644, an agonist for the L-type Ca2+ channel, stimulated the release of aspartate 2.1-fold. Immunohistochemical analyses with antibodies against aspartate and synaptophysin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the L form, although pinealocytes also contain the D form in their cytoplasm, corresponding to approximately 30% of the total free aspartate. Radiolabeled L-aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na+-dependent manner. The Na+-dependent uptake of L-aspartate was strongly inhibited by L-cysteine sulfinate, beta-hydroxyaspartate, and L-serine-O-sulfate, inhibitors for the Na+-dependent glutamate/aspartate transporter on the plasma membrane. Na+-dependent sequestration of L-aspartate was also observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that L-aspartate is released through microvesicle-mediated exocytosis from pinealocytes and is taken up again through the Na+-dependent transporter at the plasma membrane. The possible role of L-aspartate as an intercellular chemical transmitter in the pineal gland is discussed.
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PMID:L-aspartate but not the D form is secreted through microvesicle-mediated exocytosis and is sequestered through Na+-dependent transporter in rat pinealocytes. 920 28

Clostridium botulinum may produce any of seven known serotypes of neurotoxin (BoNT/A-/G), which are the most toxic bacterial proteins known. Efforts to develop a second-generation vaccine to these toxins would benefit from the isolation of hybridomas producing neutralizing monoclonal antibodies (MAbs). We hypothesized that previous efforts to isolate neutralizing MAbs against various BoNTs failed due to use of toxoided, chemically altered antigens. We employed a novel vaccination regimen employing native, active, single-chain BoNT/E (scBoNT/E). A number of the BoNT/E immunized mice were further vaccinated with lethal doses of fully active BoNT/F. MAb 7F8 consistently neutralized BoNT/F in three different assays: in vivo neutralization, passive neutralization, and neutralization of regional paralysis. There was no detectable recognition and essentially no neutralization of scBoNT/E. The epitope recognized by this MAb was denatured when treated with formalin, urea, guanidine chloride, or sodium dodecyl sulfate. Preliminary epitope mapping studies indicate that the MAb bound to a conformational epitope.
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PMID:Identification and characterization of a neutralizing monoclonal antibody against botulinum neurotoxin serotype F, following vaccination with active toxin. 938 28

Clostridium botulinum neurotoxin (NT) serotypes A, B, and E have 9, 10, and 8 Cys residues, respectively, as deduced from nucleotide sequences [Whelan et al. (1992), Appl. Environ. Microbiol. 48, 2345-2354]. Each of the 150-kDa NTs has at least one disulfide; but type B, like types A and E, may have two disulfides. Using two different chemical reagents, we studied the status of the Cys residues in these three proteins after (i) the final anion exchange chromatographic step in their purification (fresh NT), (ii) 24 hr storage at 8 degrees C, (iii) precipitation with ammonium sulfate (precipitated NT), and (iv) dissolving the precipitated NT in 6 M guanidine HCl. In all three NT serotypes the number of Cys residues titrated with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) as free -SH groups varied, depending upon the absence or presence of EDTA added to the chromatography buffer, storage condition, age, and presence of the denaturant. Titration of 9.5-10 and 5.4-6.0 -SH groups in fresh NTs type B and E, respectively, indicated total and partial absence of disulfide bonds. Fewer titratable -SH groups in the precipitated NT than in the fresh NT suggested formation of disulfide and/or inaccessibility of the -SH groups due to protein's conformational change(s). When the precipitated NTs were dissolved in 6 M guanidine HCl, in the absence of any added reducing agent, all Cys residues of types B and E, and 6.4-8.3 Cys in type A NT were titratable with DTNB. Iodoacetamide modification of precipitated NT types A, B, and E carboxymethylated 4, 2, and 2 Cys residues, respectively; these numbers rose to 6, 9.4, and 8 when these proteins were carboxymethylated after dissolving in 6 M guanidine HCl in the absence of any added reducing agent. We propose that S-S- cleavage mediated by the -SH/-S-S- exchange observed in vitro after unfolding the NTs (also unfolded by 2 M guanidine HCl or urea) possibly mimicks a similar exchange process inside the endosomes, where the NTs are thought to undergo conformational changes, resulting in the reductive cleavage of the interchain disulfide between the 50-kDa light and 100-kDa heavy chain, which in turn releases the light chain and allows its egress out of the endosomes into the cytosol.
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PMID:Status of Cys residues in the covalent structure of botulinum neurotoxin types A, B, and E. 958 42


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