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 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

Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in terminally differentiated neurons. If achieved, it would allow the study of long-lasting changes of properties of nerve cells in their functional context. To obtain evidence of such expression, we chose two proteins that produce a detectable effect even at very low intracellular concentrations. Tetanus toxin and botulinum neurotoxin fulfill this criterion, being the most potent neurotoxins known. Both toxins block neurotransmitter release at nanomolar intracellular concentrations. These di-chain proteins, consisting of a light chain and a heavy chain, have recently been sequenced. Their active sites are located (or partly located) on the light chain. mRNAs encoding the light chain of either toxin were transcribed in vitro from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic cholinergic neurons of the buccal ganglia of Aplysia californica. Depression of neurotransmitter release appeared in less than 1 hr, demonstrating successful expression of foreign mRNA injected into a neuron in situ.
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PMID:Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons. 223

Permeabilisation of PC12 cells with digitonin allowed a direct study of the intracellular action of botulinum neurotoxin A, one of a group of dichain proteins produced by Clostridium botulinum that causes the fatal neuroparalytic condition, botulism. Release of [3H]noradrenaline from these permeabilised cells could be evoked by Ca2+ and this was inhibited specifically by the neurotoxin in a dose-dependent manner (half-maximal dose approximately 2 nM under the conditions used). Inclusion of the reducing agent dithiothreitol (up to 10 mM) had no effect on the level of inhibition. Moreover, electrophoretic analysis showed that this treatment of the toxin in the native state caused negligible reduction of inter-chain disulphide bonds. Toxin-induced blockade of neurotransmitter release was incomplete and could not be overcome by increased Ca2+ concentration (100 microM). The observed toxin-insensitivity of the release from intact PC12 cells must result from inefficient toxin uptake, relative to that in peripheral cholinergic neurones. Refolded light chain alone inhibited exocytosis to the same degree and with similar potency to that of the intact neurotoxin, an effect not altered by the heavy chain. This inhibitory activity of the light chain in PC12 cells accords with observations made in permeabilised chromaffin cells [(1989) J. Biol. Chem. 264, 10354-10360; (1989) FEBS Lett. 255, 391-394] but contrasts with invertebrate neurones, where intracellular injection of the same preparations of both chains were necessary for inhibition of quantal release of acetylcholine [(1988) Proc. Natl. Acad. Sci. USA 85, 4090-4094]. These collective findings may signify an interesting difference in the release process in such diverse systems or denote a dissimilarity in the transport or processing of the toxin when applied into intact neurones or cells permeabilised by detergent or streptolysin.
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PMID:Ca2(+)-dependent noradrenaline release from permeabilised PC12 cells is blocked by botulinum neurotoxin A or its light chain. 231 61

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 flaccid paralysis in the neuromuscular disease botulism appears to depend on the coordinated roles of the approximately 50 kDa light and approximately 100 kDa heavy chain subunits of the approximately 150 kDa neurotoxic protein produced by Clostridium botulinum (J. Biol. Chem. (1987) 262, 2660 and Eur. J. Biochem. (1988) 177, 683). We observed that the light chain after separation from its conjugate heavy chain, in the presence of dithiothreitol and 2 M urea, begins to split into approximately 28 and approximately 18 kDa fragments. The other subunit-the approximately 100 kDa heavy chain following its isolation-and the parent approximately 150 kDa dichain neurotoxin do not break down under comparable conditions. This cleavage was examined in the neurotoxin serotypes A and E. The cleavage does not appear to be due to a protease. Partial amino acid sequences established that: i) the approximately 28-kDa and approximately 18-kDa fragments comprise the N- and C-terminal regions of the light chain, respectively; ii) the light chain of the neurotoxin serotypes A and E break down at precise peptide bonds; iii) the peptide bonds cleaved in serotypes A and E are five residues apart; and iv) the portions of the approximately 18 kDa fragments of serotype A and E neurotoxin sequenced so far are highly homologous to the corresponding region of tetanus neurotoxin produced by Clostridium tetani. The partial N-terminal sequence of the approximately 28 kDa fragment matches with the N-terminal sequence of the intact L chain. The 47 residues of the approximately 18-kDa fragment of type A sequenced from its N-terminal are: -Y.E.M.S.G.L.E.V.S.F.E.E.L.R.T.F.G.G.H.D.A.K.F.I.D.S.L.Q.E.N.E.F.R.L.Y.Y .Y. N.K.F.K. D.I.A.S.T.L.-. These align with those of tetanus neurotoxin beginning at its residue #259 (Tyr); the 18 underlined residues of the above 47 residues (i.e. 38%) are identical in positions between the two proteins. The 41 residues sequenced from the approximately 18 kDa fragment of type E botulinum neurotoxin are: -K.G.I.N.I.E.E.F.L. T.F.G.N.N.D.L.N.I.I.T.V.A.Q.Y.N.D.I.Y.T.N.L.L.N.D.Y.R. K.I.A.X.K. L.-.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:C. botulinum neurotoxin types A and E: isolated light chain breaks down into two fragments. Comparison of their amino acid sequences with tetanus neurotoxin. 251 79

1. The contribution of a proteolytic fragment (H2L) of botulinum neurotoxin type A (comprised of the aminoterminal region of the heavy-chain disulphide-linked to the light chain) to inhibition of neurotransmitter release was investigated, using central cholinergic synapses of Aplysia, rodent nerve-diaphragm preparations and cerebrocortical synaptosomes. 2. No reduction in neurotransmitter release was observed following external application to these preparations of highly purified H2L or after intracellular injection into Aplysia neurons. 3. The lack of activity was not the result of alteration in the light chain of H2L during preparation of the latter because (a) renaturation of this light chain with intact heavy chain produced a toxic di-chain form and (b) simultaneous application of heavy chain and light chain from H2L inhibited transmitter release in Aplysia. 4. Bath application of H2L and heavy chain together inhibited release of transmitter; however, at the neuromuscular junction the potency of this mixture was much lower than that of native toxin. A similar blockade resulted when heavy chain was applied intracellularly and H2L added to the bath, demonstrating that H2L is taken up into cholinergic neurons of Aplysia. This uptake is shown to be mediated by the amino-terminal moiety of heavy chain (H2), because bath application of light chain plus H2 led to a decrease in acetylcholine release from a neuron that had been injected with heavy chain. 5. A role within the neuron is implicated for a carboxy-terminal portion of heavy chain (H1) since intracellular injection of light chain and H2 did not affect transmitter release. Although the situation is unclear in mammalian nerves, these collective findings indicate that blockade of transmitter release in Aplysia neurons requires the intracellular presence of light chain and H1 (by inference), whilst H2 contributes to the internalization step.
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PMID:Inhibition of transmitter release by botulinum neurotoxin A. Contribution of various fragments to the intoxication process. 257 18

The effects of botulinum neurotoxins or their light and heavy chain subunits were investigated in digitonin-permeabilized adrenal chromaffin cells. Because these cells are permeable to proteins, the toxin had direct access to the cell interior. Botulinum type A neurotoxin and its light chain subunit inhibited Ca2+-dependent catecholamine secretion in a dose-dependent manner. The heavy chain subunit had no effect. Inhibition required introduction of the neurotoxin or light chain into the cell and was not seen when intact cells were incubated with these proteins. The inhibition of secretion by type A neurotoxin and light chain was incomplete, the maximal response being 65%. The inhibition was not overcome by increasing Ca2+ concentrations. The action of the light chain was irreversible and rapid. Botulinum type E neurotoxin also inhibited secretion in a dose-dependent manner. Its potency was increased 30-fold following mild trypsinization, which nicked the single chain protein to the dichain form. In contrast to the results seen with types A and E, botulinum type B neurotoxin did not inhibit secretion, while its light chain totally abolished secretion. Trypsinization of the neurotoxin produced the dichain form, which did not inhibit secretion. Reduction of the trypsinized neurotoxin with dithiothreitol produced inhibition equivalent to that seen with the purified light chain subunit. Isolated type A heavy chain had no effect on the inhibitory action of type A or B light chains. The data demonstrate that the ability of botulinum neurotoxins to inhibit secretion is confined to the light chain region of these proteins. Furthermore, while the botulinum neurotoxin types A, B, and E have similar macrostructures, they are not identical with respect to their biological activities.
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PMID:Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells. 273 26

Botulinum neurotoxins (types A and B), which are microbial proteins consisting of two disulfide-linked chains, inhibit specifically and with high potency the release of acetylcholine from peripheral nerve terminals. As a prerequisite for a long-term development of effective treatments for botulism, the internalization and inhibitory action of the toxin and its constituent chains were examined by electrophysiological methods at identified synapses in Aplysia preparations that allow both intracellular and bath application of the neurotoxins. Intracellular recordings from cholinergic cells of the buccal ganglion demonstrated that extra- or intracellular application of low doses of botulinum neurotoxin results in a specific blockade of evoked transmitter release, without changing the quantal size; an intraneuronal site of action has thus been established. In contrast, release from noncholinergic neurons of cerebral ganglion was prevented by the neurotoxin only after injection into the cell. Purified preparations of the individual renatured chains, shown to be nontoxic in a mouse bioassay, failed to affect acetylcholine release when applied extra- or intracellularly. However, inhibition of release was observed after intracellular administration of both chains or when the light chain was injected and the heavy chain was bath-applied. These findings show that both chains are required on the cytosolic side of the neuronal plasma membrane for expression of toxicity and that the cholinergic specificity of the neurotoxin is attributable to its heavy chain, which mediates targeting and subsequent neuronal uptake.
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PMID:Neurotransmitter release is blocked intracellularly by botulinum neurotoxin, and this requires uptake of both toxin polypeptides by a process mediated by the larger chain. 289 93

The action of botulinum neurotoxin type C1 on the release of acetylcholine from rat brain synaptosomes was studied by using anti-toxin heavy chain Fab and anti-toxin light chain Fab. The toxin was bound to synaptosomes at 0 degrees C for 10 min, in which [14C]acetylcholine had been accumulated previously. The toxin-binding synaptosomes were pre-incubated at 37 degrees C, and the release of acetylcholine was determined after the synaptosomes had been incubated in 25 mM KCl-incubation medium for 20 min at 37 degrees C. Inhibition of [14C]acetylcholine release from the synaptosomes was observed with increasing pre-incubation time and toxin concentration, and the maximum inhibition was seen after pre-incubation for at least 15 min, which was called the "lag time." The toxin-binding synaptosomes were reacted with anti-toxin heavy chain and anti-toxin light chain Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C. Both Fabs reversed the acetylcholine release inhibition by the toxin. However, when the Fabs were added during the pre-incubation time at 37 degrees C, they showed less restoration with increasing pre-incubation time. The restoration was completely abolished if the Fabs were added to the synaptosomes after the first half of the "lag time." On the other hand, when 125I-labeled toxin-binding synaptosomes were reacted with the Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C, anti-heavy chain Fab removed 125I-toxin from the synaptosomes, but anti-light chain Fab did not. However, if the Fabs were added to toxin-binding synaptosomes during the pre-incubation time at 37 degrees C, the Fabs could not remove 125I-toxin from the synaptosomes, and the synaptosomes retained more labeled toxin with increasing pre-incubation time. These results suggest that there are three distinct steps in the inhibition of acetylcholine release from synaptosomes by botulinum neurotoxin. The first is binding, which is reversible, temperature-independent, and mediated by the heavy chain of the toxin. The second is temperature-dependent internalization, that takes place in the first half of the "lag time," in which both the chains are internalized into synaptosomes. The third is the development of toxicity, which requires the latter half of the "lag time."
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PMID:Action of botulinum neurotoxin on acetylcholine release from rat brain synaptosomes: putative internalization of the toxin into synaptosomes. 312 13

The clostridial neurotoxins tetanus and botulinum toxin type A are known to block transmitter release from nerve terminals, probably by interfering with some essential process controlling exocytosis after the entry of Ca2+ ions. Although exocytosis occurs in many secretory cells, these toxins show a high specificity for neurones and the secretory response of cultured bovine adrenal medullary cells is not inhibited by exposure to medium containing tetanus or botulinum toxin type A (although it is by botulinum toxin type D). Here we report that when tetanus toxin and botulinum neurotoxin type A are injected intracellularly into chromaffin cells they strongly inhibit secretion, as revealed by the measurement of cell capacitance. These results indicate that these toxins are normally ineffective in chromaffin cells because they are not bound and internalized, so do not reach their site of action. Furthermore, we have localized the secretion-blocking effects of the toxin to a fragment comprising the light chain covalently linked to part of the heavy chain, suggesting that this part of the molecule contains the active site.
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PMID:Intracellularly injected tetanus toxin inhibits exocytosis in bovine adrenal chromaffin cells. 378 74


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