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)

A sensitive radioimmunoassay for the detection of botulinum toxin, produced by Clostridium botulinum, was developed. This employs homogeneous botulinum neurotoxin type A and its 125I-labelled derivative of high specific radioactivity, rather than its complex with haemagglutinin as used hitherto. The sensitivity of the assay is 1 ng of neurotoxin per ml, which is equivalent to 80 LD50 units (half-lethal doses) in mice. Neurotoxin and its complex with haemagglutinin were measurable with equal sensitivity when using antibodies against botulinum neurotoxin type A. Specificity of the assay was demonstrated by the lack of response to type B and E botulinum toxins and to heat-inactivated botulinum toxin or extracts of Clostridium sporogenes strain BL46, which contains many surface antigenic determinants common to Clostridium botulinum. Using appropriate conditions, neurotoxin added to fish extract could be quantified accurately, proportionality being observed between the amounts of standard toxin added. In addition, the amounts of toxin species produced by culturing Clostridium botulinum in canned fish was measurable; the values obtained were comparable to those observed by the mouse bioassay. Moreover, the fish samples gave a dose-response curve in the competition radioimmunoassay which was paralleled by the response of botulinum neurotoxin standards. This assay offers the most sensitive, reliable immunological method available for the quantitation of molecular forms of botulinum toxin. As the technique can be used with unpurified fish extracts, it should be widely applicable to different types of samples contaminated with botulinum toxin; furthermore, the clinical diagnosis of human botulism could be substantiated with this method.
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PMID:A sensitive and useful radioimmunoassay for neurotoxin and its haemagglutinin complex from Clostridium botulinum. 389 79

The dichain type E botulinum neurotoxin, a product of nicking the single chain protein by trypsin, is composed of a heavy and light chains. Sequence of the first 13 and 20 N-terminal residues of these two chains were determined. Also, proof is provided here that (i) the light chain of the nicked (dichain) is derived from the N-terminal one-third of the parent single chain neurotoxin, and (ii) molecular events leading to the activation, of the single chain neurotoxin cannot involve tryptic cleavage at or very close to the N-terminal of the single chain protein. The partial amino acid sequence of the light chain of botulinum type E and tetanus neurotoxins show significant similarity between the two clostridial neurotoxins.
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PMID:Partial amino acid sequences of the heavy and light chains of botulinum neurotoxin type E. 398 55

Clostridium botulinum produces botulinum neurotoxin (NT) in antigenically distinct forms. When isolated from bacterial cultures type E is a single chain, type B is a mixture of single and two-chain molecules, and type A is essentially a two-chain molecule (Mr approximately 150,000). Protease(s) in the cultures or trypsin nick single-chain NT to the two-chain form. The heavy (Mr approximately 100,000) and light (Mr approximately 50,000) chains of the two-chain molecule remain held together by -S-S-bond(s). The two chains are presumed to have different functions. NT binds to nerve cells via the heavy chain and then light chain enters the cell and blocks release of acetylcholine (Simpson, L. L. (1981) Pharmacol. Rev. 33, 155-188). We nicked single-chain NT to form the two-chain form with trypsin, minimizing secondary cleavages, then separated and purified the heavy and light chains using ion-exchange chromatography. The technique, with minor modifications, is a generalized method for types A, B, and E. These subunit chains (each a single band in sodium dodecyl sulfatepolyacrylamide gel electrophoresis) were analyzed for their complete amino acid compositions. The amino acid contents of the heavy and light chains agreed well with the parent two-chain molecule. This affirms that NT is composed of two chains. The two subunit chains are now usable for amino acid sequence and other studies. Comparison of the amino acid contents indicates more similarity among the light chains than the heavy chains of the three NT types, a similarity that agrees with our published partial amino acid sequences (first 13-18 residues) of these chains. Several (up to 9) different amino acid residues of the heavy chain (which is twice the size of the light chain) are present in double the number of corresponding residues in the light chain.
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PMID:Separation, purification, partial characterization and comparison of the heavy and light chains of botulinum neurotoxin types A, B, and E. 403 Jul 55

Blockade of neuromuscular transmission was produced in the lower hind limb of the rat by local injection of either crystalline type A botulinum toxin or purified type B botulinum neurotoxin. At 1, 3, 5 and 7 days after injection, the extensor digitorum longus nerve-muscle preparation was excised and analyzed in vitro for alterations in spontaneous and nerve stimulus-evoked quantal transmitter release. Muscles receiving type A toxin were paralyzed up to and including 7 days after injection. Muscles treated with type B toxin, although completely paralyzed at 1 and 3 days, twitched in response to nerve stimulation at 5 and 7 days after injection. Both toxins induced a marked decrease in the frequency of miniature endplate potentials but type A did so to a greater extent. The remaining population of miniature endplate potentials contained a greater frequency of potentials with small or large amplitudes and prolonged rise times compared to normal muscle. These changes were more pronounced with type A toxin than with type B toxin. In the presence of alpha-dinitrophenol (1 mM), high frequency, fast-rising miniature endplate potentials of uniform size reappeared. High K+ (20 mM) was less effective in this respect. At 3 days after toxin injection nerve impulse evoked transmitter release was reduced more for type A treated muscles than for type B. However, 3,4-diaminopyridine, an agent which increases nerve-evoked transmitter release by increasing Ca2+ influx, was more effective in reversing the paralysis in type A than in type B-treated muscles.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Different effects of types A and B botulinum toxin on transmitter release at the rat neuromuscular junction. 614 Aug 15

Acceptors for BoNT have been detected autoradiographically on the terminal membrane of motor nerves at a density of approximately 150/micron2 and shown to mediate toxin internalization, a process deemed essential for its inhibition of transmitter release. DTX, a protein with pronounced central neurotoxicity, was shown to induce convulsive states in hippocampal slices from guinea-pig. Synaptic transmission was facilitated and spontaneous epileptiform activity produced in intact cell populations. Voltage clamp analysis of hippocampal neurones revealed that DTX specifically attenuated a transient voltage-dependent K+ conductance (A-current) and this could account for the excitatory effects observed. Proteinaceous acceptors with high affinity for DTX were identified on brain synaptosomal membranes and found to contain a 65 000 Mr polypeptide. Their location in rat brain regions was established and contrasted with that of the binding sites for beta-bungarotoxin. These findings indicate the usefulness of DTX as a probe for a protein associated with one variety of K+ channel while the larger subunit of BoNT was found to interact with a membraneous component that resides at cholinergic nerve terminals and, hence, is likely to have a unique role.
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PMID:Botulinum neurotoxin and dendrotoxin as probes for studies on transmitter release. 615 94

Chloroquine and hydroxychloroquine block neuromuscular transmission in isolated tissues from mouse, rat, guinea pig and chick. Blockade is associated with depressed muscle responses to potassium and abolished muscle responses to nicotinic cholinergic agonists. Within certain time and concentration limits, the blocking effects of chloroquine and hydroxychloroquine are reversible. Both drugs antagonize the onset of paralysis caused by botulinum neurotoxin types A and B, but neither drug antagonizes tetanus toxin or beta-bungarotoxin. The ability of chloroquine and hydroxychloroquine to antagonize botulinum toxin is not due to blockade of nicotinic cholinergic receptors. At concentrations that produce neuromuscular blockade, d-tubocurarine does not antagonize botulinum toxin types A and B. Chloroquine causes botulinum toxin to remain at an antitoxin sensitive site. These data could mean that chloroquine acts at the cell membrane to inhibit toxin binding or internalization, or that it acts in the cell interior to inhibit lysosomal processing of toxin. Whatever its action, chloroquine is the most effective antagonist of botulinum toxin yet described.
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PMID:The interaction between aminoquinolines and presynaptically acting neurotoxins. 628 72

Single chain type E botulinum neurotoxin was isolated from culture fluids of Clostridium botulinum (strain Alaska E-43). The neurotoxin, which migrated as a single band in polyacrylamide gel electrophoresis with sodium dodecylsulfate, had a molecular weight of approximately 147,000. Single chain type E neurotoxin that was exposed to trypsin was converted to a dichain molecule. Pretreatment of the single chain molecule with 1,2-cyclohexanedione, a reagent that selectively modifies arginine residues, inhibited trypsin-induced generation of the dichain molecule. In dose-response experiments (10(-13) to 10(-9) M) on the isolated neuromuscular junction (phrenic nerve-hemidiaphragm preparation), the dichain neurotoxin was approximately two orders of magnitude more potent than the single chain neurotoxin. Neither specie of neurotoxin (1 pmol/mouse, in vivo; 1 X 10(-11) M, in vitro) was very effective in blocking autonomic transmission (vagus nerve-atrium preparation). The neuromuscular blocking action of the dichain molecule was divided into a sequence of three steps. There was an initial binding step that was relatively rapid, little influenced by temperature and which left the neurotoxin partially accessible to the neutralizing effects of antitoxin. There was a translocation step that was temperature dependent, antagonized by ammonium chloride and methylamine hydrochloride and which caused the neurotoxin to become inaccessible to the neutralizing effects of antitoxin. Finally, there was an intracellular lytic step, during which the toxin blocked excitation-secretion coupling.
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PMID:Botulinum neurotoxin type E: studies on mechanism of action and on structure-activity relationships. 629 75

Local blockade of transmitter release was produced by s.c. injection of purified botulinum neurotoxin (NT) types A or E above the tibialis anterior muscle of adult male rats. Extensor digitorum longus nerve-muscle preparation was examined for toxin-induced alterations in single twitch and tetanic tension (in situ) or transmitter release (in vitro). For both single twitch and tetanic tension, muscles treated with type E NT recovered from an initial partial paralysis (induced with 56 mouse LD50) or full paralysis (induced with 565 mouse LD50) by 7 days after NT injection, while those treated with only 5 mouse LD50 of type A remained either fully or partially paralysed through 10 days. Also, miniature end-plate potential frequency and mean quantal content were reduced for a longer period of time and/or to a greater extent for muscles treated with type A NT than for those treated with type E. The present results are consistent with the observed higher specific toxicity (i.p. injections in mice) for type A NT than for type E, although these differences may be exaggerated after s.c. injections. The differences in the paralytic effect between types A and E may be determined by differences in amino acid sequence, which causes type E to dissociate more easily from its site of action and/or be detoxified more rapidly. The clinical implications of these findings are discussed.
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PMID:Comparison of the effects of botulinum neurotoxin types A and E at the rat neuromuscular junction. 630 64

The procedure we have adopted to purify type E botulinum neurotoxin (mol. wt. approximately 147,000) from bacterial cultures consistently yields a pure protein (a single band in polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate). Our procedure is a modification of one of the five published procedures. Other procedures have failed to yield pure neurotoxin. To develop reliable data on the amino acid composition, three batches of the neurotoxin were analyzed, each batch isolated from a separate neurotoxin producing culture. The best estimate of the number of amino acid residues per neurotoxin molecule was: Asp240 Thr75 Ser98 Glu118 Pro45 Gly58 Ala40 Val62 CyS7 Met17 Ile123 Leu107 Tyr70 Phe62 Lys97 His15 Arg34 Trp16.
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PMID:Purification and amino acid composition of type E botulinum neurotoxin. 635 69

To develop reliable data on the amino acid composition of type F botulinum neurotoxin, three batches of the neurotoxin were analyzed. Each batch was isolated from a separate neurotoxin producing bacterial culture. Two batches had inoculum from one source and the other batch one from a different source. Two batches of the neurotoxin were purified by the same method and one was purified by a different method. The neurotoxin preparations were found comparable in purity and similar in amino acid composition. The best estimate of number of amino acid residues per neurotoxin molecule (mol. wt. 155,000) was: Asp218 Thr80 Ser105 Glu128 Pro47 Gly69 Ala47 Val72 CyS9 Met14 Ile128 Leu104 Tyr86 Phe60 Lys90 His13 Arg51 Trp23.
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PMID:Amino acid composition of Clostridium botulinum type F neurotoxin. 635 71


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