The Purification and Crystallization of
            <i>Clostridium botulinum</i>
            Type A Toxin

Lamanna, Carl; McElroy, Olive E.; Eklund, Henning W.

doi:10.1126/science.103.2681.613

Cited by 122 publications

(36 citation statements)

References 3 publications

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“…In addition, previous work reported in the literature concerning the influence of BTX on neuromuscular transmission was done either with crude, partially purified preparations or with toxin isolated and crystallized by the methods described by Lamanna, McElroy & Ecklund (1946) BOTULJNUAI TOXIN ON FROG MUSCLE 229 and Duff, Wright, Kierer, Moore & Bibler (1957). These preparations contained impurities not found in the toxin preparations used in the present studies.…”

Section: Introductionmentioning

confidence: 99%

Observations on the action of type A botulinum toxin on frog neuromuscular junctions

Boroff¹,

Castillo²,

Evoy³

et al. 1974

The Journal of Physiology

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SUMMARY1. Progressive block of neuromuscular transmission in frog sartorius and gastrocnemius preparations by haemagglutinin-free crystalline Type A botulinum toxin (BTX) was investigated by in vitro application and by injection of the toxin into living animals.2. Neuromuscular block was characterized by (a) decline in amplitude of evoked twitch contractions, (b) decline in amplitudes of end-plate potentials (e.p.p.s) and (c) changes in statistical characteristics of spontaneous miniature end-plate potentials (m.e.p.p.s).3. Progress of the block was enhanced by nerve stimulation.4. A decrease in frequency to less than 0.1/sec and decreased average amplitudes of m.e.p.p.s preceded observable impairment of neuromuscular transmission. These changes occurred as early as 3 hr after injection of the toxin into dorsal lymph sacs. 5. The amplitude distributions of m.e.p.p.s changed from a normal distribution to one that showed an increased skewness toward smaller amplitudes as the block progressed. These changes were first detectable as early as 75 min following addition of the toxin to the bath.

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Section: Introductionmentioning

confidence: 99%

Observations on the action of type A botulinum toxin on frog neuromuscular junctions

Boroff¹,

Castillo²,

Evoy³

et al. 1974

The Journal of Physiology

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“…The nucleotide and deduced protein sequences of these two type A toxins differ marginally from the sequences of the subtype A1 to A5 toxin genes (16,17). These new toxins may comprise subtypes BoNT/A6 and BoNT/A7, but their neurotoxins have not yet been purified for characterization.It has been recognized since the 1940s that BoNTs naturally form protein complexes (18,19), wherein BoNT associates with nontoxic protein components. Early attempts to isolate the BoNTs from C. botulinum cultures were made by acid precipitation of culture supernatant by adjusting the pH to 3.5 to 4 (sometimes aided by the addition of RNA), which resulted in concentration of toxicity in the precipitated material (20).…”

mentioning

confidence: 99%

“…It has been recognized since the 1940s that BoNTs naturally form protein complexes (18,19), wherein BoNT associates with nontoxic protein components. Early attempts to isolate the BoNTs from C. botulinum cultures were made by acid precipitation of culture supernatant by adjusting the pH to 3.5 to 4 (sometimes aided by the addition of RNA), which resulted in concentration of toxicity in the precipitated material (20).…”

mentioning

confidence: 99%

Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

Lin

Tepp

Bradshaw

et al. 2015

Appl Environ Microbiol

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Botulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. A Clostridium botulinum host strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both an ha silent bont/b cluster and an orfX bont/a1 cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silent bont/b cluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silent bont/b cluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected. Botulinum neurotoxins (BoNTs) are produced by anaerobic, Gram-positive, spore-forming bacteria, including Clostridium botulinum and rare strains of Clostridium baratii and Clostridium butyricum. BoNTs are the most poisonous protein toxins known in nature, and they are classified as tier 1 category A select agents due to their potential use as bioterrorism agents (1). Purified BoNT is a protein with a molecular mass of ca. 150 kDa, consisting in its active form of a heavy chain (Hc) (ϳ100 kDa) and a light chain (Lc) (ϳ50 kDa) linked by a disulfide bond and noncovalent molecular interactions (2). The heavy chain contains two functional domains, a receptor binding domain and a translocation domain (3, 4), while the light chain is responsible for catalytic activity on SNARE substrates in the neuronal cell cytosol. BoNTs are distinguished immunologically into seven serotypes designated A to G based on toxin neutralization using homologous antitoxins (5). Recently, the existence of an eighth serotype, H, in a dual-toxin-producing strain of C. botulinum has been reported (5).Of the seven established serotypes, BoNT/A is of particular interest since it is the most potent BoNT, exhibits longer duration in humans than other serotypes, and is also implicated in most cases of human botulism in the continental United States (6, 7). Within serotype A, five subtypes of BoNT/A (A1 to A5) h...

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“…Nearest decimal numbers for the amino acid residues were obtained for 100,000 g, or approximately one-ninth of the molecular weight of the crystalline protein as determined by physical methods." 1 These values in most cases are obviously beyond the accuracy of the method and are reported in this mrnner for computational reference.…”

Section: Calculattonsmentioning

confidence: 97%

“…The isolation of botulinum toxin, one of the most toxic substances known, in crystalline form 1 ' 2 has led to an interest in its chemical…”

Section: Introductionmentioning

confidence: 99%

Amino Acid Composition of Crystalline Botulinum Toxin, Type A

Stefanye¹,

Schantz²,

Spero³

1965

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The Purification and Crystallization of Clostridium botulinum Type A Toxin

Cited by 122 publications

References 3 publications

Observations on the action of type A botulinum toxin on frog neuromuscular junctions

Observations on the action of type A botulinum toxin on frog neuromuscular junctions

Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

Amino Acid Composition of Crystalline Botulinum Toxin, Type A

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