Improved purification and further characterization of <i>Clostridium perfringens</i> type A enterotoxin

Hauschild, A. H. W.; Hilsheimer, R.; Martin, William G.

doi:10.1139/m73-222

Cited by 15 publications

(11 citation statements)

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“…The amino acid composition of the enterotoxin is given in Table 1. The results are in good agreement with those of Hauschild et al ( 8 ) . The only important significant difference is the result of cysteic acid.…”

Section: Amino Acid Analysissupporting

confidence: 92%

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CHEMICAL MODIFICATION AND CHARACTERIZATION OF ENTEROTOXIN FROM CLOSTRIDIUM PERFRINGENS TYPE A

Granum¹,

Skjelkvåle²

1977

Acta Pathologica Microbiologica Scandinavica Section B Microbio

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Enterotoxin from Clostridium perfringens type A has been purified. The enterotoxin was shown to be heat‐labile, but re‐activation of heat treated enterotoxin did occur down to an activity of 15 per cent of the native enterotoxin. The molecular weight was shown to be 34,000 by ultracentrifugation, and the molecular weight did not change significantly after treatment with 0.1 M β‐mercaptoethanol and 6 M guanidine hydrochloride. It was concluded that the enterotoxin consists of one single polypeptide chain. The enterotoxin did not lose any activity after treatment with iodoacetic acid and iodoacetamide, but a complete loss of activity was observed after succinylation.

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Section: Amino Acid Analysissupporting

confidence: 92%

“…The only important significant difference is the result of cysteic acid. In this investigation it was found that the enterotoxin contained only one cysteic acid residue per molecule, compared to two cysteic acids found in the work of Hauschild et al ( 8 ) .…”

Section: Amino Acid Analysismentioning

confidence: 43%

CHEMICAL MODIFICATION AND CHARACTERIZATION OF ENTEROTOXIN FROM CLOSTRIDIUM PERFRINGENS TYPE A

Granum¹,

Skjelkvåle²

1977

Acta Pathologica Microbiologica Scandinavica Section B Microbio

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“…Using these data the compositional relatedness was performed between type A (8-6) and type A enterotoxin [18] and type delta toxin [43]. Similar results were obtained using the methods described by Cornish-Bowden [44] and Metzger et al [45].…”

Section: Amino Acid Analysismentioning

confidence: 85%

Purification and properties of an enterotoxin from a coatless spore mutant of Clostridium perfringens type A

Lindsay¹,

Sleigh²,

Ghitgas³

et al. 1985

European Journal of Biochemistry

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A method is described for isolating an enterotoxin from a coatless spore mutant (8-6) of Clostridium perfringens type A. The characteristics of this enterotoxin only slightly resembled those of previously isolated enterotoxins of C. perfringens. The type A (8-6) enterotoxin was found to be composed of two subunits of M , 18000 with isoelectric points of 3.8 and 4.3. The LD5,, for mice was 39 pg/kg with 0.10 pg corresponding to one erythemal unit. The type A (8-6) enterotoxin was inactivated by heating for 10 min at 60°C. The amino acid composition data of type A (8-6) and delta toxins was similar, but type A (8-6) and type A enterotoxins showed less similarity. This lack of similarity between type A and type A (8-6) enterotoxins was confirmed by the failure of anti-sera to type A enterotoxin to neutralize the type A (8-6) enterotoxin, in both the mouse and erythemal tests.Certain strains of Clostridium perfringens type A produce an enterotoxin that is linked to a major cause of mild food poisoning in humans [l]. The enterotoxin is a sporulationspecific gene product, whose production is correlated with late stage I1 to stage I11 of sporulation [2 -51. Several lines of evidence indicate that enterotoxin is possibly excess spore coat protein not incorporated as a structural component of the spore coat, and released from the sporangia only on lysis [6].While a large amount of data is available on the mode of action of enterotoxin [7-91 and more recently on a binding protein [lo] and membrane receptors [ll], information on structure and composition is still subject to some conjecture.The type A enterotoxin isolated by Granum and Skjelkvale [12] is a single polypeptide chain of M , 34000, however, although this molecule has now been fully sequenced [ 13 -151, the question still arises, whether this is the only enterotoxin produced by C. perfringens type A. This concern stems from some unresolved discrepancies in the literature as to the number of subunits, the M , of these subunits, their behaviour under SDS gel electrophoresis and some slight variation in amino acid composition of the enterotoxin when produced under different conditions [16 -231.In our studies on the development of spore heat resistance we have used a strain of C. perfringens type A [24] which produces a spore without a coat, although it produces coat protein(s) and heat-stable spores. Our rationale for this study was that if enterotoxin production is linked to coat protein synthesis, a coatless spore may provide enterotoxin in larger quantities than would normally be obtainable from coated spores. In this paper we report the isolation and characterization of a different enterotoxin from the coatless mutant of C. perfringens type A designated 8-6.Correspondence to J. A. Lindsay, CSIRO Division of Food Research, P. 0. Box 52, North Ryde, Sydney, New South Wales, Australia 21 13Abbreviations. i.p., intraperitoneally ; buffer A, 0.05 M phosphate buffer pH 6.7; SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performanc...

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“…Use of HPLC for gel permeation chromatography and ion-exchange c h r o m a t o g r a p h y in ptace of conventional gel permeation through Sephadex (22,24), ion-exchange c h r o m a t o g r a p h y .on a DEAE (24) .or a QAE-Sephadex column (14), or preparative electrophoresis (8) made the present method rapid and gave high resolution. The conventional methods of c h r o m a t o g r a p h y require at least one day for each step, but in the present HPLC method, one cycle of gel permeation on a G2000SW column and one cycle o.f ion-exchange on a Mono Q column require 50 and 40 min, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…The toxin is produced during sporulation (7,16) and it has been purified (1,8,12,(20)(21)(22)(23)(24)27) from sonic extracts of sporulated bacteria and shown to be a simple protein with a molecular weight of ca. 35000 (11,13,14,25). 1 Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

Purification by high performance liquid chromatography of Clostridium perfringens type a enterotoxin prepared from high toxin producers selected by a toxin-antitoxin halo

1985

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High enterotoxin-producing substrains of Clostridium perfringens type A were selected reproducibly as colonies having toxin-antitoxin haloes on agar plates of Duncan-Strong medium containing antitoxin serum. Enterotoxin from these substrains was subjected to rapid purification by high performance liquid chromatography (HPLC). For this, the toxin was extracted by sonication from sporulating bacteria grown in Duncan-Strong sporulation medium, fractionated by ammonium sulfate (40% saturation) precipitation and differential solubilization and then purified by HPLC: gel permeation chromatography through a G2000SW column and ion-exchange chromatography on a Mono Q column. Purified toxin preparations had a similar specific activity (4.2 X 10(2) mouse MLD/mg protein) and homogeneity on polyacrylamide gel-electrophoresis to preparations obtained by conventional gel permeation through a Sephadex-G200 column. By further HPLC on a Mono Q column, minor nontoxin proteins were separated from the toxin without loss of the toxicity on a protein basis. The final yield of the purified toxin was about 15% of that in the bacterial extract. The two HPLC procedures each took only one hour.

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Improved purification and further characterization of Clostridium perfringens type A enterotoxin

Cited by 15 publications

References 0 publications

CHEMICAL MODIFICATION AND CHARACTERIZATION OF ENTEROTOXIN FROM CLOSTRIDIUM PERFRINGENS TYPE A

CHEMICAL MODIFICATION AND CHARACTERIZATION OF ENTEROTOXIN FROM CLOSTRIDIUM PERFRINGENS TYPE A

Purification and properties of an enterotoxin from a coatless spore mutant of Clostridium perfringens type A

Purification by high performance liquid chromatography of Clostridium perfringens type a enterotoxin prepared from high toxin producers selected by a toxin-antitoxin halo

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