Separation of <i>E. coli</i> heat‐labile enterotoxin by preparative isotachophoresis

Möllby, R.; Hjalmarsson, Sven-Göran; Wadström, Torkel

doi:10.1016/0014-5793(75)80104-9

Cited by 9 publications

(5 citation statements)

References 15 publications

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“…There are significant differences in the molecular weight assigned to LT by various laboratories (8,10,15,18,31,35,40,49,53,56). The data presented in this report and other observations may offer an explanation.…”

Section: Discussionmentioning

confidence: 57%

“…Although several reports have appeared on the purification of LT (8,10,15,18,31,35,40,49,53,56), there is no general agreement as to the molecular weight and chemical nature of toxin as it is released from whole cells. Molecular weights ranging from 23 x 103 to over 106 have been determined, but, more importantly, the preparations noted above were 103to 106-fold less active than cholera toxin in bioassays specific for the two enterotoxins.…”

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confidence: 99%

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Purification and Chemical Characterization of the Heat-Labile Enterotoxin Produced by Enterotoxigenic Escherichia coli

1979

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Heat-labile enterotoxin (LT) produced by a human strain of enterotoxigenic Escherichia coli (286C2) was purified to homogeneity from pH extracts of fermentor-grown cells by ultrafiltration, (NH4)2SO4 fractionation, hydrophobic chromatography on norleucine-Sepharose 4B, hydroxylapatite chromatography, and Bio-Gel P-150 filtration. Purified LT preparations exhibited biological activity comparable to that of cholera toxin in four bioassays specific for the two enterotoxins (Y-1 adrenal tumor cells, Chinese hamster ovary cells, pigeon erythrocyte lysates, and skin permeability test). The overall yield of LT protein was 20%, which represented a 500-fold purification over pH extracts. A native molecular weight of 73,000 was determined by gel electrophoresis. The toxin dissociated upon treatment with sodium dodecyl sulfate, pH 7.0, into two components with molecular weights of 44,000 and 30,000. Purified LT preparations were remarkably stable over a wide range of storage conditions, temperatures, and pH's. The biological activity was increased by incubation with trypsin and completely destroyed by pronase and proteinase K, whereas deoxyribonuclease I, ribonuclease, and phospholipase D had no effect. The amino acid composition of purified LT was quite different from that of cholera toxin. Neither carbohydrate nor lipopolysaccharide was present in purified preparations. The purification scheme appeared applicable to LT produced by other human and porcine enterotoxigenic strains, but reflected the amount of LT produced by each strain. These data show that LT and cholera toxin share many common chemical and physical properties, but must be purified by different techniques. 586 on August 5, 2020 by guest http://iai.asm.org/ Downloaded from PURIFICATION AND PROPERTIES OF LT 587 strains, but the yields were about 10-fold lower.

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Section: Discussionmentioning

confidence: 57%

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confidence: 99%

Purification and Chemical Characterization of the Heat-Labile Enterotoxin Produced by Enterotoxigenic Escherichia coli

1979

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“…In our opinion, the nutritional parameters defined in this work will complement attempts to produce hypertoxigenic ENT+ mutants. There seems to be little question that the low yield of extracellular toxin has been a contributing factor to conflicting molecular weights and heterogeneity of purified preparations (11,13,14,29,34). One human strain, 286C2, produces at least 30fold more extracellular toxin than does H-10407, a strain used to study various aspects of the pathogenesis of ENT+ E. coli (12).…”

Section: Resultsmentioning

confidence: 99%

“…Preparation of media. The complex medium of Casamino Acids (CAA) and yeast extract (YE) was prepared as described by Evans et al (12) and served as a reference complex medium along with Trypticase soy broth and brain heart infusion broth, both of which have been used for LT production in other laboratories (8,14,29).…”

Section: Methodsmentioning

confidence: 99%

Nutritional Requirements for Synthesis of Heat-Labile Enterotoxin by Enterotoxigenic Strains of Escherichia coli

Gilligan

Robertson

1979

Infect Immun

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Optimal growth conditions have been established for production of heat-labile enterotoxin (LT) by both porcine and human strains of enterotoxigenic (ENT') Escherichia coli. There were no unusual growth factor requirements, and some strains produced fairly high levels of LT in a basal salts medium containing 0.5% glucose if the pH was carefully controlled. Several amino acids markedly stimulated LT synthesis when added to the basal salts-glucose medium. Methionine and lysine were the most stimulatory for both human and porcine strains. Either aspartic acid or glutamic acid further enhanced LT synthesis in the presence of methionine and lysine, with aspartic acid being more stimulatory for porcine strains and glutamic acid more stimulatory for human strains. There were no apparent vitamin requirements and no unusual cations needed for toxin synthesis except that Fe3" was slightly stimulatory for porcine strains. The stimulation by Fe3" was observed only in the presence of the three amino acids, suggesting that

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“…Although some reports (12, 16) have demonstrated that partially purified LT, isolated by column chromatography, was of molecular weight >300,000, our separation by membrane filtration indicated that the active components were smaller. lecular weights of 20,000 (7), 100,000 (5,17), 200,000 (20), and >300,000 (12,16) have been reported for LT.…”

Section: Discussionmentioning

confidence: 98%

Assay of Escherichia coli heat-labile enterotoxin with vero cells

Speirs¹,

Stavric²,

Konowalchuk³

1977

Infect Immun

115

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The continuous cell line of African green monkey kidney, Vero, showed characteristic morphological changes in response to culture filtrates from toxigenic strains ofEscherichia coli. The response compared favorably with that of Y-1 (mouse adrenal) and CHO (Chinese hamster ovary) cells. Vero cells were the simplest and most economical to maintain in the laboratory. Escherichia coli heat-labile enterotoxin (LT) acts on certain cell cultures to induce measurable biochemical changes, e.g., increased adenylate cyclase activity (6, 10, 13, 22), increased intracellular levels of cyclic adenosine 3',5'monophosphate (9, 22), and induced synthesis of A4,3-keto-steroids (1, 2, 4). With Y-1 (mouse adrenal) and CHO (Chinese hamster ovary) cells, these changes are accompanied by alterations in cell morphology and thus provide a simple diagnostic tool (3, 9, 11, 15, 18, 19). The continuous cell line Vero (African green monkey kidney) is also morphologically affected by LT and shows advantages over other assay methods. The study reported here compares the responses of Y-1 (2), CHO (9), and Vero cells to the culture filtrates of various strains ofE. coli. MATERIALS AND METHODS Enterotoxin production. E. coli enterotoxigenic human or porcine strains H10407, B2C, and B7A were obtained from H. L. DuPont (U.S.A.), and P155, P307, 339, and 711 (P307) were from C. Gyles (Canada). Nontoxigenic strains 711 and K-12 were obtained from C. Gyles and V. N. Iyer (Canada), respectively. Erlenmeyer flasks, 150 ml, containing 20 ml of Trypticase soy broth (TSB) or Evans medium (8) were inoculated and mechanically shaken at 37°C. After 16 to 20 h, the cultures were centrifuged at 17,000 x g for 30 min. The supernatants were filtered through 0.45-,um membrane filters (Millipore Corp.) and stored at 4°C until assay that same day. Filtrate dilutions were made in phosphate-buffered saline (PBS), pH 7.0. Cell culture assay. Stocks of the continuous cell lines Y-1, CHO, and Vero, purchased from the American Type Culture Collection, Rockville, Md., were passaged by trypsinization and grown as monolayers at 36°C in a 5% CO2 atmosphere. Y-1 cells were grown in Ham nutrient mixture F10 (Connaught Laboratories Ltd.), CHO cells were grown in Ham nutrient mixture F12 (Grand Island Biological Co.), and Vero cells were grown in medium 199 with

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Separation of E. coli heat‐labile enterotoxin by preparative isotachophoresis

Cited by 9 publications

References 15 publications

Purification and Chemical Characterization of the Heat-Labile Enterotoxin Produced by Enterotoxigenic Escherichia coli

Purification and Chemical Characterization of the Heat-Labile Enterotoxin Produced by Enterotoxigenic Escherichia coli

Nutritional Requirements for Synthesis of Heat-Labile Enterotoxin by Enterotoxigenic Strains of Escherichia coli

Assay of Escherichia coli heat-labile enterotoxin with vero cells

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