Isolation and Properties of Heat-Labile Enterotoxin(s) from Enterotoxigenic Escherichia coli

Finkelstein, Richard A.; LaRue, Micbael K.; Johnston, David W.; Vasil, Michael L.; Cho, Gloria J.; Jones, James R.

doi:10.1093/infdis/133.supplement_1.s120

Cited by 49 publications

(27 citation statements)

References 30 publications

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“…One of these, a high-molecularweight [223-2251, heat-labile toxin [62,218,224,, resembles choleragen in its mechanism of action [62,224, (Table VI) and probably exerts its effects by increasing intracellular cyclic AMP [62,[233][234][235]; for review, see 5,12,20,236,2371. The E coli enterotoxin has not been purified to homogeneity; it appears, however, to exist in both high-molecular-weight (2 X 1 06) and low-molecular-weight (20,000) species [223][224][225]. While thus differing from choleragen, the E coli enterotoxin does have immunologic cross-reactivity [62,224,227,235,238,, probably with the B or ganglioside-binding subunit of choleragen [241,243] .…”

Section: Mechanistic Similarities Between Choleragen a N D Eschermentioning

confidence: 99%

Mechanism of action of choleragen

Vaughan

Moss

1978

J Supramol Struct

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Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside GM1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell-free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium-dependent regulatory protein. GTP also is required for the expression of choleragen-activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol-stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP-adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell-free systems the A subunit, specifically the A1 fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A1 fragment under suitable conditions hydrolyzes NAD to ADP-ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP-ribose moiety of NAD to the guandino group of arginine (ADP-ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD-dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP-ribosylation of a specific acceptor protein. If the ADP-ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP-ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.

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Section: Mechanistic Similarities Between Choleragen a N D Eschermentioning

confidence: 99%

Mechanism of action of choleragen

Vaughan

Moss

1978

J Supramol Struct

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“…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%

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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“…Purified E. coli enterotoxin, kindly provided by F. Domer of this institute, was prepared from a porcine pathogenic E. coli strain, P 263, serotype 08:K87;88a b:H19. Relationships between this E. coli enterotoxin prepared by F. Dorner and E. coli enterotoxin prepared by R. A. Finkelstein were recently published (8). E. coli enterotoxin was radioiodinated by the procedure of Greenwood et al (10).…”

Section: Materiais and Methodsmentioning

confidence: 99%