Purification and chemical characterization of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic Escherichia coli.
Heat-stable enterotoxin (ST) produced by porcine strains of enterotoxigenic (ENT+) Escherichia coli has been purified to apparent homogeneity by sequential ultrafiltration, acetone fractionation, preparative gel electrophoresis, diethylaminoethyl Bio-Gel A ion-exchange chromatography, and Bio-Gel P-10 gel filtration. The enterotoxin, purified more than 1,500-fold, exhibited a molecular weight of 4,400, as determined by both sodium dodecyl sulfate-gel electrophoresis and gel filtration. A molecular weight of 5,100, representing 47 residues, was calculated from amino acid analysis data. The amino acid content was distinctive, with an unusually high proportion of cystines and few hydrophobic amino acids. A single amino-terminal residue, glycine, was observed. Purified ST was stable to heating (100 degrees C, 30 min) and did not lose biological activity after treatment with Pronase, trypsin, proteinase K, deoxyribonuclease, ribonuclease, and phospholipase C. Periodic acid oxidation and several organic solvents (acetone, phenol, chloroform, and methanol) had no effect on the biological activity of ST. Further, purified ST was stable to acid treatment at pH 1.0 but lost biological activity at pH values greater than 9.0. Neither lipopolysaccharide nor lipid contamination was evident in purified preparations. A characteristic absorption spectrum was observed during the course of the purification, which shifted from a maximum at 260 nm in crude preparations to 270 nm for the purified toxin. Antiserum obtained from rabbits immunized with ST or ST coupled to bovine serum albumin neutralized the action of the enterotoxin in suckling mice; however, passive hemagglutination and hemolysis titer assays suggested that ST is a poor antigen.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis; D001427:Bacterial Toxins / Q000032:analysis* / Q000276:immunology; D002241:Carbohydrates / Q000032:analysis; D004768:Enterotoxins / Q000032:analysis* / Q000276:immunology; D004926:Escherichia coli; D006358:Hot Temperature; D008970:Molecular Weight; D013056:Spectrophotometry, Ultraviolet; D013552:Swine / Q000382:microbiology
D000596:Amino Acids; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D002241:Carbohydrates; D004768:Enterotoxins