Alpha haemolysin, produced by Escherichia coli, grown in a chemically defined medium, was purified 19-fold and the endotoxin content reduced 2176-fold by ultrafiltration and glycerol-gradient ultracentrifugation. Immunodiffusion of purified α haemolysin (PH) against antiserum to crude haemolysin (CH) revealed only one precipitation line. PH was cytotoxic in nanogram amounts for mouse-fibro-blast 3T3 cells, and the cytotoxicity exhibited proportional dose-response and time-course kinetics. The cytotoxic and haemolytic activities of PH were neutralised by immunoglobulins to CH. A mutant, produced by treating the haemolytic wild type with mitomycin C, possessed all of the biochemical characteristics of the wild type with the exception that the extracellular products of the mutant were non-haemolytic and noncytotoxic.
BradfordM. M.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248
DuboisM., GillesK. A., HamiltonJ. K., RebersP. A., SmithF.1956; Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350
JorgensenS. E., HammerR. F., WuG. K.1980; Effects of a single hit from the alpha hemolysin produced by Escherichia coli on the morphology of sheep erythrocytes. Infect. Immun. 27:988
KatesM.1972 Techniques of lipidology. Isolation, analysis, and identification of lipids. Laboratory techniques in biochemistry and molecular biology3 North-Holland Publishing Co.; Amsterdam:347–353393–469
MinshewB. H., JorgensenJ., CountsG. W., FalkowS.1978a; Association of hemolysin production, hemagglutination of human erythrocytes and virulence for chicken embryos of extra-intestinal Escherichia coli isolates. Infect. Immun. 20:50
MinshewB. H., JorgensenJ., SwanstrumM., Grootes-ReuvecampG. A., FalkowS.1978b; Some characteristics of Escherichia coli strains isolated from extraintestinal infections of humans. J. infect. Dis. 137:648
MöllbyR., ThelestamM., WadströmT.1974; Effects of Clostridium perfringens phospholipase C (alpha-toxin) on the human diploid fibroblast membrane. J. membr. Biol. 16:313
OsbornM. J.1963; Studies on the gram-negative cell wall, I. Evidence for the role of 2-keto-3-deoxyoctonate in the lipopolysaccharide of Salmonella typhimurium. Proc. natn. Acad. Sci. U.S.A. 50:499
SchaefferW. I., GabliksJ., CalitisR.1967; Interference by trypsin in the interaction of staphylococcal enterotoxin B and cell cultures of human embryonic intestine. J. Bact. 93:1489
SingerR. E., DuttonW. G.1979; A comparison of the effects of endotoxin upon fibroblast proliferation and macromolecular synthesis. J. dent. Res. 58:1634
SmithH. W., LinggoodM. A.1971; Observations on the pathogenic properties of the K88, hly, and ent plasmids of Escherichia coli with particular reference to porcine diarrhoea. J. med. Microbiol. 4:467
ThelestamM., R.Möllby, WadströmT.1973; Effects of staphylococcal alpha-, beta-, delta-, and gamma-hemolysins on human diploid fibroblasts and HeLa cells: evaluation of a new quantitative assay for measuring cell damage. Infect. Immun. 8:938
WaravdekarV. S., SaslawL. D.1959; A sensitive colorimetric method for the estimation of 2-deoxy sugars with the use of the malonaldehyde-thiobarbituric acid reaction. J. biol. Chem. 234:1945
WardP. A., HillJ. H.1972; Detection of lipopolysaccharide (LPS): an improved method for isolation of the Limulus extract. Proc. Soc. exp. Biol. Med. 141:898