Treatment of Shigella dysenteriae 1 either with the antibiotic polymyxin B or by osmotic shock resulted in the release of 80 to 90% of the cytotoxin activity of the organism. Under the conditions employed, the release of toxin activity was accompanied by the appearance of a periplasmic enzyme, 5'-nucleotidase. There was no significant release of cytoplasmic contents, assessed by measurement of glucose-6-phosphate dehydrogenase activity. The release of cytotoxin and 5'nucleotidase by polymyxin B were both dependent on the duration of incubation with, and the concentration of, the antibiotic. In terms of specific activity (cytotoxin activity per milligram of protein), the polymyxin B and osmotic shock extracts were 20to 30-fold more active than crude toxin preparation derived from a whole-cell lysate. The data strongly support a periplasmic location for Shiga cytotoxin and the utility of the polymyxin B extraction to obtain starting material for toxin purification.
Materials and MethodsToxin was prepared from S. dysenteriae 1, strain 60-R, as previously described (5). This material contained enterotoxic (ileal loop secretion), neurotoxic (mouse lethal) and HeLa cell cytotoxic activities. Cytotoxicity, the most sensitive, quantitative, and reproducible assay, shows that ST may be separated by isoe|ectric focusing into two cytotoxic fractions, one with isoelectric point (pI) 7.25 (also having the enterotoxic and neurotoxic activities) and the other pI 6.1 (6, 7); both fractions are neutralized to an equivalent degree by experimental antisera versus crude ST and by convalescent human sera from natural and experimental infection (5). The studies reported herein were based entirely upon cytotoxicity assay by the quantitative micromethod of Keusch et al. (4). A single ST preparation which had not been separated by isoelectric focusing was used throughout (5), and all subsequent reference to toxin in this paper is to the cytotoxic activity.Toxin binding was measured indirectly by means of a consumption assay for toxin. HeLa cell monolayers or isolated rat liver cell membranes were exposed to toxin for varying time periods and conditions, depending on the nature of the experiment. When HeLa cells were studied the supernatant medium was aspirated from monolayers at the en~ of the experimental period. This *
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