Assay Alpha-toxin

When very small pores that are permissive for monovalent but not for divalent ions are required, alpha-toxin should be used in a concentration range of 0.5-5 ng/ml. Most cells will become perme-abilized.Note, however, that certain cells exhibit a natural resistance towards alpha-toxin and pore formation will not occur. A simple means to discern whether permeabilization has taken place is to observe whether the cells swell increases in cell volume are the consequence of an uncontrolled flux of monovalent ions and water, and can be observed microscopically or by flow cytometry. Another simple method is measurement of cellular ATP. One hour after toxin application, cells are lysed with Triton X-100, and ATP is quantified using the luciferase assay (method described in (Bhakdi ef al., 1989)). ATP depletion will always be found in cells that have been permeabilized. 

PC 12 cells were permeabilized with purified SLO as given in section 18.3.1 of this chapter. Protein content was measured using the BCA-method. Values are the mean of three samples SD. Similar results were obtained using either wild-type SLO fusion protein or its alanine mutant. The assay can also be performed using alpha-toxin for permeabilization. 

The results presented describe experiments In which the effect of glucose on the formation of extracellular proteins by two different organisms was examined. The definitive experiments were carried out at the same time using the same batches of materials and reagents and all the assays were done together on the same day. In the presence of these precautions, as shown In Figure 2, quite different results were obtained. Thus, with A. salmonlclda. In which extracellular protein accounted for 6% of the total bacterial protein output, at the most, the addition of glucose caused a virtually complete repression of exoproteln production. In the case of S. aureus. In which 30% of the protein synthesized may be secreted, there was a very marked reduction In alpha-toxin formation but only a minor modulation of the characteristic pattern of total exoproteln synthesis. 

Maurer, H.H., Schmidt, C.J., Weber, A.A., Kraemer, T. Validated electrospray LC MS assay for determination of the mushroom toxins alpha- and beta-amanitin in urine after immunoaffinity extraction. J. Chromatogr. B Biomed. Sci. Appl. 748, 125-135 (2000)... 

---