Activation of Component II

The full biological activity of C2 toxin, a mixture of components I and II, is obtained by activation of the toxin with trypsin (Miyake and Ohi-shi, 1987 Ohishi et al., 1980 Ohishi et al., 1980 Ohishi, 1987). As described in Section 9.2 (Assay method for the toxin), the full activity of the toxin is produced by a mixture of untrypsinized component I and trypsinized component II. This indicates that activation of the toxin is brought about by the molecular cleavage of component II, but not of component I, by trypsin. Therefore, to study the biological activity of botulinum C2 toxin and the effect of ADP-ribosylation of cytoplasmic actin by C2 toxin on whole cells, it is essential to prepare activated component II (trypsinized component II). 

To prepare the activated component II, incubate 10 mg of component II with 1 mg of trypsin (Sigma Chemical Company, St. Louis, Mo., USA, type lll-S) in 20 ml of 50 mM PB, pH 7.5, containing 200 mM NaCI. Apply the reaction mixture to a column of Sephacryl S-300 (2.5 X 95 cm), which is equilibrated with the buffer. 

Usually, not always, two protein peaks are eluted from the column (Ohishi, 1987). The amount of protein in the first peak from the column is always larger than that in second peak. Both have toxicity when mixed with component I. They each show a single band in SDS-PAGE. Therefore, part of the trypsin-activated component II forms an oligomer. Collect the first peak, because, if the second peak is collected, it is occasionally contaminated with trypsin and/or trypsin-digested fragments, although it depends on the gel filtration conditions. 

Transfer the collected fractions to a dialysis bag and concentrate with Ficoll 400 (Pharmacia Biotech) at 4 °C. However, care should be taken to avoid the concentration of trypsinized component II exceeding 500ng/ml, because the activated component II tends to aggregate at a higher concentration. 

5 Endocytosis of Two Nonlinked Protein Components in Cultured Cells 

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The specific-titer activity of lima-bean lectin components II and III towards type A human erythrocytes was 5,100 and 1,300, respectively, and, towards type B erythrocytes, 20 and 5.1, respectively.151 199 The hemagglutinating activity of component II is, thus, four times the activity151,199 of component III. Neither component reacted with type O human, red blood-cells, or native or trypsinized, rabbit erythrocytes.151,199... 

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