FIG. 4.

Effect of dithiothreitol on pore formation by Cry1Aa and its mutants. Each toxin was first incubated in the presence (+DTT) and absence (−DTT) of 2 mM EDTA and 5 mM dithiothreitol at a concentration of 0.5 mg/ml for a least 1 h. Vesicles (0.4 mg membrane protein/ml) isolated from fifth-instar larvae were equilibrated overnight in 10 mM HEPES-KOH (pH 7.5). Before the experiments, bovine serum albumin was added to a final concentration of 1 mg/ml. The toxin was then incubated for 1 h with the vesicles prior to the experiment, at a concentration of 150 pmol of toxin/mg of membrane protein for mutants with reduced activity (A) or 50 pmol of toxin/mg of membrane protein for those with activity near that of the wild type (B). The vesicles were then rapidly mixed with an equal volume of a solution containing 150 mM KCl, 1 mg/ml bovine serum albumin, and 10 mM HEPES-KOH (pH 7.5) directly in a cuvette by using a stopped-flow apparatus. Osmotic swelling of the vesicles was monitored by measuring scattered-light intensity at an angle of 90°. Significant differences using Student's t test were found between values obtained in the presence versus the absence of dithiothreitol (*, P < 0.05; **, P < 0.01).