FIG. 3.
Inhibition of enzyme activity of toxin B by antitoxin. (A) Toxin B was incubated with the indicated dilutions of antitoxin IgG (anti-C. difficile, anti-CDB1-546, anti-toxin A, or anti-toxin B) or buffer (con) for 15 min on ice, followed by toxin B-catalyzed 14C glucosylation of Rac1 as described in Materials and Methods. PhosphorImager data from SDS–12.5% PAGE are shown. (B) Toxin B was incubated with antitoxin IgG (anti-toxin A, anti-toxin B, or anti-CDB1-546), or buffer (control) for 15 min on ice, followed by toxin B-catalyzed glycohydrolase reaction to cleave UDP-glucose into UDP and glucose as described in Materials and Methods. UDP-glucose and cleaved products were separated by TLC, and TLC results were quantified with ImageQuant (Molecular Dynamics). Error bars indicate standard deviations.