Figure 3

Intramolecular transfer of ADP-ribose to multiple arginines in the auto-reaction. (A) Mixing marked enzymes experiments. Active Ig-CTc was mixed with inactive mutant Y493A (top panel); active full-length cholix toxin, CXT (WT), was mixed with mutant Y493A (middle panel); inactive mutant CXT(E581A) was mixed with active CTc (bottom panel) in auto-ADP-ribosylation assays. Molar concentrations shown on the top of each blot are the concentrations of active enzyme. The left blots (SA) were detected by IRDye800CW-SA; the same blots were re-probed with polyclonal rabbit anti-CTc antibody shown on right (IB). (B) Neutral hydroxylamine assays. Auto-ADP-ribosylation reaction was carried out with periplasmic fractions of E. coli lysate expressing wild type or mutant CTc. The products were subjected to 0.5 M NH₂OH (pH7.5) or 0.5 M NaCl treatment at 37°C for 2 hours. The blot shown is representative of multiple experiments. (C-E) Single amino acid substitution mutation studies. A set of representative data from the detection of biotin signals on wild type or different mutants was shown in (C). The fluorescence intensity of SA (IRDy-SA) was normalized with the Coomassie Blue intensity (CB) to obtain the IRDy-SA/CB ratio as a semi-quantitative auto-ADP-ribosylation measurement of each enzyme. Data were summarized from four sets of experiments (D). Asterisk indicates significant reduction of auto-ADP-ribosylation activity. NAD⁺ glycohydrolase activities of wild type and mutant enzymes are shown in (E).