Figure 9

ADP-ribosylation of endogenous versus exogenous substrates. (A-B) Cholix toxin catalytic fragments were pre-incubated with or without 50 μM NAD⁺ at 37°C for 1 hour. Free NAD⁺ was removed by gel filtration chromatography. The recovered enzymes were quantified. Equal concentrations of auto-ADP-ribosylated CTc (pre-incubated with NAD⁺) or control (non-auto-ADP-ribosylated CTc, processed through auto-ADP-ribosylation reaction without NAD⁺) were used in the NAD⁺ glycohydrolase activity assays and ADP-ribosylation of eEF2 in 293 lysate. (C) His-tagged oligo-L-arginine or oligo-L-asparagine peptides were incubated with purified recombinant wild type CTc, catalytically defective mutant (Y493A) or catalytically active mutant (E579Q). The samples were analyzed by a 96-well plate based ADP-ribosylation assay. Data shown are composite from two experiments with triplicates within-plate replicates. (D)Various concentrations of catalytic fragments of cholix toxin or exotoxin A (PEA) was incubated with CHO or Re1.22c cell lysate at 37°C for 1 hr. The biotin signals on the ADP-ribosylated eEF2 and auto-ADP-ribosylated enzymes were detected by IRDye800CW-SA shown on the top panel. The same blot was stripped and re-probed with anti-CTc and anti-eEF2 antibodies shown in the middle panel. The bottom panel shows the Coomassie Blue stained gel for protein loading control. (E) To detect auto-ADP-ribosylation at the presence of exogenous substrates, excess amount of CTc (6 μM) was incubated with purified flag-tagged wild type eEF2 (0.2 μM) or flag-tagged eEF2 (H715R) mutant (0.2 μM) at the presence of 50 μM biotinyl-NAD⁺ for various periods of time.