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Figure 6 | BMC Biochemistry

Figure 6

From: ADP-Ribosylargininyl reaction of cholix toxin is mediated through diffusible intermediates

Figure 6

Auto-ADP-ribosylation results a mixture of α - and β-anomeric ADP-ribosylargininyl adducts. (A) Auto-ADP-ribosylated wild type CTc or M8 derived mutants or the ADP-ribosylated exoT substrate was incubated with purified hADPRH for various periods of time. The biotinyl-ADP-ribosylation remaining was detected by blotting with IRDye800CW-SA and quantified by Odyssey Infrared imaging system. The data shown in (A) are representative blots from multiple experiments. Five independent experiments were quantified and summarized in (B). Data from 3 different experiments of hydrolysis of auto-ADP-ribosylated M8K525R, M8K543R and M8K566R were summarized in (C). The error bar shows standard error of mean (SEM). Hydrolysis index is the ratio of normalized biotin signals remained on the hADPRH treated versus untreated substrates. (D) Auto-ADP-ribosylation of catalytic fragments of exotoxin A (PEA) and cholix toxin (CTc) was carried out with various concentrations of biotinyl-NAD+ in the ADP-ribosylation buffer containing 3 μM of purified enzymes shown in top panel. The bottom panel shows the Coomassie blue stained bands for protein loading control.

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