Purification of a nitric oxide-stimulated ADP-ribosylated protein using biotinylated beta-nicotinamide adenine dinucleotide

ADP-ribosylation, involving the transfer of an ADP-ribose moiety from NAD to proteins, is mediated by several bacterial toxins and endogenous ADP-ribosyltransferases. We report here the synthesis of biotinylated NAD and its use to label and purify biotinyl-ADP-ribosylated proteins. We demonstrate that biotinylated NAD can be used by diphtheria toxin to biotinylate elongation factor 2. Using avidin affinity chromatography, we have purified a protein whose ADP-ribosylation is enhanced by nitric oxide and which has been identified as glyceraldehyde-3-phosphate dehydrogenase.