Use of biotinylated NAD to label and purify ADP-ribosylated proteins.
Biotin- or digoxigenin-conjugated NAD has been used successfully to label EF-2 by diphtheria toxin, an alpha subunit of G protein by pertussis toxin, and poly(ADP-ribose) synthase through auto-poly(ADP-ribosyl)ation (J. Zhang, unpublished result, 1996). It is likely that many other ADP-ribosyl-transferases are capable of using modified NAD as substrates. Compared to radioactive labeling, biotinylation has several advantages. Commercially available precursors make synthesis of biotinylated NAD simple and economic. No extensive purification of the product is required. Because biotinylated NAD can be separated from NAD readily, there is no dilution, in contrast to [32P]NAD, in which only a small proportion of the NAD molecules are radioactive. Once purified, biotinylated NAD can be stored for a long time without decay (unlike radioactive NAD, which does decay). Most importantly, the system described here may afford an efficient means for purifying and identifying ADP-ribosylated proteins. Biotinylated NAD can be used for in situ labeling to study the cellular localization and tissue distribution of the ADP-ribosylated proteins.
Zhang
J
J
Guilford Pharmaceuticals, Inc., Baltimore, Maryland 21224, USA.
D000246:Adenosine Diphosphate Ribose / Q000302:isolation & purification*; D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D002846:Chromatography, Affinity / Q000379:methods*; D019204:GTP-Binding Proteins / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification; D009243:NAD / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000302:isolation & purification*
C107266:8-(N-(N-biotinyl)-8-aminohexylamino)NAD; C107265:N(6)-(N-(N-biotinyl)-6-aminohexylcarbamoylmethyl)NAD; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D011506:Proteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D001710:Biotin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011065:Poly(ADP-ribose) Polymerases; D019204:GTP-Binding Proteins