Synthesis and purification of deoxyribose analogues of NAD+ by affinity chromatography and strong-anion-exchange high-performance liquid chromatography.
Two different chemical analogues of NAD+, containing either 2'-deoxyribose (2'-dNAD+) or 3'-deoxyribose (3'-dNAD+) were synthesized enzymatically with oxidized nicotinamide mononucleotide (NMN+)-adenyl transferase (E.C. 2.7.7.18). These compounds were efficiently purified by affinity chromatography on a boronate gel, followed by strong-anion-exchange high-performance liquid chromatography under isocratic conditions. These chromatographic steps resulted in the elimination of unreacted deoxyadenosine triphosphates (dATP) and NMN+, respectively. The purified dNAD+ isomers were examined as possible substrates of homogeneous poly(ADP-ribose) polymerase, purified from calf thymus. 3'-dNAD+ was effectively utilized as a substrate by the polymerase, which catalyzed the formation of protein-bound poly(3'-dADP-ribose) during automodification. However, 2'-dNAD+ was not a substrate for the automodification reaction catalyzed by this DNA-dependent enzyme. Instead, 2'-dNAD+ was a potent non-competitive inhibitor of NAD+ in the elongation reaction.
Alvarez-Gonzalez
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Biomedical Division, Samuel Roberts Noble Foundation, Inc., Ardmore, OK 73402.
D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003855:Deoxyribose / Q000138:chemical synthesis* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D009243:NAD / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000302:isolation & purification; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification
D009243:NAD; D003855:Deoxyribose; D011065:Poly(ADP-ribose) Polymerases