Selective probing of ADP-ribosylation reactions with oxidized 2'-deoxynicotinamide adenine dinucleotide

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Abstract
A homogeneous preparation of an arginine-specific mono(ADP-ribosyl)transferase from turkey erythrocytes effectively utilized 2''-deoxy-NAD+ for the 2''-deoxy(ADP-ribose) modification of arginine methyl ester with an apparent Km of 27.2 .mu.M and a Vmax of 36.4 .mu.mol min-1 (mg of protein)-1. The adduct formed was also used as a substrate by an avian erythrocyte arginine (ADP-ribose)-specific hydrolase that generated free 2''-deoxy(ADP-ribose). In contrast, 2''-deoxy-NAD+ was not a substrate in the initiation or elongation reaction catalyzed by highly purified poly(ADP-ribose) polymerase from calf thymus. However, 2''-deoxy-NAD+ was a potent noncompetitive inhibitor of NAD+ in the elongation reaction catalyzed by the polymerase, with an apparent Ki of 32 .mu.M. These results indicate that 2''-deoxy-NAD+ may be utilized to specifically identify protein acceptors for endogenous mono(ADP-ribosyl)transferases in complex biological systems that may contain a high activity of poly(ADP-ribose) polymerase, i.e., cell nuclei preparations.

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