DEOXYNAD AND DEOXYADP-RIBOSYLATION OF PROTEINS

Recently, two deoxyribose analogs of beta NAD(+) (2'-deoxy and 3'-deoxyNAD(+)) have been synthesized and purified in this laboratory. Whereas 2'-deoxyNAD(+) was an efficient substrate for arg-specific mon(ADP-ribosyl) transferases, it was not a substrate for poly(ADP-ribose) polymerase (PARP). Instead, it was a non-competitive inhibitor of beta NAD(+) in the ADP-ribose polymerization reaction catalyzed by PARP. Thus, 2'-deoxyNAD(+) has been utilized to distinguish between mono(ADP-ribose) and poly(ADP-ribose) acceptor proteins. 2'-deoxyNAD(+) has also been used to characterize the arg-specific mono(2'-deoxyADP-ribosyl)ation reaction of PARP with cholera toxin or avian mono(ADP-ribosyl)transferase. By contrast, 3'-deoxyNAD(+) can effectively be utilized as a substrate by PARP. However, while the estimated Km and Keat of polymerization with 3'-deoxyNAD(+) were 20 mu M and 0.11 moles/sec, the Km and Keat with beta NAD(+) as a substrate were 59 mu M and 1.29 moles/sec, respectively. Determination of the average size of 3'-deoxyADP-ribose polymers indicated that chains no larger than four residues are synthesized with this substrate. Thus, the utilization of 3'-deoxyNAD(+) has facilitated the electrophoretic identification of poly(ADP-ribose) acceptor proteins in mammalian chromatin.