NAD(-RIBOSYL) TRANSFERASE() ANALOGS SUBSTITUTED IN THE PURINE BASE ASSUBSTRATES FOR POLY(ADP)

Poly(ADP-ribosyl) transferase (pADPRT) catalyzes the transfer of the A DP-ribose moiety from NAD(+) onto proteins as well as onto protein-bou nd ADP-ribose. As a result, protein-bound polymers of ADP-ribose are f ormed, pADPRT itself contains several acceptor sites for ADP-ribose po lymers and may attach polymers to itself (automodification). In this s tudy the influence of substitutions in the purine base of NAD(+) on th e polymerization reaction was investigated. The adenine moiety of NAD( +) was replaced by either guanine, hypoxanthine or 1,N-6-ethenoadenine . These analogs served as substrates for polymer synthesis as judged f rom the extent of automodification of the enzyme and the sizes of the polymers formed. Time course experiments revealed that 1,N-6-etheno NA D(+) (epsilon-NAD(+)) and nicotinamide hypoxanthine dinucleotide (NHD) were rather poor substrates as compared to NAD(+). Synthesis of GDP- ribose polymers from nicotinamide guanine dinucleotide (NGD(+)) was mo re efficient, but still significantly slower than poly(ADP-ribosyl)ati on of the enzyme using NAD(+). The size of the different polymers appe ared to correlate with these observations. After 30 min of incubation in the presence of 1 mM substrate, polymers formed from epsilon-NAD(+) or NHD+ contained up to 30 epsilon-ADP-ribose or IDP-ribose units, re spectively. Using NGD(+) as substrate polymers consisted of more than 60 GDP-ribose units, an amount similar to that achieved by poly(ADP-ri bosyl)ation in the presence of only 0.1 mM NAD(+) as substrate. These results suggest that the presence of an amino group in the purine base of NAD(+) may facilitate catalysis. Substitution of the nicotinamide moiety of NAD(+) with 3-acetylpyridine had no detectable effect on pol ymer formation. Oligomers of GDP-ribose and epsilon-ADP-ribose exhibit ed a slower mobility in polyacrylamide gels as compared to ADP-ribose or IDP-ribose oligomers. This feature of the two former analogs as wel l as their markedly attenuated polymerization by pADPRT provide valuab le tools for the investigation of the enzymatic mechanism of this prot ein. Moreover, polymers of epsilon-ADP-ribose may be useful for studyi ng enzymes degrading poly(ADP-ribose) owing to the fluorescence of thi s analog. Digestion of epsilon-ADPR polymers with snake venom phosphod iesterase was accompanied by a significant fluorescence enhancement.