SYNTHESES OF PHOTOACTIVE ANALOGS OF ADENOSINE-DIPHOSPHATE (HYDROXYMETHYL)PYRROLIDINEDIOL AND PHOTOAFFINITY-LABELING OF POLY(ADP-RIBOSE) GLYCOHYDROLASE

TWO isomeric azidoadenosyl analogues of adeno sine diphosphate (hydrox ymethyl)pyrrolidinediol [ADP-HPD; Slama, J. T., et al. (1995) J, Med. Chem. 38, 389-393] were synthesized as photoaffinity labels for poly(A DP-ribose) glycohydrolase. 8-Azidoadenosine diphosphate (hydroxymethyl )pyrrolidinediol (8-N-3-ADP-HPD) inhibited the enzyme activity by 50% at ca. 1 mu M, a concentration 80-fold lower than that where the isome ric 2-azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol did. [ alpha-P-32]-8-N-3-ADP-KPD was therefore synthesized and used to photod erivatize poly(ADP-ribose) glycohydrolase, Irradiation of recombinant poly(ADP-ribose) glycohydrolase and low concentrations of [alpha-P-32] -8-N-3-ADP-HPD with short-wave UV light resulted in the covalent incor poration of the photoprobe into the protein, as demonstrated by gel el ectrophoresis followed by autoradiography or acid precipitation of the protein followed by scintillation counting. No photoincorporation occ urred in the absence of UV light. The photoincorporation saturated at low concentrations of the photoprobe and photoprotection was observed in the presence of low concentrations of ADP-HPD, an indication of the specificity of the photoinsertion reaction. These results demonstrate that [alpha-P-32]-8-N-3-ADP-HPD can be used to specifically covalentl y photoderivatize the enzyme to characterize the polypetides that cons titute the ADP-HPD binding site of poly(ADP-ribose) glycohydrolase. Th e photoincorporation reaction was further used to determine the abilit y of ADP-ribose polymers of varying size to compete with [alpha-P-32]- 8-N-3-ADP-HPD for binding to the enzyme. Photoincorporation of [alpha- P-32]-8-N-3-ADP-HPD was inhibited by 80% in the presence of low concen trations of short, unbranched ADP-ribose oligomers (5-15 ADP-ribose un its in length). No similar photoprotection was afforded by the additio n of a high-molecular weight highly branched polymer. These results in dicate that the photolabel shares a binding site with the short, linea r polymer, but not with the long, highly branched polymer.