title: Syntheses of photoactive analogues of adenosine diphosphate (hydroxymethyl)pyrrolidinediol and photoaffinity labeling of poly(ADP-ribose) glycohydrolase.
abstract: Two isomeric azidoadenosyl analogues of adenosine diphosphate (hydroxymethyl)pyrrolidinediol [ADP-HPD; Slama, J. T., et al. (1995) J. Med. Chem. 38, 389-393] were synthesized as photoaffinity labels for poly(ADP-ribose) glycohydrolase. 8-Azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol (8-N3-ADP-HPD) inhibited the enzyme activity by 50% at ca. 1 microM, a concentration 80-fold lower than that where the isomeric 2-azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol did. [alpha-32P]-8-N3-ADP-HPD was therefore synthesized and used to photoderivatize poly(ADP-ribose) glycohydrolase. Irradiation of recombinant poly(ADP-ribose) glycohydrolase and low concentrations of [alpha-32P]-8-N3-ADP-HPD with short-wave UV light resulted in the covalent incorporation of the photoprobe into the protein, as demonstrated by gel electrophoresis followed by autoradiography or acid precipitation of the protein followed by scintillation counting. No photoincorporation occurred 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-32P]-8-N3-ADP-HPD can be used to specifically covalently photoderivatize the enzyme to characterize the polypetides that constitute the ADP-HPD binding site of poly(ADP-ribose) glycohydrolase. The photoincorporation reaction was further used to determine the ability of ADP-ribose polymers of varying size to compete with [alpha-32P]-8-N3-ADP-HPD for binding to the enzyme. Photoincorporation of [alpha-32P]-8-N3-ADP-HPD was inhibited by 80% in the presence of low concentrations of short, unbranched ADP-ribose oligomers (5-15 ADP-ribose units in length). No similar photoprotection was afforded by the addition of a high-molecular weight highly branched polymer. These results indicate that the photolabel shares a binding site with the short, linear polymer, but not with the long, highly branched polymer.
authors.affiliation: Department of Medicinal and Biological Chemistry, College of Pharmacy, University of Toledo, Ohio 43606, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ramsinghani
authors.forename: D W
authors.initials: DW
authors.lastname: Koh
authors.forename: J C
authors.initials: JC
authors.lastname: Amé
authors.forename: M
authors.initials: M
authors.lastname: Strohm
authors.forename: M K
authors.initials: MK
authors.lastname: Jacobson
authors.forename: J T
authors.initials: JT
authors.lastname: Slama
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010761:Phosphorus Radioisotopes; D019920:Photoaffinity Labels / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D011759:Pyrrolidines / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D010761:Phosphorus Radioisotopes; D019920:Photoaffinity Labels; D011759:Pyrrolidines; C096272:adenosine diphosphate (hydroxymethyl)pyrrolidinediol; D000244:Adenosine Diphosphate; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase