Ramsinghani S, Koh D W, Amé J C, Strohm M, Jacobson M K, Slama J T
Department of Medicinal and Biological Chemistry, College of Pharmacy, University of Toledo, Ohio 43606, USA.
Biochemistry. 1998 May 26;37(21):7801-12. doi: 10.1021/bi9730386.
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.
合成了二磷酸腺苷（羟甲基）吡咯烷二醇[ADP-HPD；斯拉马，J. T.等人（1995年）《药物化学杂志》38卷，389 - 393页]的两种异构叠氮腺苷类似物，作为聚（ADP - 核糖）糖苷水解酶的光亲和标记物。8 - 叠氮二磷酸腺苷（羟甲基）吡咯烷二醇（8 - N3 - ADP - HPD）在约1微摩尔浓度时可抑制该酶活性50%，此浓度比异构的2 - 叠氮二磷酸腺苷（羟甲基）吡咯烷二醇低80倍。因此合成了[α - 32P] - 8 - N3 - ADP - HPD并用于对聚（ADP - 核糖）糖苷水解酶进行光衍生化。用短波紫外光照射重组聚（ADP - 核糖）糖苷水解酶和低浓度的[α - 32P] - 8 - N3 - ADP - HPD，光探针共价掺入蛋白质中，这通过凝胶电泳后进行放射自显影或蛋白质酸沉淀后进行闪烁计数得以证明。在无紫外光时未发生光掺入。光掺入在低浓度光探针时达到饱和，并且在低浓度ADP - HPD存在下观察到光保护作用，这表明光插入反应具有特异性。这些结果表明，[α - 32P] - 8 - N3 - ADP - HPD可用于特异性地将该酶共价光衍生化，以表征构成聚（ADP - 核糖）糖苷水解酶ADP - HPD结合位点的多肽。光掺入反应还进一步用于确定不同大小的ADP - 核糖聚合物与[α - 32P] - 8 - N3 - ADP - HPD竞争结合该酶的能力。在低浓度短的、无分支的ADP - 核糖寡聚物（长度为5 - 15个ADP - 核糖单元）存在下，[α - 32P] - 8 - N3 - ADP - HPD的光掺入被抑制80%。添加高分子量高度分支的聚合物未提供类似的光保护作用。这些结果表明，光标记物与短的线性聚合物共享一个结合位点，但与长的高度分支的聚合物不共享。
