Chain length analysis of ADP-ribose polymers generated by poly(ADP-ribose) polymerase (PARP) as a function of beta-NAD+ and enzyme concentrations.
Chain length analysis of ADP-ribose polymers generated by poly(ADP-ribose) polymerase (PARP) as a function of beta-NAD+ and enzyme concentrations.
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聚（ADP-核糖）聚合酶（PARP）生成的 ADP-核糖聚合物的链长分析作为 β-NAD 和酶浓度的函数。
DOI：
10.1080/713803695
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Alvarez-Gonzalez,R
中科院分区：
文献类型：
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作者：
Mendoza-Alvarez,H;Chavez-Bueno,S;Alvarez-Gonzalez,R
聚（ADP-核糖）聚合酶（PARP）的双反应物自聚（ADP-核糖基）化（EC 2.4. 2.30）通过在固定的蛋白质浓度下使用增加浓度的β-NAD [sup+]（供体底物）或在固定的β-NAD [sup+]浓度下使用增加浓度的PARP（受体底物）进行。合成的[[sup 32] P] ADP-核糖聚合物通过Glu的羧酸酯部分和聚合物之间的单酯键的碱性水解从PARP化学分离。然后通过高分辨率聚丙烯酰胺凝胶电泳和放射自显影分析核酸样聚合物。观察到的ADP-核糖链长度显示底物浓度依赖性延长（从0.2 θM至2 mM β-NAD [sup+]）。在4.5、9、18、27和36 nM PARP的固定浓度下观察到相似结果。因此，我们得出结论，ADP-核糖供体底物的浓度决定了合成的聚合物的平均链长。相反，当PARP浓度在4.5至18 nM之间变化时，在固定的β-NAD [sup+]浓度下，聚合物尺寸不变。然而，当PARP浓度> 18 nM时，产生的单体ADP-核糖的总量明显减少。因此，我们得出结论，高浓度的PARP导致ADP-核糖链起始反应水平的受体底物抑制。
Bireactant autopoly (ADP-ribosyl) ation of poly (ADP-ribose) polymerase (PARP)(EC 2.4. 2.30) was carried out by using either increasing concentrations of β-NAD [sup+](donor substrate) at a fixed protein concentration or increasing concentrations of PARP (acceptor substrate) at a fixed β-NAD [sup+] concentration. The [[sup 32] P] ADP-ribose polymers synthesized were chemically detached from PARP by alkaline hydrolysis of the monoester bond between the carboxylate moiety of Glu and the polymer. Nucleic acid-like polymers were then analyzed by high-resolution polyacrylamide gel electrophoresis and autoradiography. The ADP-ribose chain lengths observed displayed substrate concentration-dependent elongation from 0.2 θM to 2 mM β-NAD [sup+]. Similar results were observed at fixed concentrations of 4.5, 9, 18, 27, and 36 nM PARP. Therefore, we conclude that the concentration of the ADP-ribose donor substrate determines the average chain length of the polymer synthesized. In contrast, the polymer size was unaltered when the concentration of PARP was varied from 4.5 to 18 nM at a fixed β-NAD [sup+] concentration. However, when PARP concentrations > 18 nM were used, the total amount of monomeric ADP-ribose produced was noticeably less. Therefore, we conclude that high concentrations of PARP lead to acceptor substrate inhibition at the level of the ADP-ribose chain initiation reaction.