2 DIFFERENT TYPES OF BONDS LINKING SINGLE ADP-RIBOSE RESIDUES COVALENTLY TO PROTEINS - QUANTIFICATION IN EUKARYOTIC CELLS
2 DIFFERENT TYPES OF BONDS LINKING SINGLE ADP-RIBOSE RESIDUES COVALENTLY TO PROTEINS - QUANTIFICATION IN EUKARYOTIC CELLS
复制标题
DOI：
10.1111/j.1432-1033.1978.tb12730.x
复制
发表时间：
1978-01-01
期刊：
影响因子：
--
通讯作者：
HILZ, H
中科院分区：
文献类型：
--
作者：
BREDEHORST, R;WIELCKENS, K;HILZ, H
用中性羟胺处理大鼠肝和埃利希腹水瘤细胞的蛋白质，使与蛋白质共价结合的单个ADP-核糖残基成为酸溶性的。将酸溶性提取物与1 M NaOH在56 ℃下温育。C将释放的ADP-核糖选择性地转化为5“-AMP，然后可以通过对5”-AMP的高度特异性放射免疫测定来定量。用碱直接处理酸不溶性组分产生了比NH 2 OH处理更高量的ADP-核糖当量，这表明释放了额外的ADP-核糖残基，其通过NH 2 OH抗性、碱不稳定的连接与受体连接，如已经在体外观察到的。在成年大鼠肝中，由NH_2 OH不稳定键连接的单（ADP-核糖）残基为5300 pmol/mg DNA，NaOH释放的单（ADP-核糖）残基为12，600 pmol，埃利希腹水癌细胞（生长稳定期）的相应值分别为480 pmol和1660 pmol。与体外发现的比率相反，尽管总可释放单（ADP-核糖）残基的差异接近10倍，但两种组织均表现出较高水平的ADP-核糖残基与受体结合的NH 2 OH-抗性连接比NH 2 OH-敏感键连接的残基。在真核细胞中，大部分ADP-核糖基化的蛋白质显然是由单个ADP-核糖而不是由聚（ADP-核糖）链修饰的。在测定ADP-核糖残基之前用高氯酸提取组蛋白H1表明，组蛋白H1在体内仅携带非常小的一部分总蛋白结合单（ADP-核糖）残基。
Single ADP-ribose residues covalently bound to protein of rat liver and Ehrlich ascites tumor cells were rendered acid soluble by treatment with neutral hydroxylamine. Incubation of the acid-soluble extract with 1 M NaOH at 56.degree. C converted the released ADP-ribose selectively to 5''-AMP, which could then be quantified by a highly specific radioimmunoassay for 5''-AMP. Direct treatment of the acid-insoluble fractions with alkali yielded a higher amount of ADP-ribose equivalents than NH2OH treatment, indicating the release of additional ADP-ribose residues linked to the acceptors by an NH2OH-resistant, alkali-labile linkage, as already observed in vitro. In adult rat liver 5300 pmol mono(ADP-ribose) residues/mg DNA linked by NH2OH-labile bonds, and 12,600 pmol mono(ADP-ribose) residues released by NaOH were found. The corresponding values for Ehrlich ascites tumor cells (stationary growth phase) were 480 pmol and 1660 pmol, respectively. In contrast to the ratios found in vitro, and in spite of the nearly 10-fold difference in total releasable mono(ADP-ribose) residues, both tissues exhibited higher levels of ADP-ribose residues bound to the acceptors by the NH2OH-resistant linkage than residues linked by NH2OH-susceptible bonds. The bulk of the ADP-ribosylated proteins in eukaryotic cells is apparently modified by single ADP-ribose rather than by poly(ADP-ribose) chains. Extraction of histone H1 with perchloric acid prior to the determination of ADP-ribose residues indicated that histone H1 in vivo carried only a very small fraction of the total protein-bound mono(ADP-ribose) residues.