Okayama H, Ueda K, Hayaishi O
Proc Natl Acad Sci U S A. 1978 Mar;75(3):1111-5. doi: 10.1073/pnas.75.3.1111.
Nuclear proteins modified by mono or poly ADP-ribosylation were selectively isolated and purified by covalent chromatography on a dihydroxyboryl polyacrylamide bead column that specifically interacts with cis-diol-containing compounds. From rat liver nuclei that had been incubated with NAD+, histones and some nonhistone proteins were extracted with 0.25 M HCl. Approximately 60% of the ADP-ribose incorporated into 20% trichloroacetic acid-precipitable material was recovered in this extract. The ADP-ribosylated material was then isolated from the extract by covalent chromatography on a borate gel column and further purified by carboxymethylcellulose column chromatography. As judged by electrophoretic mobilities in various gel systems and by amino acid compositions, approximately 50% of the ADP-ribose recovered in the carboxymethylcellulose fractions was associated with several nonhistone proteins with molecular weights of 2-6 x 10(4), while 35% aand 15% were associated with histones H2B and H1, respectively. Since the average chain length of the polymer bound to any of these proteins was less than two ADP-ribos-l units, the percentage distribution reflects the number of ADP-ribosylated sites rather than the chain length.
通过在二羟基硼基聚丙烯酰胺珠柱上进行共价层析，选择性地分离和纯化了经单或多聚ADP - 核糖基化修饰的核蛋白，该柱能与含顺式二醇的化合物特异性相互作用。用0.25M HCl从与NAD +孵育过的大鼠肝细胞核中提取组蛋白和一些非组蛋白。在该提取物中回收了约60%掺入到20%三氯乙酸可沉淀物质中的ADP - 核糖。然后通过在硼酸盐凝胶柱上进行共价层析从提取物中分离出ADP - 核糖基化物质，并通过羧甲基纤维素柱层析进一步纯化。根据在各种凝胶系统中的电泳迁移率和氨基酸组成判断，在羧甲基纤维素级分中回收的约50%的ADP - 核糖与几种分子量为2 - 6×10⁴的非组蛋白相关，而35%和15%分别与组蛋白H2B和H1相关。由于与这些蛋白质中任何一种结合的聚合物的平均链长小于两个ADP - 核糖单元，因此百分比分布反映的是ADP - 核糖基化位点的数量而非链长。
