Adams R L, Gardiner K, Rinaldi A, Bryans M, McGarvey M, Burdon R H
Biochim Biophys Acta. 1986 Oct 16;868(1):9-16. doi: 10.1016/0167-4781(86)90080-1.
We have purified the DNA methylase from mouse ascites tumour cells to a specific activity of 11,500 units per mg protein using denatured Micrococcus luteus DNA as methyl acceptor. Methyl groups are transferred to cytosines almost exclusively in CpG dinucleotides. The purified enzyme contains two polypeptides of molecular mass 185 and 160 kDa, and an antiserum raised in a rabbit to the purified enzyme specifically reacts with these two proteins in crude extracts. The two proteins can be partially separated by affinity chromatography when activity is associated with the 185 kDa protein which can be proteolytically degraded to give polypeptides of 170 and later 100 and 50 kDa. Only the 185 kDa methylase is lost when cells are treated with azadeoxycytidine and this is the predominant form firmly bound in the nucleus of dividing cells. Antibody bound to the 185 kDa band in protein blots will itself bind native DNA methylase, which can be detected by its binding 14C-labelled, azacytosine-containing DNA.
我们使用变性的藤黄微球菌DNA作为甲基受体，从小鼠腹水肿瘤细胞中纯化出了DNA甲基转移酶，其比活性达到每毫克蛋白质11,500单位。甲基几乎只转移到CpG二核苷酸中的胞嘧啶上。纯化后的酶含有分子量分别为185 kDa和160 kDa的两种多肽，用纯化酶免疫兔子产生的抗血清能与粗提物中的这两种蛋白质发生特异性反应。当活性与185 kDa蛋白质相关联时，这两种蛋白质可以通过亲和层析进行部分分离，185 kDa蛋白质可被蛋白酶降解为170 kDa的多肽，随后又可降解为100 kDa和50 kDa的多肽。当细胞用氮杂脱氧胞苷处理时，只有185 kDa的甲基转移酶会丧失，并且这是在分裂细胞的细胞核中牢固结合的主要形式。与蛋白质印迹中185 kDa条带结合的抗体本身会结合天然DNA甲基转移酶，这可以通过其与含14C标记的氮杂胞嘧啶的DNA结合来检测。
