Burtseva N N, Gimadutdinov O A, Vaniushin B F
Biokhimiia. 1987 Feb;52(2):290-302.
Using cell extract fractionation with ammonium sulfate and subsequent chromatography on DEAE- and DNA-cellulose and Blue Sepharose, two cytosine DNA-methylases were isolated from blood lymphocytes of cows suffering from lympholeukosis; one cytosine DNA-methylase was isolated from blood lymphocytes of healthy animals. The DNA-methylases from normal lymphocytes was purified 383-fold; the enzyme has a specific activity of 2.3 u./mg, Mr of 114 000 Da and a pH optimum of 7.6. The molecular mass of DNA-methylases from leukemic lymphocytes is about 130,000 Da. The enzymes isolated from leukemic lymphocytes, i.e., DNA-methylase I (568-fold purification, specific activity 14.2 u./mg) and DNA-methylase II (524-fold purification, specific activity 13.1 u./mg) possess different action optima at pH 7.8 and 6.7, respectively. The total DNA-methylase activity of leukemic lymphocytes is about 4 times that of normal lymphocytes. All the DNA-methylases under study methylate in vitro bacterial and animal DNAs of different base composition; bacterial DNAs are the best (GC content is approximately 70%), while homologous DNAs- the worst acceptors of CH3-groups. Heat-denaturated DNAs are methylated more intensively than initial DNAs. The optimal NaCl concentration in the reaction mixture is approximately 50 mM; EDTA (greater than 10 mM) inhibits the reaction. DNA-methylase I of leukemic and normal lymphocytes show the same pH optimum and specificity of action. In vitro methylation of bacterial DNA by these DNA-methylases in the presence of [3H-methyl]S-adenosylmethionine results in the similar type of label distribution among pyrimidine isopliths in the DNA. DNA-methylase II from leukemic lymphocytes methylates about two times more Pu-C-Pu sequences in th same DNA than does DNA-methylase I from leukemic and normal lymphocytes and thus reveals a different specificity. The changes in the type of DNA methylation as well as the appearance of an additional DNA-methylase possessing a different specificity of action in leukemic lymphocytes may be responsible for cell transformation and transcriptional changes in chronic lympholeukosis.
通过硫酸铵进行细胞提取物分级分离，随后在DEAE - 纤维素、DNA - 纤维素和蓝色琼脂糖上进行层析，从患有淋巴白血病的奶牛血液淋巴细胞中分离出两种胞嘧啶DNA甲基化酶；从健康动物的血液淋巴细胞中分离出一种胞嘧啶DNA甲基化酶。正常淋巴细胞中的DNA甲基化酶纯化了383倍；该酶的比活性为2.3 u./mg，分子量为114000 Da，最适pH为7.6。白血病淋巴细胞中DNA甲基化酶的分子量约为130000 Da。从白血病淋巴细胞中分离出的酶，即DNA甲基化酶I（纯化568倍，比活性14.2 u./mg）和DNA甲基化酶II（纯化524倍，比活性13.1 u./mg），分别在pH 7.8和6.7时具有不同的作用最适值。白血病淋巴细胞的总DNA甲基化酶活性约为正常淋巴细胞的4倍。所有研究的DNA甲基化酶在体外可甲基化不同碱基组成的细菌和动物DNA；细菌DNA是最佳底物（GC含量约为70%），而同源DNA是最差的CH3基团受体。热变性DNA比初始DNA甲基化更强烈。反应混合物中的最佳NaCl浓度约为50 mM；EDTA（大于10 mM）会抑制反应。白血病和正常淋巴细胞的DNA甲基化酶I表现出相同的最适pH和作用特异性。在[3H - 甲基]S - 腺苷甲硫氨酸存在下，这些DNA甲基化酶对细菌DNA进行体外甲基化，导致DNA中嘧啶异质体之间的标记分布类型相似。白血病淋巴细胞中的DNA甲基化酶II在同一DNA中甲基化的Pu - C - Pu序列比白血病和正常淋巴细胞中的DNA甲基化酶I多约两倍，因此显示出不同的特异性。DNA甲基化类型的变化以及白血病淋巴细胞中出现具有不同作用特异性的额外DNA甲基化酶可能是慢性淋巴细胞白血病中细胞转化和转录变化的原因。
