[Properties and specificity of action of DNA methylases from blood lymphocytes of healthy cattle and cattle with chronic lymphoid leukemia]

Abstract

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.