Purification and Characterization of DNA Methylase From HL-60 Cells

A solid leukemia sarcoma has been successfully developed after subcutaneous inoculation of the cultured human promyelocytic leukemia cells (HL-60 cells) into unde mice. The solid leukemia sarcoma is a more plantiful source than the cultured cells for enzymatic study and its growing environment is closer to that of the human body than the cultured cells.We establish an efficient procedure of purifying HL-60 cells DNA methylase which includes: disruption of HL-60 cells by homogenization and sonication, removing the cell fragments and cellular particles by centrifuge and ultracentrifuge (105.000 g); removing endogenous DNA by streptomycin sulfate, salting out by (NH4)2SO4, ion exchange chromatography on DEAE-cellulose (DE-52), gel filtration over Sephadex G-100 column.The DNA methylase from HL-60 cells has been purified 204 fold by this procedure. The purified enzyme shows a single-band on PG-PAGE. A 479-kD molecular weight of this enzyme is measured by PG-PAGE. The enzyme properties of HL-60 DNA methylase     

甲基化作用增强了盐效应对M.L.DNA大分子构象的影响

本文用Hha I DNA甲基化酶为工具酶,制备了甲基化水平为2.39％的Micrococcus luteus DNA(天然的M.L.DNA是完全未甲基化的)。又以CD光谱、UV光谱及微量热法等手段,比较了甲基化和未甲基化的M.L.DNA在不同MgCl_2浓度下的[θ]值、A值及释热值的变化。发现随着MgCl_2浓度的增加,两种M.L.DNA的[θ]_(275)值均减少;而—[θ]_(220-250)值加大;A_(260)值则呈明显的减色效应;释热值却相应增加。这都说明MgCl_2的作用使M.L.DNA大分子在空间上趋于缩拢,且这个过程均持续约10min。同时发现:甲基化水平为2.39％的M.L.DNA对MgCl_2效应的敏感性要比末甲基化的M.L.DNA高50—70倍。     

5-氮胞苷(5-aza-CR)及其类似物对HL-60细胞分化及DNA甲基化的影响

本文报道了5-aza-CR和5-aza-CdR对HL-60细胞分化及DNA甲基化作用的影响。用NBT染色法测定了加药处理前后HL-60细胞分化程度的变化;用[~3H-SAM]为甲基供体,以同位素参入法测定了加药前后HL-60细胞中甲基化酶活力的变化;并用HPLC法测定了加药前后HL-60细胞DNA的甲基化水平。结果表明:在5-aza-CR或5-aza-CdR的使用浓度下处理HL-60细胞沿粒细胞系分化程度增加,DNA甲基化酶活力下降,DNA甲基化水平也下降。并且这些变化均与5-aza-CR的浓度相关;5-aza-CR的影响较5-aza-CdR小,因此后者是更为有效的诱导物。本项研究中还以不同甲基化水平的HL-60细胞DNA为模板,在体外测定了E.Coli RNA聚合酶的活力。发现:随着HL-60细胞DNA甲基化水平的降低,RNA聚合酶的体外转录活力升高。     

ENHANCEMENT OF THE SALT EFFECT ON THE CONFORMATION OF M. L. DNA MOLECULES BY METHYLATION

Methylated Micrococcus luteus DNA (M. L. DNA) in which the DNA methylation level was 2.39% was prepared by using Hhal DNA methylase. (The natural M. L. DNA is completely unmethylated.) The [θ]_(275) value of M. L. DNA was decreased and the —[θ]_(220-250) value was increased with increasing concentration of MgC1_2. The UV spectra of M. L. DNA at different concentration of MgCl_2 showed that a hypochromisity happens at 260nm with increasing concentration of MgC1_2. The heat-releasing of M. L. DNA also increased with increasing concentration of MgC1_2. A11 these results showed that the M. L. DNA molecules tend to shrink in space, The difference between methylated DNA and unmethylated DNA was compared. The sensitivity of methylated DNA to MgC1_2 is 40—70 fold higher than that of the unmethylated M. L. DNA.     

HL-60细胞DNA甲基化酶的纯化和性质

通过将HL-60细胞移植到裸鼠体内获得实体肿瘤。以此为材料确立了一个简便的提纯DNA甲基化酶程序,它包括:细胞的超声破碎、超离心、去核酸、硫酸铵盐析、DEAE-纤维素柱层析及Sephadex G100凝胶过滤。用梯度-PAGE得到一条电泳均一的区带,测得该酶的分子量为479kD,该酶的最适pH为8.2,对钾、钠离子敏感,对热不稳定;用L-B作图法测得其对底物S-腺苷酰-L-甲硫氨酸(SAM)的K_m为2.94×10~(-5)mol/L;SAH是该酶的竞争性抑制剂。在1mmol/L S-腺苷酰-L-高丰胱氨酸(SAH)存在下,其K_i为4.17×10~(-4)mol/L,同时也测定了该酶对DNA底物的专一性及几种代谢相关物对该酶的调节作用。     

等电聚焦——分离蛋白质的新技术

引言等电聚焦(Isoelectric focusing)或简称电聚焦(electrofocusing),这一名称是自1967年以来普遍采用的,在此之前也称为等电分离,等电划分,等电聚集,等电分析,聚焦电泳或稳定电解。在六十年代初期,报导了关于这一方法的理论探讨和实际工作。1966年合成了载体两性电解质,并成功地用于肌红蛋白的分离,使这方法具有了实用价值。近几年来这一技术得到迅速推广。关于这方面的会议集和综述也有不少。     

大肠杆菌L-天门冬酰胺酶的酪氨酸微区

本文用圆二色光谱、微量热法研究了大肠杆菌L-天门冬酰胺酶的构象变化与活力的关系。进一步证明了效应物L-半胱氨酸对该酶的可逆抑制作用,是由于其诱发了该酶分子中酪氨酸微区的微环境改变所致。并用分光光度滴定、化学修饰、氨基酸定量分析等方法,对酪氨酸微区的空间构象进行了初步探讨。作者还用邹承鲁作图法证明了该酶分子表面有两个酪氨酸残基为必需基团。