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以Lineweave-Burk plot双倒数作图法测得该酶对底物S-腺苷酰甲硫氨酸(SAM)的K_m=7.69×10~(-6)mol/L,在1mmol/LS-腺苷酰高半胱氨酸(SAH)存在下,Ki=7.33×10~(-4)mol/L,两条直线相交于纵轴,证明SAH是该酶的竞争性抑制剂。该酶最适pH为7.8,对热不稳定。同时还测定了该酶对不同DNA底物的专一性及盐浓度、代谢相关物’两价阳离子、某些酸根等对该酶调节性质的影响。以碘代乙酰胺修饰该酶的SH基’及用二硫苏糖醇(DTT)和巯基乙醇(MSH)保护该酶SH基所作的实验表明SH基是该酶活性中心所必需的,用高效液相色谱(HPLC)法证明该酶所甲基化的碱基为刘氏小球菌(M·L、DNA)分子中的胞嘧啶,且求得甲基化30min后所得甲基化水平为2.39%。同时也证明当用该酶将λDNA甲基化后,可使BamHI限制性核酸内切酶对甲基化后的λDNA丧失切割作用。 相似文献
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通过硫酸铵盐析,DEAE-纤维素柱层析,磷酸纤维素亲和层析及SephadexG-100凝胶过滤法,从噬淀粉芽孢杆菌HI(Bacillus amyloliguefaciens HI)提纯了DNA甲基化酶。用聚丙烯酰胺凝胶电泳检查,已达电泳均一,比活力提高了326倍。并用聚丙烯酰胺梯度凝胶电泳和Sephadex G-100凝胶过滤法测得其天然酶的分子量为273000,又用SDS聚丙烯酰胺凝胶电泳测得它的亚基分子量为34500,故该酶有8个分子量相同的亚基。用凝胶电聚焦法测得其pI_(22 c)=9.0。 相似文献
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鼠肝细胞癌变中DNA甲基化作用的研究 总被引:4,自引:0,他引:4
Activity of DNA methylase and DNA methylation level were measured from normal mouse liver, mouse liver charged with H22a ascitic hepatoma and H22a ascitic hepatoma cell by measuring incorporation of H3-methyl. S-Adenosyl-3H-methyl-methionine (3H-SAM) was used as methyl donor. DNA methylation level of different cells were measured by HP-LC. DNA methylase activity and DNA methylation level of H22a ascitic hepatoma, mouse liver charged with H22a ascitic hepatoma are lower than normal mouse liver. Treatments of antitumor drugs lead to a rising of DNA methylase activity of tumor cell, however, the DNA methylation level of tumor cell has not rised after such treatments. 相似文献
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本文用~3H标记的S-腺苷酰甲硫氨酸(~3H-SAM)为甲基供体,以同位素掺入法测定了正常小鼠肝细胞、H_(223)腹水癌细胞及荷癌肝细胞的DNA甲基化酶活力。并用HPLC法测定了上述细胞的DNA甲基化水平。发现:H_(223)腹水癌细胞及荷癌肝细胞的DNA甲基化酶活力和DNA甲基化水平明显低于正常肝细胞。当以抗肝癌药物去甲斑蝥素和斑蝥酸钠处理荷癌小鼠6天后,可使H_(223)腹水癌细胞及荷癌肝细胞的DNA甲基化酶活力回升,但并未检出DNA甲基化水平的回升。 相似文献
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