鼠肝DNA甲基化酶的纯化及物理化学性质的研究

以Ｗｉｓｔａｒ大鼠肝为材料，确立了一个简便的纯化鼠肝ＤＮＡ甲基化酶的程序，包括：细胞的超声破碎、去内源核酸、硫酸铵盐析、磷酸纤维素亲和层析、ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０柱层析及ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤。用不同浓度聚丙烯酰胺凝胶电泳和孔梯度凝胶电泳检测，纯化后的酶已达电泳均一，且酶的比活力提高１１２倍。以聚丙烯酰胺孔梯度凝胶电泳测得其天然酶的分子量为３６５ｋＤ，以ＳＤＳ－聚丙烯酰胺凝胶电泳测得该酶有两种亚基，大亚基为９５ｋＤ，小亚基为８５ｋＤ，推测该酶由两个大亚基和两个小亚基组成。

Purification and Physicochemical Properties of DNA Methylase from Rat Liver

A simple and efficient procedure for purifying DNA methylase from rat liver has been established. The purification steps include: disruption of rat liver cells by homogenization and sonication; removing the cell fragments and cellular particles by centrifuge; removing the endogeneous DNA by streptomycin sulfate; salting out by(NH_4)_2SO_4; affinity chromatography on phospho-cellulose(P11); ion-exchange chromatography on DEAE-Sephadex A-50; gel filtration over Sephadex G-150 column. The DNA methylase from rat liver has been purified for 112 folds by this procedure. The electrophoretogram of the DNA methylase revealed a single band on PAGE which are in diffcrcnt concentration and PG-PAGE. The approximate molecular weight of the enzyme is 365 kD as ditermined by PG-PAGE. The result of SDS-PAGE demonstrates that this enzyme is assembled by two types of subunits, which are 95 kD and 85 kD. Considering the symmetry of molecule, thus it was presumed that this enzyme consists of two large subunits(95 kD) and two small subunits(85 kD).