Gundelfinger E, Saumweber H, Dallendörfer A, Stein H
Eur J Biochem. 1980 Oct;111(2):395-401. doi: 10.1111/j.1432-1033.1980.tb04953.x.
DNA-dependent RNA polymerase III, which is usually highly resistant to alpha-amanitin, has been purified from Drosophila hydei pupae. The enzyme is insensitive to alpha-amanitin concentrations up to 0.1 microM; at 100 microM alpha-amanitin the enzyme activity is inhibited by approximately 10%. The enzyme was extracted at low ionic strength and purified to homogeneity by six purification steps; 0.1--0.2 mg enzyme/kg pupae could be obtained. The subunit composition of the enzyme was determined after sucrose-gradient centrifugation by sodium dodecyl sulphate electrophoresis in polyacrylamide gels. The enzyme was resolved into putative subunits of molecular weight 154 000, 135 000, 62 000, 58 000, 38 000, 32 000, 31 000, 27 200, 26 500, 21 500 and 17 500. This subunit composition is in general accord with that of eucaryotic class III enzymes. The catalytic properties (salt-activation profile, ratio of activity with denatured DNA to that with native DNA) and the order of elution of the enzyme from DEAE-cellulose with respect to RNA polymerase II agree with the classification of the isolated enzyme as an RAN polymerase III.
通常对α-鹅膏蕈碱具有高度抗性的DNA依赖性RNA聚合酶III已从海德果蝇蛹中纯化出来。该酶对高达0.1微摩尔的α-鹅膏蕈碱浓度不敏感；在100微摩尔α-鹅膏蕈碱时，酶活性受到约10%的抑制。该酶在低离子强度下提取，并通过六个纯化步骤纯化至同质；每千克蛹可获得0.1 - 0.2毫克酶。通过在聚丙烯酰胺凝胶中进行十二烷基硫酸钠电泳，在蔗糖梯度离心后测定了该酶的亚基组成。该酶被分解为推定的分子量为154000、135000、62000、58000、38000、32000、31000、27200、26500、21500和17500的亚基。这种亚基组成总体上与真核生物III类酶的组成一致。该酶的催化特性（盐激活曲线、变性DNA与天然DNA的活性比）以及该酶相对于RNA聚合酶II从DEAE - 纤维素上洗脱的顺序与分离出的酶作为RAN聚合酶III的分类一致。
