Schiebel W, Haas B, Marinković S, Klanner A, Sänger H L
Max-Planck-Institut für Biochemie, Martinsried, Germany.
J Biol Chem. 1993 Jun 5;268(16):11851-7.
An RNA-directed RNA polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue was purified to electrophoretic homogeneity. A terminal transferase activity that was found to cofractionate with RdRP from DEAE-Sepharose and DNA-cellulose columns was removed by chromatography on a Mono Q column. The highly purified RdRP exhibits a specific activity of 500 nmol x mg-1 x 30 min-1, which corresponds to a 100,000-fold enrichment of the enzyme. In buffer containing 50% glycerol, its activity decreased by about 15%/month. RdRP activity coincided with the silver staining intensity of a single 128-kDa polypeptide when the fractions eluted from the Mono Q column were analyzed by electrophoresis in a SDS-polyacrylamide gel. Its molecular mass and its sedimentation coefficient of 6.6 S indicate that RdRP is a nearly globular molecule. The catalytic activity of RdRP is resistant to alpha-amanitin and actinomycin D. In tomato leaves systemically infected with potato spindle tuber viroid, the activity of RdRP was found to be increased about 3-fold compared with RdRP isolated from healthy leaf tissue.
从番茄叶片组织中纯化出一种RNA依赖性RNA聚合酶（RdRP，EC 2.7.7.48），使其达到电泳纯。一种与来自DEAE-琼脂糖柱和DNA-纤维素柱的RdRP共分级分离的末端转移酶活性，通过在Mono Q柱上进行层析被去除。高度纯化的RdRP表现出500 nmol·mg⁻¹·30 min⁻¹的比活性，这相当于该酶有100,000倍的富集。在含有50%甘油的缓冲液中，其活性每月下降约15%。当对从Mono Q柱洗脱的级分进行SDS-聚丙烯酰胺凝胶电泳分析时，RdRP活性与一条128 kDa单一多肽的银染强度一致。其分子量和6.6 S的沉降系数表明RdRP是一个近乎球状的分子。RdRP的催化活性对α-鹅膏蕈碱和放线菌素D具有抗性。在被马铃薯纺锤块茎类病毒系统感染的番茄叶片中，发现RdRP的活性与从健康叶片组织中分离的RdRP相比增加了约3倍。
