Purification, subunit structure, and immunological properties of chromatin-bound ribonucleic acid polymerase I from cauliflower inflorescence.
Purification, subunit structure, and immunological properties of chromatin-bound ribonucleic acid polymerase I from cauliflower inflorescence.
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花椰菜花序中染色质结合核糖核酸聚合酶 I 的纯化、亚基结构和免疫学特性。
DOI：
10.1021/bi00567a004
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发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
Guilfoyle,TJ
中科院分区：
文献类型：
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作者：
Guilfoyle,TJ
Tom J. Guilfoyle摘要：大规模纯化的脱氧核糖核酸（DNA）依赖的核糖核酸（RNA）聚合酶I从花椰菜花序已实现以下的酶从染色质模板的溶解。在高离子强度下通过肝素-琼脂糖色谱法纯化酶的初始步骤防止了染色质蛋白的再聚集，如果盐浓度降低，则可能发生染色质蛋白的再聚集。酶经磷酸纤维素层析、二乙氨基乙基（DEAE-Sephadex）层析、DEAE-cellulose层析和甘油密度梯度离心进一步纯化。纯化的RNA聚合酶Ⅰ的比活为400- 450 nmol UMP掺入RNA的时间为20 min/mg蛋白。从1 kg组织中可获得约300 g纯化酶，总收率> 30%。将酶纯化至均一需要约4-5天。总组织的总纯化倍数约为10000倍，分离染色质的总纯化倍数约为250倍。通过该方法纯化的花椰菜RNA聚合酶I由190000、170000、125000、38000、25000、22000和17500道尔顿的七种多肽组成，如通过一个（十二烷基）确定的。真核细胞含有三种不同类型的核DNA 1依赖性RNA聚合酶，它们通常被称为RNA聚合酶I、II、III、III和III。和三.每一类RNA聚合酶都具有特征性的色谱和催化性质、转录功能、细胞内定位和亚基结构[Roeder（1979）综述]。RNA聚合酶I定位于核仁中并转录核糖体DNA。RNA聚合酶II存在于核质中，催化前体的合成，
Tom J. Guilfoyle abstract: The large-scale purification of deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase I from cauliflowerinflorescence has been achieved following solubilization of the enzyme from the chromatin template. The initial step of purification of the enzyme by heparin-Sepharose chromatography at high ionic strength prevents reaggregation of chromatinproteins which can occur if salt concentrations are reduced. The enzyme was further purified by phosphocellulose, diethylaminoethyl (DEAE)-Sephadex, and DEAE-cellulose chromatography and glycerol density gradient cen-trifugation. The purifiedRNA polymerase I has a specific activity of 400-450nmol of UMP incorporated into RNA in 20 min per mg of protein. From 1 kg of tissue,~ 300 gg of purified enzyme is obtained with an overall yield of> 30%. The purification of the enzyme to homogeneity takes about 4-5 days. The overall purification is~10000-fold from total tissue and~ 250-fold from isolated chromatin. Cauliflower RNA polymerase I purified by this procedure consists of seven polypeptides of 190000, 170000, 125 000, 38 000, 25 000, 22 000, and 17 500 daltons as determined by one-(dodecyl^ Eukaryotic cells contain three distinct classes of nuclear DNA1-dependent RNA polymerases which are generally re-ferred to as RNA polymerases I, II, and III. Each class of RNA polymerase has characteristic chromatographic and catalytic properties, transcriptional functions, intracellular localizations, and subunit structures [reviewed by Roeder (1979)]. RNA polymerase I is localized in the nucleolus and transcribes ribosomal DNA. RNA polymerase II is found in the nucleoplasm and catalyzes the synthesis of precursors to