PURIFICATION AND CHARACTERIZATION OF ADENOSINE-TRIPHOSPHATE - RIBONUCLEIC-ACID ADENYLTRANSFERASE FROM ESCHERICHIA-COLI
PURIFICATION AND CHARACTERIZATION OF ADENOSINE-TRIPHOSPHATE - RIBONUCLEIC-ACID ADENYLTRANSFERASE FROM ESCHERICHIA-COLI
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DOI：
10.1111/j.1432-1033.1973.tb02953.x
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发表时间：
1973-01-01
期刊：
影响因子：
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通讯作者：
SIPPEL, AE
中科院分区：
文献类型：
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作者：
SIPPEL, AE
使用 ATP 作为底物（ATP：RNA 腺苷酸转移酶）以高产量从大肠杆菌中分离出引物依赖性聚 (A) 聚合活性，并纯化至表观同质性。为此目的，必须使用限制多种推断酶活性的测定系统。由于酶在低盐条件（<0.4 M NaCl）下会聚集成大分子细胞成分或沉淀，因此有必要在高盐条件下进行整个纯化过程。这是通过使用高盐核糖体上清液、聚乙二醇-葡聚糖-氯化钠相分配步骤和非常有效的最终高盐磷酸纤维素色谱来完成的。在 0.5 M NaCl 下，该酶的分子量约为 58000。十二烷基硫酸盐凝胶电泳显示分子量 × 50000 的单条多肽链。该酶对 ATP 作为底物具有高度偏好。锰离子作为辅助因子显示出比镁离子更高的活性。所有类别的天然 RNA 均用作引物。需要 RNA 的游离 3' 末端羟基。该酶不能催化焦磷酸解或磷酸解反应。结果表明，ATP：RNA 腺苷酸转移酶优先合成附着在 RNA 引物上的相当长的聚 (A) 链。无法检测到酶蛋白与 DNA 依赖性 RNA 聚合酶亚基之间的关系。
A primer‐dependent poly (A)‐polymerizing activity using ATP as substrate (ATP: RNA adenyltransferase) was isolated in high yield fromEscherichia coliand purified to apparent homogeneity. For this purpose an assay system had to be used which restricted a variety of infering enzyme activities. Since the enzyme aggregates to macromolecular cell components or precipitates when kept in low salt conditions (<0.4 M NaCl), it was necessary to perform the entire purification procedure in high salt conditions. This was accomplished by using a high salt ribosomal supernatant, a polyethylenglycol‐dextran‐NaCl phase partition step and a very efficient final high salt phosphocellulose chromatography. At 0.5 M NaCl the enzyme has a molecular weight of approximately 58000. Dodecylsulfate gel electrophoresis shows a single polypeptide chain of molecular weight × 50000. The enzyme has a high preference for ATP as substrate. Manganese ions show higher activity as cofactors than magnesium ions. All classes of natural RNAs are used as primers. The free 3′terminal hydroxyl group of the RNA is required. The enzyme is unable to catalyze a pyrophosphorolysis or a phosphorolysis reaction. It is shown that ATP: RNA adenyl‐transferase preferentially synthesizes rather long chains of poly(A) attached to the RNA primers. No relation between the enzyme protein and subunits of the DNA‐dependent RNA polymerase could be detected.