Rose K M, Jacob S T
Eur J Biochem. 1976 Aug 1;67(1):11-21. doi: 10.1111/j.1432-1033.1976.tb10626.x.
Poly(A) polymerase was extracted from isolated nuclei of rat liver and a rapidly growing solid tumor (Morris hepatoma 3924A). The enzyme from each tissue was purified by successive chromatography on DEAE-Sephadex, phosphoecllulose, hydroxyapatite and QAE-Sephadex. Purified enzyme from both liver and tumor was essentially homogeneous as judged by polyacrylamide gel electrophoresis. Under nondenaturing conditions, enzyme activity corresponded to visible protein and, upon denaturation, a single polypeptide was detected. The enzymes had absolute requirements for Mn2+ as the divalent ion, ATP as the substrate and an oligonucleotide or polynucleotide as the primer. Both enzymes were inhibited by sodium pyrophosphate, N-ethylmaleimide, Rose Bengal, cordycepin 5'-triphosphate and several rifamycin derivatives. The reactions were unaffected by potassium phosphate, alpha-amanitin and pancreatic ribonuclease. However, the liver and hepatoma enzymes differed from each other with respect to apparent Km, primer saturation levels and sensitivity to pH changes. The most striking differences between the enzymes were in their calculated molecular weights (liver, 48000; hepatoma, 60000) and amino acid compositions. Finally, the level of the hepatoma enzyme relative to that of the liver enzyme was at least 1.5-fold higher when expressed per mg DNA.
多聚腺苷酸聚合酶是从大鼠肝脏和一种快速生长的实体瘤（莫里斯肝癌3924A）的分离细胞核中提取的。通过在DEAE-葡聚糖、磷酸纤维素、羟基磷灰石和QAE-葡聚糖上连续层析，对来自每个组织的酶进行了纯化。通过聚丙烯酰胺凝胶电泳判断，来自肝脏和肿瘤的纯化酶基本均一。在非变性条件下，酶活性与可见蛋白相对应，变性后，检测到一条单一的多肽。这些酶对二价离子Mn2+、作为底物的ATP以及作为引物的寡核苷酸或多核苷酸有绝对需求。两种酶都受到焦磷酸钠、N-乙基马来酰亚胺、孟加拉玫瑰红、虫草素5'-三磷酸和几种利福霉素衍生物的抑制。这些反应不受磷酸钾、α-鹅膏蕈碱和胰核糖核酸酶的影响。然而，肝脏和肝癌的酶在表观Km、引物饱和水平和对pH变化的敏感性方面彼此不同。这些酶之间最显著的差异在于它们计算出的分子量（肝脏，48000；肝癌，60000）和氨基酸组成。最后，以每毫克DNA表示时，肝癌酶相对于肝脏酶的水平至少高1.5倍。
