小鼠骨髓瘤DNA聚合酶γ多肽组分的鉴定

Akio Matsukage , Kazushi Tanabe , Masamitsu Yamaguchi , Yukarin N. Taguchi , Miwako Nishizawa , Taijo Takahashi , Toshitada Takahashi
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引用次数: 8

摘要

以(rA)n·(dT) 12-18为模板引物,对小鼠骨髓瘤DNA聚合酶γ进行了广泛纯化,最终比活性为每mg蛋白156 000单位(每小时nmol dTMP结合)。通过DNA-纤维素柱层析获得的蛋白质组分的十二烷基硫酸钠聚丙烯酰胺凝胶电泳显示Mr = 47000的多肽的数量随DNA聚合酶γ活性成比例地变化。Mr = 14 000的一个小多肽似乎也随着酶的活性而变化,但其他多肽没有变化。通过125i标记的肽图谱分析表明,小鼠骨瘤DNA聚合酶γ制剂中的Mr 47000多肽在结构上与鸡胚DNA聚合酶γ的Mr 47000多肽相关(Yamaguchi, M., Matsukage, A. and Takahashi, T. (1980) J. Biol。化学,255,7002 - 7009)。一种针对鸡胚DNA聚合酶γ的抗体与小鼠酶发生交叉反应,表明禽和鼠酶之间存在结构关系。由于Mr 47000多肽占纯化制剂中总蛋白的31.4%,因此估计每mg Mr 47000多肽的比活性约为490 000单位。纯化酶的聚(dT)延伸率为每分钟1260个核苷酸。该值与周转率(每个酶分子每分钟1530个核苷酸)的范围相同,周转率是根据mr47000多肽的“预期”比活性和分子量(假设mr47000多肽的四聚体结构,Mr = 188000)计算得出的。结果表明,mr47000多肽是小鼠骨髓瘤DNA聚合酶γ的一个组成部分。
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Identification of a polypeptide component of mouse myeloma DNA polymerase γ

Mouse myeloma DNA polymerase γ was extensively purified to a final specific activity of 156 000 units (nmol dTMP incorporation per h) per mg protein on (rA)n · (dT)12–18 as a template primer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of protein fractions obtained by DNA-cellulose column chromatography revealed that the amount of a polypeptide of Mr = 47 000 changed proportionally with DNA polymerase γ activity. A minor polypeptide of Mr = 140 000 also seemed to change with the enzyme activity, but other polypeptides did not. Analysis by 125I-labeled peptide mapping indicates that the Mr 47 000 polypeptide in the mouse myeloma DNA polymerase γ preparation is structurally related to the Mr 47 000 polypeptide of chick embryo DNA polymerase γ (Yamaguchi, M., Matsukage, A. and Takahashi, T. (1980) J. Biol. Chem. 255, 7002–7009). An antibody against chick embryo DNA polymerase γ cross-reacted with the mouse enzyme, indicating a structural relationship between avian and murine enzymes. Since the Mr 47 000 polypeptide accounts for 31.4% of total protein in the purified preparation, the specific activity is estimated to be about 490 000 units per mg of the Mr 47 000 polypeptide. The rate of poly(dT) elongation by the purified enzyme was 1 260 nucleotides per min. This value is in the same range as the turnover number (1 530 nucleotides per min per enzyme molecule) which is calculated from the “expected” specific activity with respect to the Mr 47 000 polypeptide and the molecular weight (Mr = 188000 on the assumption of a tetrameric structure of the Mr 47 000 polypeptide). Results indicate that the Mr 47 000 polypeptide is a component of the mouse myeloma DNA polymerase γ.

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