来自长春花（长春花）冠瘿瘤细胞的高分子量脱氧核糖核酸聚合酶。

High molecular weight deoxyribonucleic acid polymerase from crown gall tumor cells of periwinkle (Vinca rosea).

作者信息

Gardner J M, Kado C I

出版信息

Biochemistry. 1976 Feb 10;15(3):688-97. doi: 10.1021/bi00648a037.

Abstract

A high molecular weight (6 S) plant DNA polymerase from axenic Vinca rosea tissue culture cells has been purified 2200-fold and characterized. The enzyme has a molecular weight of 105 000 (+/-5000). Sodium dodecyl sulfate-acrylamide gel electrophoresis of the purified enzyme yields polypeptide subunits having molecular weights of 70 000 and 34 000. The purified enzyme has a pH optimum of 7.5; a cation requirement optimum of 6 mM Mg2+ or 0.5 mM Mn2+; an apparent requirement for Zn2+; a Km of 1 muM for dTTP; and a 3.5-fold stimulation by 50 mM KCl. The enzyme is sensitive to N-ethylmaleimide (1 mM), heparin (0.1 muM), ethanol (5%), pyrophosphate (0.05 muM), and o-phenanthroline (0.1 mM) but is insensitive to rifamycin. Denatured DNA is found to be the best natural template, and only negligible activity can be demonstrated with the ribopolymer templates poly(dT)n-poly(rA)n and p(dT)10-poly(rA)n. In addition to the polymerization reaction, the enzyme catalyzes a pyrophosphate exchange reaction. Antibody to calf thymus 6-8S DNA polymerase does not inhibit DNA polymerase from Vinca rosea, suggesting no antigenic relationships between the mammalian and plant enzymes.

摘要

从无菌长春花组织培养细胞中纯化出一种高分子量（6S）的植物DNA聚合酶，并对其进行了表征，纯化倍数达2200倍。该酶的分子量为105000（±5000）。纯化后的酶经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，产生分子量分别为70000和34000的多肽亚基。纯化后的酶最适pH为7.5；阳离子需求最适值为6 mM Mg2+或0.5 mM Mn2+；明显需要Zn2+；对dTTP的Km值为1 μM；50 mM KCl可使其活性提高3.5倍。该酶对N-乙基马来酰胺（1 mM）、肝素（0.1 μM）、乙醇（5%）、焦磷酸（0.05 μM）和邻菲罗啉（0.1 mM）敏感，但对利福平不敏感。发现变性DNA是最佳天然模板，而对于核糖聚合物模板聚（dT）n-聚（rA）n和聚（dT）10-聚（rA）n，仅能证明其活性可忽略不计。除聚合反应外，该酶还催化焦磷酸交换反应。抗小牛胸腺6-8S DNA聚合酶的抗体不抑制长春花的DNA聚合酶，这表明哺乳动物和植物的酶之间不存在抗原关系。