Stalker D M, Mosbaugh D W, Meyer R R
Biochemistry. 1976 Jul 13;15(14):3114-21. doi: 10.1021/bi00659a027.
Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) FROM THE Novikoff hepatoma has been purified over 200 000-fold (based on the increase in specific activity), by ammonium sulfate fractionation and chromatography on DEAE-Sephadex, phosphocellulose, hydroxylapatite, and DNA-cellulose. The enzyme is remarkably stable through all stages of purification until DNA-cellulose chromatography when it must be kept in buffers containing 0.5 M NaCl and 1 mg/ml bovine serum albumin for stability. The enzyme appears to be homogeneous as evidenced by a single stainable band when subjected to electrophoresis in polyacrylamide gels of different porosity. The stainable band corresponds to the DNA polymerase as determined by slicing sister gels and assaying for enzyme activity. The specific activity of the homogeneous preparation is about 60 000 units/mg. The enzyme lacks detectable exonuclease or endonuclease activity. It has a molecular weight of 32 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. In sucrose gradients, the molecular weight is estimated at 31 000. The isoelectric point of the hydroxylapatite fraction enzyme is 8.5. The Novikoff beta-polymerase requires all four deoxyribonucleoside triphosphates, primer-template, and a divalent cation for maximal activity. The apparent Km for total deoxyribonucleoside triphosphate is 7-8 muM and for DNA 125 mug/ml. Activated DNA, rendered 7% acid soluble by DNase I, is the preferred primer-template, although a number of synthetic polynucleotides can by efficiently utilized, particularly in the presence of Mm2+ optimum is 7 mM; the Mn2+ optimum is 1 mM. The pH optimum is 8.4 in Tris-HCl or 9.2 in glycine buffer. The beta-polymerase is sstimulated about twofold by NaCl or KCl at an optimum of 50-100 MM, and the enzyme maintains considerable activity at high ionic strengths. The DNA polymerase is inhibited by ethanol, acetone, and a variety of known polymerase inhibitors. Glycols stimulate the enzyme as does spermine or spermidine. Unlike most beta-polymerases, the Novikoff enzyme is moderately sensitive to N-ethylmaleimide.
从诺维科夫肝癌组织中提取的脱氧核糖核酸聚合酶β（EC 2.7.7.7），通过硫酸铵分级分离以及在DEAE - 葡聚糖、磷酸纤维素、羟基磷灰石和DNA - 纤维素上进行层析，已纯化了200000倍以上（基于比活性的增加）。在纯化的各个阶段，该酶都非常稳定，直到进行DNA - 纤维素层析时，必须将其保存在含有0.5M氯化钠和1mg/ml牛血清白蛋白的缓冲液中以保持稳定性。当在不同孔隙率的聚丙烯酰胺凝胶中进行电泳时，该酶呈现出单一的可染色带，这表明它似乎是均质的。通过切割姐妹凝胶并测定酶活性确定，该可染色带对应于DNA聚合酶。均质制剂的比活性约为60000单位/毫克。该酶缺乏可检测到的核酸外切酶或核酸内切酶活性。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定，其分子量为32000。在蔗糖梯度中，分子量估计为31000。羟基磷灰石级分酶的等电点为8.5。诺维科夫β - 聚合酶需要所有四种脱氧核糖核苷三磷酸、引物 - 模板和二价阳离子才能达到最大活性。总脱氧核糖核苷三磷酸的表观Km为7 - 8μM，DNA的表观Km为125μg/ml。经DNA酶I处理后7%可酸溶的活化DNA是首选的引物 - 模板，不过许多合成多核苷酸也能被有效利用，特别是在存在镁离子的情况下；镁离子的最佳浓度为7mM；锰离子的最佳浓度为1mM。在Tris - HCl中pH最佳值为8.4，在甘氨酸缓冲液中为9.2。β - 聚合酶在50 - 100mM的最佳浓度下，受氯化钠或氯化钾刺激约两倍，并且该酶在高离子强度下仍保持相当的活性。DNA聚合酶受到乙醇、丙酮和各种已知的聚合酶抑制剂的抑制。二醇类物质以及精胺或亚精胺会刺激该酶。与大多数β - 聚合酶不同，诺维科夫酶对N - 乙基马来酰胺中度敏感。
