Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92717.
Proc Natl Acad Sci U S A. 1984 Apr;81(8):2354-8. doi: 10.1073/pnas.81.8.2354.
A DNA polymerase has been purified >3,000-fold from the chloroplasts of pea plants by chromatography on DEAE-cellulose, phosphocellulose, single-stranded DNA-agarose, and sedimentation in a glycerol gradient. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate indicates that the final fraction contained a single discernible protein band of 90,000 daltons. Gel filtration on Sephacryl S-200 and glycerol gradient sedimentation under nondenaturing conditions demonstrate that the chloroplast DNA polymerase has a native molecular mass of approximately 87,000 daltons. The purified polymerase lacks any associated nuclease activity. The enzyme activity is inhibited by N-ethylmaleimide (74% at 1.0 mM) and ethidium bromide (90% at 0.23 mM) and is resistant to aphidicolin. The purified enzyme is totally dependent on the presence of added DNA, has an absolute requirement for Mg(2+) (12 mM optimal), is stimulated by K(+) (120 mM optimal), and requires all four deoxynucleoside triphosphates for maximum activity. Native DNA which has been degraded to a limited extent with DNase I is the most efficient template.
一种 DNA 聚合酶已通过 DEAE-纤维素、磷酸纤维素、单链 DNA-琼脂糖层析和甘油梯度沉降从豌豆植物的叶绿体中被纯化超过 3000 倍。在含有十二烷基硫酸钠的聚丙烯酰胺凝胶电泳分析表明，最后部分含有一种单一可辨别的 90000 道尔顿的蛋白质带。在非变性条件下的 Sephacryl S-200 凝胶过滤和甘油梯度沉降表明，叶绿体 DNA 聚合酶具有约 87000 道尔顿的天然分子量。纯化的聚合酶缺乏任何相关的核酸酶活性。该酶的活性被 N-乙基马来酰亚胺（在 1.0 mM 时抑制 74%）和溴化乙锭（在 0.23 mM 时抑制 90%）抑制，并且对 aphidicolin 有抗性。纯化的酶完全依赖于添加的 DNA 的存在，对 Mg(2+)（最佳 12 mM）有绝对要求，被 K(+)（最佳 120 mM）刺激，并需要所有四种脱氧核苷三磷酸才能达到最大活性。用 DNA 酶 I 有限降解的天然 DNA 是最有效的模板。
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