Elie C, De Recondo A M, Forterre P
Groupe de Biologie et Génétique Moléculaires, Centre National de la Recherche Scientifique, Villejuif, France.
Eur J Biochem. 1989 Jan 2;178(3):619-26. doi: 10.1111/j.1432-1033.1989.tb14490.x.
We have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a polypeptide of 100 kDa. On the basis of a Stokes radius of 4.2 nm and a sedimentation coefficient of 6 S, the purified enzyme has an estimated molecular mass of 109 kDa. These results are consistent with the enzyme being a monomer of 100 kDa. In addition a polyclonal antiserum, obtained by injection of the electroeluted 100-kDa polypeptide into a rabbit, specifically neutralized the DNA-polymerase activity. The enzyme is sensitive to both N-ethylmaleimide and 2',3'-dideoxyribosylthymine triphosphate and resistant to aphidicolin. The purified DNA polymerase has neither exonuclease nor primase activities. In our in vitro conditions, the enzyme is thermostable up to 80 degrees C and is active between 55 degrees C and 85 degrees C in the presence of activated calf-thymus DNA.
我们已将嗜热嗜酸古细菌嗜酸热硫化叶菌中的一种DNA聚合酶纯化至接近同质。纯化后的酶进行十二烷基硫酸钠凝胶电泳显示有一条100 kDa的多肽。根据4.2 nm的斯托克斯半径和6 S的沉降系数，纯化后的酶估计分子量为109 kDa。这些结果与该酶为100 kDa的单体一致。此外，通过将电洗脱的100 kDa多肽注射到兔子体内获得的多克隆抗血清可特异性中和DNA聚合酶活性。该酶对N-乙基马来酰亚胺和2',3'-二脱氧核糖基胸腺嘧啶三磷酸均敏感，对阿非科林有抗性。纯化后的DNA聚合酶既无核酸外切酶活性也无引发酶活性。在我们的体外条件下，该酶在高达80℃时具有热稳定性，在存在活化的小牛胸腺DNA的情况下，在55℃至85℃之间具有活性。
