来自浆细胞瘤MOPC 104E和艾贝尔森鼠白血病病毒的DNA聚合酶的纯化与特性分析

Purification and characterization of DNA polymerases from the plasmacytoma MOPC 104E and Abelson murine leukemia viruses.

作者信息

Weimann B J, Schmidt J, Takacs B

出版信息

Eur J Biochem. 1978 Apr 17;85(2):571-9. doi: 10.1111/j.1432-1033.1978.tb12272.x.

DOI:10.1111/j.1432-1033.1978.tb12272.x

PMID:206440

Abstract

RNA-dependent DNA polymerases of intracisternal A particles from the mouse plasma cell tumor MOPC 104E and of Abelson murine leukemia virus (A-MuLV) were isolated from particle preparations by Nonidet P40 and ultrasonic treatment and purified by column chromatography on DEAE-cellulose and phosphocellulose, followed by centrifugation in linear sucrose gradients. Both DNA polymerases were very similar in their elution patterns from phospho and DEAE-cellulose, template specificities, requirements for optimum activity and inactivation by anti-(reverse transcriptase) antiserum. They are associated with ribonuclease H activity. For molecular weight determinations, antibody-precipitated enzymes were bound to staphylococcal-protein-A-Sepharose, solubilized and run on dodecylsulfate/polyacrylamide gels. Their apparent molecular weight was estimated to be 80000.

摘要

从小鼠浆细胞瘤MOPC 104E的脑内A颗粒以及Abelson鼠白血病病毒（A-MuLV）中分离出的RNA依赖性DNA聚合酶，通过NP-40和超声处理从颗粒制剂中提取，并先后经DEAE-纤维素柱层析和磷酸纤维素柱层析纯化，随后在线性蔗糖梯度中离心。两种DNA聚合酶在从磷酸纤维素和DEAE-纤维素上的洗脱模式、模板特异性、最佳活性的需求以及抗（逆转录酶）抗血清的失活作用方面都非常相似。它们与核糖核酸酶H活性相关。为了测定分子量，将抗体沉淀的酶与葡萄球菌蛋白A-琼脂糖结合，溶解后在十二烷基硫酸钠/聚丙烯酰胺凝胶上进行电泳。其表观分子量估计为80000。