Cao Q P, Pitt S, Leszyk J, Baril E F
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Biochemistry. 1994 Jul 19;33(28):8548-57. doi: 10.1021/bi00194a021.
A 150-kDa DNA-dependent ATPase composed of 83/68-kDa subunits was previously reported to cofractionate with a 21S complex of enzymes for DNA synthesis from HeLa cells (Vishwanatha, J. K., & Baril, E. F. (1990) Biochemistry 29, 8753-8759). The DNA-dependent ATPase was purified to electrophoretic homogeneity from a HeLa cell homogenate by a modified procedure that involves subcellular fractionation, poly(ethylene-glycol) precipitation of the combined nuclear extract/cytosol, and chromatography on Q-Sepharose and native and denatured DNA/celluloses followed by Mono-S fast protein liquid chromatography. The purified enzyme showed equimolar amounts of 83- and 68-kDa polypeptides following polyacrylamide gel electrophoresis under denaturing conditions. Sequence analysis of peptide fragments derived from the separated 83- and 68-kDa polypeptides showed 90-100% homology with the corresponding 80- and 70-kDa subunits of human Ku protein. Immunoblot analysis of the ATPase during the course of its purification and immunoprecipitation with antibodies to the 80- and 70-kDa subunits of human Ku protein confirmed the relationship of the 83- and 68-kDa polypeptides of the human DNA-dependent ATPase to the subunits of human Ku protein. Both the 83- and 68-kDa polypeptides are phosphorylated by a DNA-dependent protein kinase that cofractionates with the ATPase. The DNA-dependent ATPase activity is up regulated by phosphorylation.
先前有报道称，一种由83/68 kDa亚基组成的150 kDa DNA依赖性ATP酶与来自HeLa细胞的DNA合成酶21S复合物共分离（Vishwanatha, J. K., & Baril, E. F. (1990) Biochemistry 29, 8753 - 8759）。通过一种改良方法从HeLa细胞匀浆中纯化该DNA依赖性ATP酶至电泳纯，该方法包括亚细胞分级分离、合并的核提取物/胞质溶胶的聚乙二醇沉淀，以及在Q-Sepharose和天然及变性DNA/纤维素上进行色谱分离，随后进行Mono-S快速蛋白质液相色谱。在变性条件下进行聚丙烯酰胺凝胶电泳后，纯化的酶显示出等摩尔量的83 kDa和68 kDa多肽。对从分离的83 kDa和68 kDa多肽衍生的肽片段进行序列分析，结果显示与人Ku蛋白相应的80 kDa和70 kDa亚基具有90 - 100%的同源性。在ATP酶纯化过程中对其进行免疫印迹分析，并用针对人Ku蛋白80 kDa和70 kDa亚基的抗体进行免疫沉淀，证实了人DNA依赖性ATP酶的83 kDa和68 kDa多肽与人Ku蛋白亚基的关系。83 kDa和68 kDa多肽均被一种与ATP酶共分离的DNA依赖性蛋白激酶磷酸化。DNA依赖性ATP酶活性通过磷酸化上调。
