Purification and characterization of an apurinic/apyrimidinic endonuclease from HeLa cells.
Purification and characterization of an apurinic/apyrimidinic endonuclease from HeLa cells.
复制标题
DOI：
10.1016/s0021-9258(19)69623-7
复制
发表时间：
1981-04
期刊：
影响因子：
--
通讯作者：
C. Kane;S. Linn
中科院分区：
文献类型：
--
作者：
C. Kane;S. Linn
从HeLa细胞作用于脱嘌呤/脱嘧啶（AP）网站的脱氧核糖核酸内切酶已被纯化到明显的同质性，通过十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳判断。在整个纯化过程中，Triton X-100的存在对于活性的稳定和刺激是必要的。核酸内切酶具有通过分子筛测定的32，000的表观天然分子量和通过其在SDS-聚丙烯酰胺凝胶中的电泳迁移率判断的41，000的表观亚基分子量。该活性对Mg 2+或Mn 2+具有绝对需求，并且在6.7和9.0之间具有宽的pH最适值，最大活性接近pH 7.5。该酶对未处理的双链体或单链DNA没有可检测的核酸外切酶活性，也没有任何核酸内切酶活性。它被腺嘌呤、次黄嘌呤、腺苷、AMP、ADP-核糖和NAD+抑制，但不受咖啡因、嘧啶碱基、ADP、ATP或NADH的影响。使用各种受损的DNA底物并没有表明该酶作用于AP位点以外的其他位点。该酶似乎切割AP DNA，以便在5'末端留下脱氧核糖-5-磷酸，在3'末端留下3 '-OH;它还从在3'末端具有脱氧核糖的AP DNA中除去脱氧核糖-5-磷酸。特异性抗体已在兔中产生，它只与纯化的酶（可能是酶本身）中存在的41，000-道尔顿蛋白质相互作用，以及与来自人胎盘和成纤维细胞的部分纯化的AP内切核酸酶组分相互作用。
An endodeoxyribonuclease from HeLa cells acting on apurinic/apyrimidinic (AP) sites has been purified to apparent homogeneity as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The presence of Triton X-100 was necessary throughout the purification for stabilization and stimulation of activity. The endonuclease has an apparent native molecular weight of 32,000 determined by molecular sieving and an apparent subunit molecular weight of 41,000 as judged by its electrophoretic mobility in SDS-polyacrylamide gels. The activity has an absolute requirement for Mg2+ or Mn2+ and a broad pH optimum between 6.7 and 9.0 with maximal activity near pH 7.5. The enzyme has no detectable exonuclease activity, nor any endonuclease activity on untreated duplex or single-stranded DNA. It is inhibited by adenine, hypoxanthine, adenosine, AMP, ADP-ribose, and NAD+, but it is unaffected by caffeine, the pyrimidine bases, ADP, ATP, or NADH. The use of a variety of damaged DNA substrates provided no indication that the enzyme acts on other than AP sites. The enzyme appears to cleave AP DNA so as to leave deoxyribose-5-phosphate at the 5' terminus and a 3'-OH at the 3' terminus; it also removes deoxyribose-5-phosphate from AP DNA which has deoxyribose at the 3' terminus. Specific antibody has been produced in rabbits which interacts only with a 41,000-dalton protein present in the purified enzyme (presumably the enzyme itself), as well as with partially purified AP endonuclease fractions from human placenta and fibroblasts.