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二维聚丙烯酰胺凝胶上小牛胸腺DNA聚合酶α、β及末端脱氧核苷酸转移酶的分析

Analysis of calf thymus DNA polymerases alpha and beta and terminal deoxynucleotidyl transferase on two-dimensional polyacrylamide gels.

作者信息

Koiwai O, Masaki S, Kaneda T, Yoshida S

出版信息

J Biochem. 1982 Aug;92(2):433-9. doi: 10.1093/oxfordjournals.jbchem.a133950.

Abstract

Calf thymus DNA polymerases alpha and beta [EC 2.7.7.7] and terminal deoxynucleotidyl transferase [EC 2.7.7.31] were analyzed on two-dimensional gel slabs. DNA polymerase beta appeared as a single spot on two-dimensional gel at the position of 40,000 daltons and pI 8.0 using non-equilibrium pH gradient gel electrophoresis for the first-dimensional run. By overlapping gel slabs, it was possible to identify the distinct spot of DNA polymerase beta among many polypeptide spots of a crude enzyme fraction. 10S DNA polymerase alpha showed two clusters of polypeptide spots on two-dimensional gel slab. One cluster was composed of three large polypeptides of 140,000-150,000 daltons and another was composed of four smaller polypeptides of 46,000-50,000 daltons. All these spots were arranged in a narrow pI range (6.5-6.8) although each spot showed a distinct pI value. Purified terminal deoxynucleotidyl transferase showed three polypeptides of 57,000, 42,000, and 33,000 daltons at similar pI values (7.0-7.2). Each polypeptide consisted of plural spots which differed slightly in pI but were the same in molecular weight. These results suggest a microheterogeneity of polypeptides of terminal deoxynucleotidyl transferase as well as those of 10S DNA polymerase alpha.

摘要

在二维凝胶板上分析了小牛胸腺DNA聚合酶α和β[EC 2.7.7.7]以及末端脱氧核苷酸转移酶[EC 2.7.7.31]。使用非平衡pH梯度凝胶电泳进行第一维电泳时,DNA聚合酶β在二维凝胶上呈现为一个单一斑点,位于40,000道尔顿和pI 8.0的位置。通过重叠凝胶板,可以在粗酶组分的许多多肽斑点中识别出DNA聚合酶β的独特斑点。10S DNA聚合酶α在二维凝胶板上显示出两簇多肽斑点。一簇由三个140,000 - 150,000道尔顿的大多肽组成,另一簇由四个46,000 - 50,000道尔顿的小多肽组成。所有这些斑点都排列在一个狭窄的pI范围内(6.5 - 6.8),尽管每个斑点都显示出一个独特的pI值。纯化的末端脱氧核苷酸转移酶在相似的pI值(7.0 - 7.2)下显示出57,000、42,000和33,000道尔顿的三种多肽。每个多肽由多个斑点组成,这些斑点在pI上略有不同,但分子量相同。这些结果表明末端脱氧核苷酸转移酶以及10S DNA聚合酶α的多肽存在微异质性。

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