Purification and characterization of human 3-methyladenine-DNA glycosylase.
Purification and characterization of human 3-methyladenine-DNA glycosylase.
复制标题
人 3-甲基腺嘌呤-DNA 糖基化酶的纯化和表征。
DOI：
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发表时间：
1993
影响因子：
14.9
通讯作者：
Timothy R. O'Connor
中科院分区：
文献类型：
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作者：
Timothy R. O'Connor
3-甲基腺嘌呤-DNA 糖基化酶的人类 cDNA 编码序列在大肠杆菌中表达。除了全长 3-甲基腺嘌呤-DNA 糖基化酶编码序列外，还表达了源自该序列的两个其他序列（由差异 RNA 剪接和截短的 anpg cDNA 产生）。所有三种蛋白质均被纯化至物理同质性，并且它们的 N 端氨基酸序列与核酸序列预测的序列相同。全长蛋白质有 293 个氨基酸，编码分子量为 32 kDa 的蛋白质。针对其中一种蛋白质的多克隆抗体与其他两种蛋白质和鼠 3-甲基腺嘌呤-DNA 糖基化酶发生反应，但不与其他几种大肠杆菌 DNA 修复蛋白发生反应。所有三种蛋白质都会从 DNA 中切除 3-甲基腺嘌呤、7-甲基鸟嘌呤和 3-甲基鸟嘌呤以及乙基化碱基。蛋白质在离子强度（最佳 100 mM KCl）、pH（最佳 7.6）以及 3-甲基腺嘌呤和 7-甲基鸟嘌呤切除动力学（平均值：3-甲基腺嘌呤：Km 9 nM 和 kcat 10 min-1，7-甲基鸟嘌呤：Km 29 nM 和 kcat 0.38 min-1）方面的活性具有可比性。然而，与这些结果相反，全长和剪接变体蛋白质在 50 摄氏度下的热稳定性低于截短的蛋白质。
A human cDNA coding sequence for a 3-methyladenine-DNA glycosylase was expressed in Escherichia coli. In addition to the full-length 3-methyladenine-DNA glycosylase coding sequence, two other sequences (resulting from differential RNA splicing and the truncated anpg cDNA) derived from that sequence were also expressed. All three proteins were purified to physical homogeneity and their N-terminal amino acid sequences are identical to those predicted by the nucleic acid sequences. The full-length protein has 293 amino acids coding for a protein with a molecular mass of 32 kDa. Polyclonal antibodies against one of the proteins react with the other two proteins, and a murine 3-methyladenine-DNA glycosylase, but not with several other E. coli DNA repair proteins. All three proteins excise 3-methyl-adenine, 7-methylguanine, and 3-methylguanine as well as ethylated bases from DNA. The activities of the proteins with respect to ionic strength (optimum 100 mM KCl), pH (optimum 7.6), and kinetics for 3-methyladenine and 7-methylguanine excision (average values: 3-methyladenine: Km 9 nM and kcat 10 min-1, 7-methylguanine: Km 29 nM and kcat 0.38 min-1) are comparable. In contrast to these results, however, the thermal stability of the full-length and splicing variant proteins at 50 degrees C is less than that of the truncated protein.