重组人类dihydroorotate脱氢酶:表达、纯化和表征催化地功能性截断的酶。
文章的细节
-
引用
复制到剪贴板 -
科普兰RA,戴维斯JP,道林RL,伦巴都D,墨菲KB,帕特森助教
重组人类dihydroorotate脱氢酶:表达、纯化和表征催化地功能性截断的酶。
拱生物化学Biophys。1995年10月20日,323 (1):79 - 86。
- PubMed ID
-
7487077 (在PubMed]
- 文摘
-
人类的氨基端截断cDNA dihydroorotate脱氢酶(DHODase)被诱导T7 lac启动子的控制下在一个嘧啶营养缺陷型的菌株大肠杆菌缺乏内源性酶。诱导基因表达了媒体缺乏外生嘧啶的增长。重组酶的纯化是同质性洗涤剂提取的细菌膜由两个色谱步骤。由此产生的酶的纯度是判定为> 95%基于sds - page Coomassie染色。酶显示ca的表观分子量。40 kDa sds - page和ca 120 kDa。本机凝胶排阻层析法,表明multimeric原生酶。重组DHODase显示一个特定的活动和公里为dihydroorotate类似酶的牛和人类肝脏组织。重组酶的活动对pH值的依赖关系是同样类似于酶从人类肝脏和可能表明催化营业额的一个关键组氨酸残基的参与;只有8个组氨酸残基留在这里DHODase的截断版本使用。重组酶的催化活性是剂量依赖性地抑制由histidine-selective diethylpyrocarbonate修改代理。这些结果进一步表明组氨酸的潜在作用酶营业额。 Brequinar sodium, an experimental drug which has been shown to be a nanomolar noncompetitive inhibitor of mammalian DHODases, inhibited the activity of the purified recombinant enzyme with a Ki value similar to that for enzyme derived from human liver tissue. The recombinant DHODase thus displays enzymatic behavior similar to the 50-kDa full-length human liver enzyme, illustrating that the catalytically essential structural features of the enzyme, as well as the site of Brequinar binding, are contained within the 40-kDa truncated version of the enzyme that was expressed here.