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Regulation of gene expression and function of cytochrome b5

细胞色素 b5 基因表达和功能的调控

基本信息

批准号：
09044092
负责人：
YUBISUI Toshitsugu
金额：
$ 1.6万
依托单位：
Kochi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

In these studies we investigated the regulation of gene expression and function of cytochrome b5.1. Yubisui has succeeded in cloning 2 cDNAs from tunicates. One of them obtained from Polyandrocarpa misakiensis has about 1.8 kbp including the coding regions of 405 bp for 135 amino acids, and the other from Ciona savignyi has about 790 bps including the coding region of 402 bp for 134 amino acids. Both amino acid sequences have about 75% homology to those of mammalian cytochronie b5s.2. Steggles also cloned a cDNA for swine cytochrome bS, which contained a 24 bp insertion after 96th codon to code for G1u97, Pro98 and stop99 to produce the soluble form of cytochrome b5. Regulation of gene expression of the soluble and membrane-bound form depends on the functin of promotors.3. Porter constructed a co-expression system for cytochrome b5, P450, and P450 reductase in Eschericlzia coli. Cytochrome b5 effectively accerelated the drug metabolism by cytochrome P450/P450 reductase system.4. Campbell investigated the function of cytochrome b5 moiety in nitrate reductase. The cytochrome b5 and flavoprotein domains of the nitrate reductase were expressed as a fusion protein, and crystalyzed. Based on the crystalyzed structure he succeeded in changing the NADPH-dependent enzyme to NADH-specific enzyme by site-directed mutagenesis.

在这些研究中，我们研究了细胞色素b5.1基因表达和功能的调控。Yubisui已经成功地从被囊动物身上克隆了2个cdna。其中，从misakipolyandrocarpa misakiensis中获得的一个编码区约为1.8 kbp，包含135个氨基酸的405 bp编码区；从Ciona savignyi中获得的另一个编码区约为790 bps，包含134个氨基酸的402 bp编码区。这两种氨基酸序列与哺乳动物细胞编年体b5s的同源性约为75%。Steggles还克隆了猪细胞色素b5的cDNA，该cDNA在第96个密码子后插入24 bp，用于编码G1u97、Pro98和stop99，从而产生细胞色素b5的可溶性形式。可溶性和膜结合形式的基因表达调控取决于启动子的功能。Porter构建了细胞色素b5、P450和P450还原酶在大肠杆菌中的共表达系统。细胞色素b5通过细胞色素P450/P450还原酶系统有效加速药物代谢。Campbell研究了细胞色素b5片段在硝酸还原酶中的作用。硝酸还原酶的细胞色素b5和黄素蛋白结构域以融合蛋白的形式表达并结晶。基于结晶结构，他成功地通过位点定向诱变将nadh依赖酶改变为nadh特异性酶。