Study on the uronate-cycle oxidoreductases that are expressed highly in kidney

肾脏高表达糖醛酸循环氧化还原酶的研究

基本信息

批准号：
13672290
负责人：
HARA Akira
金额：
$ 2.3万
依托单位：
Gifu Pharmaceutical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Five percent of glucose metabolized in the uronate cycle, xylitol produced in this cycle has been suggested to be involved in development of diabetic complications. However, the structure and properties of enzymes that produce and metabolize xylitol have not been well studied. In this study, we characterized the following three enzymes of this cycle to elucidate roles other than sugar metabolism.1. L-Xylulose reductase (XR). The cDNAs for XRs of human and four rodents were isolated, and the properties of the recombinant enzymes were studied. Mammalian XRs are homotetramers belonging to the short-chain dehydrogenase/reductase family, are identical to diacetyl reductase, and reduce various dicarbonyl compounds, some aldehydes and ketones. The enzyme was distributed in many mammalian tissues, in which liver and kidney show high contents of the enzyme. The enzyme was localized on brush-border membranes of renal tubular cells. Human XR was crystallized and determined its crystal structure, … More which had a intramolecular disulfide bond per subunit. In addition, three residues involving in the catalytic mechanism, five substrate-binding residues and structural factors of cold inactivation were elucidated by site-directed mutagenesis. Some specific and selective inhibitors of XR were found by screening and computer-modeling based on the crystal structure, and their permeability into the cell was tested using XR-overexpressed cells. Physiological effects of the XR-gene transgenic and knockout mice were examined.2. L-Xylitol dehydrogenase (XDH). XDH was highly distributed in liver and kidney of many mammalian tissues. Purification of cytosolic and mitochondrial XDHs of guinea pig liver revealed that they are identical and are sorbitol dehydrogenase that belongs to the medium-chain dehydrogenase/reductase family. The enzyme oxidized several aromatic alcohols.3. L-Gulonate dehydrogenase (GDH). The tissue distribution of GDH was also the same as those of XR and XDH. The enzymes purified from rabbit liver was dimeric, and its amino acid sequence analysis suggested that this enzyme belongs to the 3-hydroxyacyl CoA dehydrogenase family. GDH accepts several no-sugar compounds as the substrates. Less

百分之五的葡萄糖在糖醛酸循环中代谢，该循环中产生的木糖醇被认为与糖尿病并发症的发展有关。然而，在这项研究中，产生和代谢木糖醇的酶的结构和特性尚未得到充分研究。我们表征了该循环中的以下三种酶，以阐明除 L-木酮糖还原酶 (XR) 之外的作用。人类和四种啮齿动物的 XR 的 cDNA 如下。分离并研究了该重组酶的性质，该酶属于短链脱氢酶/还原酶家族的同源四聚体，与二乙酰还原酶相同，并且还原多种二羰基化合物、一些醛和酮。哺乳动物组织中，肝脏和肾脏显示出高含量的酶，该酶位于肾小管细胞的刷状缘膜上。结晶并确定了其晶体结构，其中每个涉及的亚基都有一个分子内二硫键，此外，通过定点诱变阐明了催化机制中的三个残基、五个底物结合残基和冷失活的结构因素。通过基于晶体结构的筛选和计算机建模，发现了XR的选择性抑制剂，并利用XR过表达的细胞测试了它们对细胞的渗透性的生理作用。对XR基因转基因小鼠和敲除小鼠进行了检测。 2．XDH在许多哺乳动物组织的肝脏和肾脏中高度分布。属于中链脱氢酶/还原酶家族的山梨糖醇脱氢酶，该酶氧化多种芳香醇。3． GDH的组织分布也与XR和XDH相同，从兔肝脏中纯化的酶为二聚体，其氨基酸序列分析表明该酶属于3-羟酰辅酶A脱氢酶家族。 GDH 接受多种无糖化合物作为底物。