Purification and structure analysis of endothelin-converting enzyme.

内皮素转换酶的纯化及结构分析。

• 批准号: 04671488
• 负责人: TAKAYANAGI Ryoichi
• 金额: $ 1.34万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04671488/
• 关键词: Endothelin; Endothelin-converting enzyme; Endothelial cell; Big endothelin; ECE; ET

1. Purification and characterization of endothelin-converting enzyme (ECE).ECE was solubilized with Lubrol PX from membrane fraction of porcine aortic endothelium and was purified by sequential chromatography on DEAE-agarose, Ricinus communis agglutinin 120-agarose, peanut agglutinin-agarose, Mono Q,and TSK3000SW_<XL> columns. Apparent MW was 120 kDa when analyzed on SDS-PAGE under reducing conditions. The C-terminal sequence of His^<27> to Gly^<34> and Trp^<21> at P^1 position of big ET-1 are essential for its substrate activity.2. cDNA cloning of ECE.Two types of ECE cDNA,ECE-1alpha and ECE-1beta, were cloned from human umbilical vein endothelial cells. ECE-1beta encodes 758 amino acid residues, which has a single putative transmembrane domain (amino acids 57-77). ECE belongs to NEP and Kell blood-group protein. Further physiological characteristics of ECE has been studied by the expression of these cloned ECE-1cDNAs.

1.内皮素转化酶（ECE）的纯化和表征用来自猪主动脉内皮的膜级分的Lubrol PX溶解ECE，并通过在DEAE-琼脂糖、蓖麻凝集素120-琼脂糖、花生凝集素-琼脂糖、Mono Q和TSK 3000 SW_柱上的连续色谱法纯化ECE<XL>。在还原条件下进行SDS-PAGE分析时，表观MW为120 kDa。大ET-1的C-末端P^1位的His^<27>→ Gly^<34>→ Trp^<21>→序列是其底物活性所必需的.从人脐静脉内皮细胞中克隆了ECE-1 α和ECE-1 β两种类型的ECE cDNA。ECE-1 β编码758个氨基酸残基，具有单一的跨膜结构域（氨基酸57-77）。ECE属于NEP和Kell血型蛋白。通过表达这些克隆的ECE-1cDNA，进一步研究了ECE的生理特性。

项目成果

R.Takayanagi: "Molecular cloning,sequence analysis and expression of a cDNA encoding human type‐1 angiotensin receptor" Biochem.Biophys.Res.Commun.183. 910-916 (1992)

R. Takayanagi：“编码人 1 型血管紧张素受体的 cDNA 的分子克隆、序列分析和表达”Biochem.Biophys.Res.Commun.183 (1992)。

大中佳三: "ヒトアンジオテンシン 受容体の分子生物学" 日本臨床. 51. 1568-1574 (1992)

Keizo Ohnaka：“人血管紧张素受体的分子生物学”日本临床。 51. 1568-1574 (1992)

高柳涼一: "血管系におけるエンドセリン受容体サブタイプの機能分化" 最新医学. 47. 699-702 (1992)

Ryoichi Takayanagi：“血管系统内皮素受体亚型的功能分化”现代医学 47. 699-702 (1992)。

Ohnaka K. et al.: "Partial purification of phosphoramidon-sensitive endothelin-converting enzyme in porcine aortic endothelial cells : high affinity for ricinus communis agglutinin" Biochem. Biophys. Res. Commun.185. 611-616 (1992)

Ohnaka K. 等人：“猪主动脉内皮细胞中磷酰胺敏感性内皮素转化酶的部分纯化：对蓖麻凝集素的高亲和力”Biochem。

高柳涼一、他: "ブタ大動脈内膜エンドセリン変換酵素の精製と解析" 日本臨床代謝学会記録. 31. 82-83 (1994)

Ryoichi Takayanagi 等：“猪主动脉内膜内皮素转换酶的纯化和分析”日本临床代谢学会记录 31. 82-83 (1994)。