Purification and structure analysis of endothelin-converting enzyme.

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。

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.