Analysis of the processing mechanism of endothelin-converting enzyme and the development of its inhibitors as novel therapeutics for vascular complications.

1.大内皮素-1类似物对内皮素转换酶-1（ECE-1）具有抑制活性。通过对大内皮素-1（big ET-1）进行不同的氨基酸取代，发现了两种对ECE-1具有抑制活性的大内皮素-1（big ET-1）类似物，[F^<21>]big ET-1（18 -34）和[A^<31>]big ET-1（18-34）。动力学分析表明，[F^<21>]big ET-1（18-34）为竞争性抑制剂，[A^<31>]big ET-1（18-34）为非竞争性抑制剂，提示ECE-1以不同的方式识别P1位点和C端部分.阐明大ET-1转化的细胞内位点。了解大ET-1在细胞中的何处转化对于药物设计是重要的，然而，大ET-1转化的细胞内位点一直存在争议。在本研究中，使用ECE-1和绿色荧光蛋白（GFP）的嵌合体，在活细胞中可见信号，揭示了内源性大ET-1在细胞内转化，而不是在细胞表面.通过ECE-1cDNA的突变分析ECE-1的性质：人cDNA的突变表明ECE-1胞外C端结构域的糖基化是酶活性所必需的，而跨膜结构域在正常糖基化的完成中起重要作用。

1. Big ET-1 analogues with the inhibitory activity on endothelin-converting enzyme-1 (ECE-1). Two big endothelin-1(big ET-1) analogues with the inhibitory activity on ECE-1, [F^<21>]big ET-1(18-34) and [A^<31>]big ET-1(18-34), were found by various amino acid substitution of big ET-1. Kinetics analyses showed that [F^<21>]big ET-1(18-34) is a competitive inhibitor and [A^<31>]big ET-1 (18-34) is a non-competitive inhibitor, suggesting that ECE-1 recognizes the P1 position and the C-terminal portion in a different fashon.2. Clarification of intracellular site for big ET-1-conversion. It is important for the drug design to know where in the cell the big ET-1 is converted, however, the intracellular site for the conversion of big ET-1 has been controversial. In the present study, using a chimera of ECE-1 and green fluorescent protein (GFP), visible signal in living cells, it was revealed that endogenous big ET-1 is converted intracellularly, not on the cell surface.3. Analyses of the properties of ECE-1 by mutagenesis of ECE-1 cDNA.Mutagenesis of human cDNA revealed that glycosylation of the extracellular C-terminal domain of ECE-1 is essential to the enzymatic activity, and that the transmembrane domain plays an important role in the completion of normal glycosylation.