Purification and chraracterization of a novel endothelin-degrading enzyme using protein-free culture system

我们发现了一种能降解内皮素的新型蛋白酶。我们首先将这种蛋白酶命名为内皮素降解酶（EDE）。该酶还能裂解其他肽激素，如胰高血糖素、血管活性肠肽（VIP）和分泌素，但不能裂解EGF、胃泌素、胰岛素、生长抑素、P物质、神经紧张素或生长激素。我们将这种酶重新命名为胰高血糖素降解酶（GDE），因为它对胰高血糖素的亲和力高于内皮素。该蛋白是从无蛋白培养的人胰腺癌细胞系、HPC-YO调节培养基中通过离子交换、凝胶过滤和羟基磷灰石柱层析纯化得到的。SDS-PAGE测定GDE分子量为83,000。阻断GDE的n端氨基酸序列，确定仅基内肽酶切得到的5个部分氨基酸序列为NLTEEYDVS-DGELELLYEK、VETYYDLLFEK、LYWFLDEAK、SNSTSYVK和YYASTSYDDTYK。在已知蛋白质中未发现相同或同源的氨基酸序列，表明GDE是一种新型肽酶。此外，我们可以澄清GDE cDNA的开放阅读框（ORF），没有发现相同或同源的DNA序列。ORF由2.6 kbp组成。我们制备了几种GDE抗体，并在各种细胞系的条件培养基中建立了GDE微量检测系统。在不久的将来，我们将测量健康对照者和患者血清中的GDE水平。

We have found a novel proteinase which degrades endothelin. We designated this proteinase as endothelin-degrading enzyme (EDE) at first. This enzyme also cleaves other peptide hormones such as glucagon, vasoactive intestinal peptide (VIP) and secretin, however, it did not cleave EGF,gastrin, insulin, somatostatin, substance P,neurotensin, or growth hormone. We renamed this enzyme as glucagon-degrading enzyme (GDE), since the affinty to glucagon was higher than endothelin. This was purified from protein-free cultured human pancreatic cancer cell line, HPC-YO conditioned-medium by a combination of ion-exchange, gel filtration and hydroxlapatite column chromatographies. The molecular weight of GDE is 83,000, as determined on SDS-PAGE.The N-terminal amino acid sequence of GDE was blocked, and the five partial amino acid sequences obtained onlysyl-endopeptidase digestion were determined to be NLTEEYDVS-DGELELLYEK,VETYYDLLFEK,LYWFLDEAK,SNSTSYVK,and YYASTSYDDTYK.The same or homologous amino acid sequences have not been found in known proteins, demonstrating that GDE is a novel peptidase. Furthermore, We could clarified open reading frame (ORF) of GDE cDNA,and the same or homologous DNA sequences have not found. The ORF was consisted of 2.6 kbp. We produced several kinds of antibodies to GDE,and established the microassay system of GDE in the conditioned media of various kind of cell lines. In near future, we will measure GDE levels in sera of healthy controls and patitents.