Purification and cloning of carp nephrosin, a secreted zinc endopeptidase of the astacin family
Purification and cloning of carp nephrosin, a secreted zinc endopeptidase of the astacin family
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DOI：
10.1074/jbc.272.21.13772
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发表时间：
1997-05-23
影响因子：
4.8
通讯作者：
Chang, GD
中科院分区：
文献类型：
--
作者：
Hung, CH;Huang, HR;Chang, GD
通过活性蓝72-琼脂糖亲和层析和FPLC Mono-P柱层析，从鲤鱼头肾中连续柱层析纯化出一种23 kDa的分泌型蛋白水解酶。鲤鱼头肾的这种蛋白水解酶的分泌可以被高浓度的钾刺激。由于鲤鱼蛋白主要存在于头肾、肾和脾(均为淋巴造血器官)中，故将其命名为肾素。鲤鱼肾素对金属螯合剂最敏感，但对其他类型的蛋白水解酶的抑制剂不敏感。用纯化的肾素多克隆抗血清免疫筛选，从鲤鱼头肾cDNA文库中分离到一个克隆。该基因全长1086个碱基对，开放阅读框编码273个氨基酸的蛋白质，包括19个氨基酸的信号肽和56个氨基酸的前肽。推导的氨基酸序列与青蒿素HCE1(52.5%)、青蒿素LCE(50.7%)、小龙虾ASTIN(33.2%)、小鼠Meprin-α(34%)和小鼠Meprin-β(33.5%)有中等程度的同源性，这些都是ASTIN锌内肽酶家族的成员。Nephrosin是在淋巴造血组织中发现的第一个ASTIN家族成员。
We have purified a secreted proteinase of 23 kDa from carp head kidney by sequential column chromatography on a Reactive Blue 72-agarose dye affinity column and an FPLC Mono-P column. The secretion of this proteinase from carp head kidney can be stimulated by high concentrations of potassium. Since the carp proteinase is present mainly in the head kidney, kidney, and spleen (all of which are lymphohematopoietic organs), it is named nephrosin. The carp nephrosin is most sensitive to metal chelators, but not to inhibitors specific for other classes of proteinases. A cDNA clone has been isolated from a carp head kidney cDNA library by immunoscreening with a polyclonal antiserum raised against purified nephrosin. The cloned cDNA is 1086 base pairs in length and has an open reading frame encoding a protein of 273 amino acids, including a 19-amino acid signal peptide and 56-amino acid propeptide. The deduced amino acid sequence shows moderate levels of identity to medaka HCE1 (52.5%), medaka LCE (50.7%), crayfish astacin (33.2%), murine meprin-alpha (34%), and murine meprin-beta (33.5%), all members of the astacin family of zinc endopeptidases. Nephrosin is the first member of the astacin family found in lymphohematopoietic tissues.