Isolation and characterizaion of renin processing enzyme from renin granule

肾素颗粒中肾素加工酶的分离和表征

基本信息

批准号：
04670567
负责人：
IKEDA Masaharu
金额：
$ 1.28万
依托单位：
University of Occupational & Environmental Health
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-04670567/
关键词：
Renin Prorenin Processing Renin Granule Subtilisin プレセシング

项目摘要

Processing mechanism of prorenin to renin is still controversial. It is postulated that renin is synthesized and processed in the rough endoplasmic reticulum and the Golgi complex and stored in the renin granules and secreted by exocytosis. Recently several processing enzymes which process prohormones or protein precursors were found and identified as intrinsic processing enzymes.The purpose of this study was to clarify the nature of the intrinsic renin processing enzyme in the subcellular fraction of the kidney cortex. Generally prohormone convertases or protein precursor processing enzymes were reported to a common catalytic domain homologous to that of the subtilisin serine protease family.Hence in the first step of the present study, we investigated whether subtilisin per se can process prorenin to renin. When radiolabelled[^<35>S]-human recombinant prorenin was incubated with subtilisin BNP^-, human recombinant prorenin which showed a molecular weight of 43,000 dalton was cleaved … More to renin, a molecular weight of 38,000 dalton. In addition, when semipurified human amniotic prorenin(or inactive renin) was incubated with subtilisin, we confirmed the activation of prorenin by subtilisin by detection of active renin which was quantitated by a specific immunoradiometric assay system. These results indicate that subtilisin per se can process and activate prorenin to active form.In the next study, we investigated whether renin granule fraction of the kidney can process recombinant human prorenin to renin by cleaving prosegment of the prorenin. Human prorenin (molecular weight 43,000) was cleaved to renin (molecular weight 38,000) by renin granule fraction. This result suggests that renin granule fraction contained prorenin processing enzyme. In this study, we tried to isolate the prorenin processing enzyme from renin granule fraction, but we could noto isolate or identified prorenin processing enzyme in the renin granule because of loss of activity probably due to its lability during isolation. Less

肾素原对肾素的加工机制仍存在争议，推测肾素在粗面内质网和高尔基复合体中合成和加工，并储存在肾素颗粒中并通过胞吐作用分泌。发现并鉴定为内在加工酶。本研究的目的是阐明肾皮质亚细胞部分中内在肾素加工酶的性质。据报道，转化酶或蛋白质前体加工酶具有与枯草杆菌蛋白酶丝氨酸蛋白酶家族同源的共同催化结构域。因此，在本研究的第一步中，我们研究了放射性标记时枯草杆菌蛋白酶本身是否可以将肾素原加工成肾素。 35>S]-人重组肾素原与枯草杆菌蛋白酶BNP^-（人重组肾素原）一起孵育，其分子量为43,000 道尔顿被裂解为肾素，分子量为 38,000 道尔顿此外，当半纯化的人羊膜肾素（或无活性肾素）与枯草杆菌蛋白酶一起孵育时，我们通过检测定量的活性肾素证实了枯草杆菌蛋白酶对肾素原的激活。这些结果表明枯草杆菌蛋白酶本身可以加工肾素原并将其激活为活性物质。在下一项研究中，我们研究了肾脏的肾素颗粒部分是否可以通过切割肾素原片段将重组人肾素原加工成肾素。人肾素原（分子量43,000）被肾素颗粒部分切割成肾素（分子量38,000）。这一结果表明肾素颗粒部分含有肾素原加工酶。在本研究中，我们试图分离出肾素原加工酶。从肾素颗粒级分中分离出肾素原加工酶，但我们无法分离或鉴定肾素颗粒中的肾素原加工酶，因为其活性丧失可能是由于其在分离过程中的不稳定性。