Studies on Renin Activation and Prorenin Receptor

肾素激活和肾素原受体的研究

基本信息

批准号：
01570507
负责人：
IKEDA Masaharu
金额：
$ 1.34万
依托单位：
Fukuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01570507/
关键词：
Prorenin Renin Processing Recombinant Renin レコンビナントプロレニン

项目摘要

To clarify the possible conversion of prorenin in renin granules where conversion reportedly occurred, we investigated whether the renin granule fraction of the kidney could activate or process prorenin to the active form.Renin granules were isolated from the dog kidney cortex by discontinuous sucrose density gradient centrifugation. Inactive renin from human amniotic fluid was incubated with the subcellular fraction of the dog kidney cortex. Human active renin was quantified by immunoradiometric assay. [35S]-prorenin which was expressed by COS cell using human renin cDNA was used as a substrate for processing. [35S]-prorenin was incubated with renin granule fraction. Processing of renin was estimated by change of molecular size using SDS-polyacrylamide gel electrophoresis.The renin granule fraction that showed the highest renin activity stimulated the inactive renin to become the active form. The membrane preparation retained the activity of renin activation. Other subcellular fractions showed less renin activation. The optimal pH for renin activation by the membrane was pH 5 to 6. The activation was inhibited by N-ethylmaleimide but not by EDTA or serine protease inhibitors.In addition, [35S]-prorenin incubated with renin granule membrane revealed the conversion of prorenin to active form by estimation of the change of molecular size. This conversion was inhibited by leupeptin,N-ethylamide but not by EDTA or serine protease inhibitors.These results suggest that renin is processed by a membrane bound protease in renin granules.

为了澄清据报道发生转化的肾素颗粒中肾素原的可能转化，我们研究了肾脏的肾素颗粒部分是否可以激活肾素原或将肾素原加工成活性形式。通过不连续蔗糖密度梯度离心从狗肾皮质中分离肾素颗粒。将人羊水中的无活性肾素与狗肾皮质的亚细胞部分一起孵育。通过免疫放射测定法对人活性肾素进行定量。使用人肾素cDNA由COS细胞表达的[35S]-肾素原作为处理的底物。 [35S]-肾素原与肾素颗粒级分一起孵育。使用SDS-聚丙烯酰胺凝胶电泳通过分子大小的变化来估计肾素的加工。表现出最高肾素活性的肾素颗粒级分刺激非活性肾素变成活性形式。膜制剂保留了肾素激活的活性。其他亚细胞部分表现出较少的肾素激活。膜激活肾素的最佳pH为pH 5至6。该激活被N-乙基马来酰亚胺抑制，但不被EDTA或丝氨酸蛋白酶抑制剂抑制。此外，[35S]-肾素原与肾素颗粒膜一起孵育揭示了肾素原的转化通过估计分子大小的变化转化为活性形式。这种转化被亮肽素、N-乙酰胺抑制，但不被 EDTA 或丝氨酸蛋白酶抑制剂抑制。这些结果表明，肾素是由肾素颗粒中的膜结合蛋白酶加工的。