Studies on Renin Activation and Prorenin Receptor

为了阐明prorentin在肾素颗粒中可能发生的转化，我们研究了肾脏的肾素颗粒部分是否可以激活或将prorentin加工成活性形式。采用不连续蔗糖密度梯度离心法从狗肾皮质中分离肾素颗粒。从人羊水中提取的失活肾素与狗肾皮质的亚细胞部分孵育。采用免疫放射法定量测定人活性肾素。以COS细胞利用人肾素cDNA表达的[35S]-prorenin为底物进行加工。[35S]-prorenin与肾素颗粒部分孵育。用sds -聚丙烯酰胺凝胶电泳法测定肾素的分子量变化。表现出最高肾素活性的肾素颗粒组分刺激无活性的肾素变为活性形式。膜的制备保留了肾素的活化活性。其他亚细胞部分显示肾素活化较少。膜活化肾素的最佳pH为pH 5 ~ 6。n -乙基马来酰亚胺可抑制其活化，而EDTA或丝氨酸蛋白酶抑制剂则无抑制作用。此外，[35S]-prorenin与肾素颗粒膜孵育，通过估计分子大小的变化揭示了prorenin向活性形态的转化。白细胞介素、n -乙胺能抑制这种转化，但EDTA或丝氨酸蛋白酶抑制剂不能抑制这种转化。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。

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.