Isolation and characterizaion of renin processing enzyme from renin granule

prorentin对肾素的加工机制仍有争议。肾素在粗内质网和高尔基复合体中合成和加工，储存在肾素颗粒中，通过胞吐作用分泌。近年来发现了几种加工激素前体或蛋白质前体的加工酶，并将其鉴定为内在加工酶。本研究的目的是阐明肾皮质亚细胞部分内在肾素加工酶的性质。通常，激素原转化酶或蛋白质前体加工酶被报道具有与枯草杆菌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，我们研究枯草杆菌素本身是否可以将prorentin加工成肾素。将放射性标记的[^<35>S]-人重组prorentin与枯草杆菌素BNP^-孵育后，分子量为43,000道尔顿的人重组prorentin被裂解为分子量为38,000道尔顿的肾素。此外，当半纯化的人羊水原肾素（或失活肾素）与枯草菌素孵育时，通过检测活性肾素，我们证实了枯草菌素对原肾素的激活作用，活性肾素是用一种特定的免疫放射测定系统定量测定的。这些结果表明枯草杆菌素本身可以加工和激活促凝素为活性形式。在接下来的研究中，我们研究了肾素颗粒是否可以通过切割原肾素的前片段将重组人原肾素加工成肾素。人原肾素（分子量43,000）被肾素颗粒裂解为肾素（分子量38,000）。提示肾素颗粒中含有促肾素加工酶。在本研究中，我们试图从肾素颗粒中分离出促肾素加工酶，但由于其在分离过程中的不稳定性而失去了活性，我们无法从肾素颗粒中分离或鉴定促肾素加工酶。少

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