Shinagawa T, Do Y S, Baxter J D, Carilli C, Schilling J, Hsueh W A
Department of Medicine, Los Angeles County/University of Southern California Medical Center 90033.
Proc Natl Acad Sci U S A. 1990 Mar;87(5):1927-31. doi: 10.1073/pnas.87.5.1927.
Using pure recombinant human prorenin as a substrate, we have identified an enzyme in human kidney that accurately processes prorenin to active renin (EC 3.4.23.15). In the crude homogenate, the predominant activity of this potential renin-processing enzyme (RPE) converted the Mr 47,000 inactive prorenin to Mr 44,000 active renin and had a pH optimum of approximately 6. The activity was blocked by cysteine protease inhibitors, but not by pepstatin, EDTA, or serine protease inhibitors. This RPE activity was not detected in a similarly prepared homogenate of human chorion decidua tissue, which produces primarily prorenin, or in human plasma. The activity was purified 100-fold by ammonium sulfate precipitation, p-chloromercuribenzoate affinity chromatography, and chromatofocusing. The partially purified enzyme has a Mr of approximately 27,000 and an isoelectric point in the pH 4.8-5.6 range. The activity in the purified RPE preparation had the same pH optimum as that in crude homogenate, cleaved the prosegment at the same site used by the kidney in vivo based on amino-terminal sequencing of the processed renin, and did not degrade prorenin or renin. These data suggest that the cysteine protease we have isolated is a candidate for authentic renal RPE.
以纯重组人肾素原作为底物，我们在人肾脏中鉴定出一种酶，它能准确地将肾素原加工成活性肾素（EC 3.4.23.15）。在粗匀浆中，这种潜在的肾素加工酶（RPE）的主要活性将分子量为47,000的无活性肾素原转化为分子量为44,000的活性肾素，其最适pH约为6。该活性被半胱氨酸蛋白酶抑制剂阻断，但不被胃蛋白酶抑制剂、乙二胺四乙酸（EDTA）或丝氨酸蛋白酶抑制剂阻断。在主要产生肾素原的人绒毛膜蜕膜组织的类似制备匀浆或人血浆中未检测到这种RPE活性。通过硫酸铵沉淀、对氯汞苯甲酸亲和层析和色谱聚焦将该活性纯化了100倍。部分纯化的酶分子量约为27,000，等电点在pH 4.8 - 5.6范围内。纯化的RPE制剂中的活性与粗匀浆中的活性具有相同的最适pH，根据加工后肾素的氨基末端测序，在体内肾脏使用的相同位点切割前肽段，并且不降解肾素原或肾素。这些数据表明我们分离出的半胱氨酸蛋白酶是真正的肾RPE的候选者。
