Do Y S, Shinagawa T, Tam H, Inagami T, Hsueh W A
J Biol Chem. 1987 Jan 25;262(3):1037-43.
Renin was completely purified from human kidney cortex employing a rapid three-step procedure which included homogenization and ammonium sulfate precipitation, aminohexyl-pepstatin affinity chromatography, and affinity chromatography using a synthetic octapeptide renin inhibitor (H-77) with a reduced peptide bond (-CH2-NH- instead of -CO-NH-) between Leu5-Leu6, Three kg of cortex dissected from 10 kg of human cadaver kidney yielded 1.7 +/- 0.5 mg of protein (mean +/- S.E. for five procedures) with a specific activity of 1094 +/- 166 Goldblatt units/mg of protein and an overall recovery of 52 +/- 2%. Both gel filtration high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 44,000, although Mr = 22,000 and 18,000 bands were also identified by SDS-PAGE. The pH optima with sheep angiotensinogen were 5.5 and 7.8 and the Km was 0.31 microM. With pure human substrate the pH optimum was 6.0 and the Km was 1.15 microM. Enzyme activity was inhibited by two different anti-human renal renin antibodies. Amino-terminal sequencing demonstrated a leucine residue at the 1-position. Sequencing of 15 additional amino acids agreed with that predicted from the gene sequence and indicated that prorenin is converted to renin following cleavage at the carboxyl end of two basic residues, Lys-2 Arg-1. As with SDS-PAGE analysis, high performance liquid chromatography in the presence of 6 M urea demonstrated Mr = 44,000, 22,000, and 18,000 bands. Immunoblot studies revealed that all of these bands cross-reacted with antihuman renin antibody. Amino-terminal sequencing indicated the Mm = 22,000 band is the amino terminus and the Mr = 18,000 band the carboxyl terminus of Mr = 44,000 renin. In the aqueous phase, these subunits bound to H-77 suggesting that they represent components of the active enzyme complex. Unlike mouse renin, there was no evidence of disulfide bonds. These results raise the question of whether human renin circulates as a subunit aggregation as well as a single chain protein. This may serve as a possible mechanism to regulate renin activity in plasma and tissues.
采用快速三步法从人肾皮质中完全纯化肾素，该方法包括匀浆和硫酸铵沉淀、氨基己基胃蛋白酶抑制剂亲和层析，以及使用一种合成八肽肾素抑制剂（H-77）的亲和层析，该抑制剂在亮氨酸5 - 亮氨酸6之间具有还原肽键（-CH2-NH-而非-CO-NH-）。从10千克人尸体肾脏中切取3千克皮质，得到1.7±0.5毫克蛋白质（五次操作的平均值±标准误），比活性为1094±166戈德布拉特单位/毫克蛋白质，总回收率为52±2%。凝胶过滤高效液相色谱和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE）均显示分子量为44,000，不过SDS-PAGE也鉴定出了分子量为22,000和18,000的条带。以绵羊血管紧张素原作为底物时，最适pH值为5.5和7.8，米氏常数为0.31微摩尔。以纯人底物时，最适pH值为6.0，米氏常数为1.15微摩尔。酶活性被两种不同的抗人肾肾素抗体抑制。氨基末端测序显示第1位为亮氨酸残基。另外15个氨基酸的测序结果与基因序列预测一致，表明肾素原在两个碱性残基赖氨酸 - 2和精氨酸 - 1的羧基末端裂解后转化为肾素。与SDS-PAGE分析一样，在6 M尿素存在下的高效液相色谱显示分子量为44,000、22,000和18,000的条带。免疫印迹研究表明，所有这些条带均与抗人肾素抗体发生交叉反应。氨基末端测序表明分子量为22,000的条带是分子量为44,000肾素的氨基末端，分子量为18,000的条带是其羧基末端。在水相中，这些亚基与H-77结合，表明它们代表活性酶复合物的组成成分。与小鼠肾素不同，没有证据表明存在二硫键。这些结果提出了一个问题，即人肾素是否以亚基聚集体以及单链蛋白的形式在循环中存在。这可能是调节血浆和组织中肾素活性的一种可能机制。
