Takii Y, Figueiredo A F, Inagami T
Hypertension. 1985 Mar-Apr;7(2):236-43. doi: 10.1161/01.hyp.7.2.236.
Identification of inactive prorenin in the kidney has been difficult due to rapid proteolytic conversion of the inactive zymogen to its active form in the tissue or during homogenization and purification. Immunochemical methods, Western blotting, direct radioimmunoassay, and immunoaffinity chromatography were used to isolate and identify rat kidney renin and prorenin and to determine their molecular weights without complete purification. Antisera to pure rat renin were raised in rabbits. A specific reaction between the antisera and rat renin was demonstrated by double immunodiffusion, inhibition of enzyme activity, and competitive radioimmunoassay. The anti-rat renin IgG did not cross-react with purified human renin or rat spleen or kidney cathepsin D. The IgG showed binding affinity to both inactive renin as well as active enzyme. A combination of affinity chromatographies consisting of pepstatin-Sepharose, IgG-Sepharose, and Affi-Gel Blue permitted rapid and complete separation of inactive renin from active renin in rat kidney extract. Neither inactive nor active renin preparations exhibited aspartyl protease activity on hemoglobin used as substrate. The apparent molecular weight of inactive renin was estimated as 50,000 by gel filtration. Electrophoresis of partially purified inactive renin in sodium dodecyl sulfate (SDS) polyacrylamide gel followed by transblotting of proteins to a nitrocellulose sheet and immunochemical staining with anti-renin IgG showed a single protein band with a molecular weight of 48,000. Activation of inactive renin by trypsin was accompanied by the reduction of the 48,000-dalton native protein to a 39,000-dalton protein as determined by the SDS polyacrylamide gel electrophoresis and the transblotting.(ABSTRACT TRUNCATED AT 250 WORDS)
由于在组织中或匀浆及纯化过程中无活性的肾素原迅速被蛋白水解转化为其活性形式，因此在肾脏中鉴定无活性肾素原一直很困难。免疫化学方法、蛋白质印迹法、直接放射免疫测定法和免疫亲和层析法被用于分离和鉴定大鼠肾脏肾素和肾素原，并在未完全纯化的情况下测定它们的分子量。用纯大鼠肾素免疫家兔制备抗血清。通过双向免疫扩散、酶活性抑制和竞争性放射免疫测定法证明了抗血清与大鼠肾素之间的特异性反应。抗大鼠肾素IgG与纯化的人肾素或大鼠脾脏及肾脏组织蛋白酶D无交叉反应。该IgG对无活性肾素和活性酶均显示出结合亲和力。由胃蛋白酶抑制剂 - 琼脂糖、IgG - 琼脂糖和Affi - Gel Blue组成的亲和层析组合能够快速、完全地从大鼠肾脏提取物中分离无活性肾素和活性肾素。无论是无活性还是活性肾素制剂，以血红蛋白为底物时均未表现出天冬氨酸蛋白酶活性。通过凝胶过滤法估计无活性肾素的表观分子量为50,000。在十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶中对部分纯化的无活性肾素进行电泳，随后将蛋白质转印到硝酸纤维素膜上，并用抗肾素IgG进行免疫化学染色，结果显示出一条分子量为48,000的单一蛋白带。经SDS聚丙烯酰胺凝胶电泳和转印测定，胰蛋白酶激活无活性肾素会伴随着48,000道尔顿的天然蛋白降解为39,000道尔顿的蛋白。（摘要截短至250字）
