Pure human renin. Identification and characterization and of two major molecular weight forms.
Pure human renin. Identification and characterization and of two major molecular weight forms.
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纯人肾素。
DOI：
10.1016/s0021-9258(18)43403-5
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
H. Strout
中科院分区：
文献类型：
--
作者：
E. Slater;H. Strout
用DEAE-纤维素层析、胃抑素亲和层析、凝胶过滤和以人工合成的八肽肾素抑制剂(D-Leu6)或抗肾素免疫球蛋白为配基的亲和层析两种方法中的任何一种方法从正常肾脏中纯化人肾肾素。用这两种方法均可获得约50万倍的提纯倍数和1毫克蛋白质的产量或7%的酶活(10公斤)。最大比活度为1170个金布拉特单位/毫克。测定了氨基酸组成和动力学性质。用纯化的血管紧张素原底物，最适pH为5.5~6.0，Km值为1.54×10~(-6)M。纯化和鉴定了两种具有相似酶和免疫学性质但表观分子大小和电荷不同的主要肾素形式。其中一种形式是抗体亲和层析后得到的主要形式，经十二烷基硫酸钠凝胶电泳法测得其表观分子尺寸为50kodalton，在pH 7.8的聚丙烯酰胺圆盘凝胶电泳法上迁移较慢(Rf=0.32)。另一种形式的表观分子尺寸为39千道尔顿，在聚丙烯酰胺盘状凝胶上迁移得更快(Rf=0.76)。这种较小的形式在允许在整个纯化过程中持续存在酸性蛋白酶活性的方案中占主导地位。此外，在这种酶的存在下，肾素的分子大小被证明从50到40千道尔顿变化，随后从肾素纯化的倒数第二步中分离出来，并被初步鉴定为肾组织蛋白酶D。这些发现有助于协调其他人获得的纯人肾素的某些不同特征，解释了人酶的显著不稳定性，并表明人肾素的表观分子尺寸比以前报道的要大一些。
Human renal renin was purified from normal kidney by either of two protocols which combined sequential DEAE-cellulose chromatography, pepstatin affinity chromatography, gel filtration, and a final step of affinity chromatography using either the synthetic octapeptide renin inhibitor (D-Leu6] or antirenin immunoglobulin as ligand. An approximate 500,000-fold purification and a yield of 1 mg of protein or 7% enzymatic activity from 10 kg were obtained by either method. Maximum specific activity was 1170 Goldblatt units/mg. Amino acid composition and kinetic properties were determined. Using purified angiotensinogen substrate, optimum pH was 5.5-6.0 and the Km was 1.54 X 10(-6) M. Two major forms of renin possessing similar enzymatic and immunologic properties, but differing in apparent molecular size and charge were purified and characterized. One form, the major form obtained after antibody affinity chromatography, had an apparent molecular size of 50 kilodaltons by sodium dodecyl sulfate-gel electrophoresis and migrated more slowly (RF = 0.32) on polyacrylamide disc gel electrophoresis at pH 7.8. The other form had an apparent molecular size of 39 kilodaltons and migrated more rapidly (RF = 0.76) on polyacrylamide disc gels. This smaller form predominated in protocols which allowed the persistent presence of acid protease activity throughout purification. Moreover, renin molecular size was demonstrated to change from 50 to 40 kilodaltons in the presence of this protease, which was subsequently isolated from the penultimate step of renin purification and tentatively identified as a renal cathepsin D. These findings help reconcile certain disparate characteristics for pure human renin obtained by others, explain the marked instability of the human enzyme, and suggest that the apparent molecular size of human renin is somewhat larger than had been previously reported.