Purification of human kidney angiotensin I converting enzyme using reverse-immunoadsorption chromatography.
Purification of human kidney angiotensin I converting enzyme using reverse-immunoadsorption chromatography.
复制标题
使用反向免疫吸附色谱法纯化人肾血管紧张素 I 转换酶。
DOI：
10.1016/0003-2697(82)90451-1
复制
发表时间：
1982
影响因子：
2.9
通讯作者：
Erdös,EG
中科院分区：
文献类型：
--
作者：
Weare,JA;Gafford,JT;Lu,HS;Erdös,EG
本文报道了一种从人肾组织中快速、高效纯化血管紧张素Ⅰ转换酶的方法。胰蛋白酶溶解后，酶被部分纯化的DEAE-纤维素和羟基磷灰石层析。最后一步是在柱上进行“反向免疫吸附”，该柱是通过将抗污染蛋白的抗血清与溴化氰活化的琼脂糖CL-6 B偶联而制备的。从600 g肾组织开始，以Hip-His-Leu为底物，获得6.1 mg酶，比活为108 U/mg，纯化3400倍，总收率为26%。该制剂在7.5%SDS-尿素凝胶上呈单一条带，在交叉免疫电泳中与不纯酶的抗血清呈单一弧形。通过丹磺酰化检测单个N-末端氨基酸（亮氨酸）。该程序允许使用人酶开始结构研究。“反向免疫吸附”可以是用于蛋白质纯化的通常有用的方法。
A rapid and highly efficient procedure for purification of angiotensin I converting enzyme from human kidney has been developed. Following tryptic solubilization, the enzyme was partially purified by DEAE-cellulose and hydroxylapatite chromatography. The final step consisted of “reverse immunoadsorption” on a column prepared by coupling antisera raised against contaminating proteins to CNBr-activated Sepharose CL-6B. Starting with 600 g kidney tissue, 6.1 mg of enzyme was obtained with a specific activity of 108 U/mg using Hip-His-Leu as substrate, a 3400-fold purification with an overall yield of 26%. The preparation gave a single band on 7.5% SDS-urea gels and a single arc against antisera to impure enzyme in crossed immunoelectrophoresis. A single N-terminal amino acid (leucine) was detected by dansylation. This procedure has allowed the initiation of structural studies with the human enzyme. “Reverse immunoadsorption” may be a generally useful method for protein purification.