Skidgel R A, Davis R M, Erdös E G
Anal Biochem. 1984 Aug 1;140(2):520-31. doi: 10.1016/0003-2697(84)90203-3.
A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.
一种能从肽链上切割碱性C末端氨基酸的羧肽酶，通过三步法从浓缩人尿中纯化得到：在Affi-Gel Blue上进行色谱分离、精氨酸-琼脂糖亲和色谱分离以及在TSK-G3000SW柱上通过高效液相色谱进行凝胶过滤。尿羧肽酶纯化了406倍，产率为11%，以苯甲酰甘氨酰精氨酸为底物时比活性为49 μmol/min/mg。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳中，它迁移为一条分子量为75,700的单一谱带。它切割苯甲酰甘氨酰精氨酸、苯甲酰甘氨酰赖氨酸、苯甲酰甘氨酰精氨酸、苯甲酰丙氨酰赖氨酸和苯甲酰苯丙氨酰赖氨酸的相对速率与人类血浆羧肽酶N、羧肽酶N的48,000分子量活性亚基或人胰羧肽酶B不同。尿羧肽酶不水解羧肽酶A的底物苯甲酰甘氨酰苯丙氨酸，但能轻易切割缓激肽，其米氏常数为46 μM，催化常数为32 min⁻¹。其活性被氯化钴增强，被醋酸镉、邻菲罗啉或DL-2-巯甲基-3-胍基乙基硫代丙酸抑制。该酶的最适pH为7.0，在pH 6.0时其活性下降60%。在37℃（pH 8.0）下至少稳定2小时，但在室温且pH低于4.5时不稳定。尿羧肽酶的分子量、电泳迁移率和活性不受胰蛋白酶影响。pH和稳定性的影响进一步将尿羧肽酶与其他人类羧肽酶区分开来。通过“蛋白质免疫印迹”技术分析时，尿羧肽酶在免疫上与羧肽酶N不同。因此，人尿中含有一种与已知羧肽酶不同的碱性羧肽酶，它可能由肾脏释放到尿液中。在这里它可以使激肽和其他含有碱性C末端氨基酸的肽失活。
