Skidgel R A, Johnson A R, Erdös E G
Biochem Pharmacol. 1984 Nov 1;33(21):3471-8. doi: 10.1016/0006-2952(84)90122-9.
Carboxypeptidase N, purified to homogeneity from human plasma, rapidly hydrolyzed Lys6- or Arg6-enkephalins when measured by high pressure liquid chromatography. Comparison of the kinetics of hydrolysis of the enkephalin hexapeptides and bradykinin by carboxypeptidase N revealed the following values for the Km and kcat: Arg6-Met5-enkephalin, 49 microM, 1024 min-1; Arg6-Leu5-enkephalin, 57 microM, 375 min-1; Lys6-Met5-enkephalin, 216 microM, 6204 min-1; bradykinin, 19 microM, 58 min-1. Thus, while bradykinin had the lowest Km, the specificity constants (kcat/Km) for all the enkephalin hexapeptides were higher than that of bradykinin due to their high turnover numbers. Preincubation of the enzyme with 0.1 mM CoCl2 increased both the kcat and Km of bradykinin and Arg6-Met5-enkephalin. Similar results were obtained when the above experiments were conducted with the active 48,000 dalton subunit of carboxypeptidase N. Basic carboxypeptidase activity was found in the amniotic fluid, in membrane fractions of various human and bovine tissues, and in cultured cells in the following order of decreasing specific activity: human placental microvilli, human kidney, human amniotic fluid, human lung, bovine lung, bovine pulmonary artery, human foreskin fibroblasts, human pulmonary arterial endothelial cells, and human lung fibroblasts. The membrane-bound carboxypeptidase activity had a neutral pH optimum and behaved similarly to plasma carboxypeptidase N in the presence of various inhibitors and activators. It was different from the carboxypeptidase activity in bovine adrenal chromaffin granules which had an acid pH optimum and was inhibited by sulfhydryl reagents. These studies show that human carboxypeptidase N, an enzyme found in high concentration in blood, readily hydrolyzes Arg6- or Lys6-enkephalins. It could thus control the levels of these peptides if they are released into the circulation from the adrenal gland. In addition, a membrane-bound carboxypeptidase N-like enzyme in various tissues may regulate the local levels of biologically active peptides containing C-terminal basic amino acids such as hexapeptide enkephalins, kinins, anaphylatoxins or fibrinopeptides.
从人血浆中纯化至同质的羧肽酶N，通过高压液相色谱法测定时，能快速水解Lys6-或Arg6-脑啡肽。羧肽酶N对脑啡肽六肽和缓激肽的水解动力学比较显示，其米氏常数（Km）和催化常数（kcat）如下：Arg6-Met5-脑啡肽，49微摩尔，1024分钟⁻¹；Arg6-Leu5-脑啡肽，57微摩尔，375分钟⁻¹；Lys6-Met5-脑啡肽，216微摩尔，6204分钟⁻¹；缓激肽，19微摩尔，58分钟⁻¹。因此，虽然缓激肽的Km最低，但由于所有脑啡肽六肽的周转数高，其特异性常数（kcat/Km）高于缓激肽。酶与0.1毫摩尔氯化钴预孵育增加了缓激肽和Arg6-Met5-脑啡肽的kcat和Km。用羧肽酶N的活性48,000道尔顿亚基进行上述实验时得到了类似结果。在羊水、各种人和牛组织的膜组分以及培养细胞中发现了碱性羧肽酶活性，其比活性按以下顺序降低：人胎盘微绒毛、人肾、人羊水、人肺、牛肺、牛肺动脉、人包皮成纤维细胞、人肺动脉内皮细胞和人肺成纤维细胞。膜结合的羧肽酶活性在中性pH时最适宜，在存在各种抑制剂和激活剂时表现与血浆羧肽酶N相似。它与牛肾上腺嗜铬颗粒中的羧肽酶活性不同，后者在酸性pH时最适宜且被巯基试剂抑制。这些研究表明，人羧肽酶N是一种在血液中高浓度存在的酶，能轻易水解Arg6-或Lys6-脑啡肽。因此，如果这些肽从肾上腺释放到循环中，它可以控制其水平。此外，各种组织中膜结合的羧肽酶N样酶可能调节含有C端碱性氨基酸的生物活性肽的局部水平，如六肽脑啡肽、激肽、过敏毒素或纤维蛋白肽。
