Rossier J, Barrès E, Hutton J C, Bicknell R J
Laboratoire de Physiologie Nerveuse, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Anal Biochem. 1989 Apr;178(1):27-31. doi: 10.1016/0003-2697(89)90350-3.
Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.
羧肽酶H，酶学委员会编号为3.4.17.10，也被称为脑啡肽转换酶、羧肽酶E和嗜铬羧肽酶B，是一种参与生物活性肽生物合成的重要酶。为了测定该酶，将组织在至少20体积（毫升/克）的0.025M Tris-HCl缓冲液（pH 8）中匀浆，缓冲液中含有5毫克/毫升的牛血清白蛋白。离心后，将上清液调至pH 5.6并再次离心。在2mM氯化钴中预孵育20分钟后，将上清液与0.1mM（终浓度）的放射性底物[3H]苯甲酰-Phe-Ala-Arg一起孵育。通过加入680微升乙腈/0.25M盐酸（0.7/1）终止100微升的测定。将1.5毫升的试管转移到闪烁瓶中，并用4毫升Econofluor（一种与水不混溶的闪烁液）冲洗。在有机相中回收的产物[3H]苯甲酰-Phe-Ala可直接计数，不受水相中残留底物的干扰。空白值低于1%。以每毫克组织每分钟纳摩尔数表示，大鼠可溶性酶的活性在纹状体中为0.34，在胰岛中为21.0，在前叶垂体中为16.6，在中叶垂体中为46.0，在神经垂体中为10.9。在每种情况下，25 microM胍基乙硫基琥珀酸（羧肽酶H的活性位点导向抑制剂）都能完全抑制活性。
