Lipperheide C, Otto K
Biochim Biophys Acta. 1986 Feb 19;880(2-3):171-8. doi: 10.1016/0304-4165(86)90077-2.
Lysosomal carboxypeptidase B (peptidyl-L-amino-acid hydrolase, EC 3.4.18.1) from bovine spleen purified to apparent homogeneity was found to have a molecular weight of 52 000 in the absence and of 25 000 in the presence of urea, determined by gel filtration, indicating the existence of two subunits of identical size. The amount of approx. 15% carbohydrate estimated after cleavage by endoglycosidase H was shown to be insignificant for enzymatic activity. The isoelectric focusing separated lysosomal carboxypeptidase B into several protein bands - each enzymatically active - with a range of isoelectric points between 4.6 and 5.2. The titration of the sulphydryl group in the active site of the enzyme with the proteinase inhibitor E-64 yielded one thiol group per molecule. A maximum of activation was achieved by the addition of selenocystamine together with dithioerythritol and EDTA in the incubation solution. Under these conditions the carboxypeptidase hydrolyzed benzoylglycylarginine (80 kat/mol enzyme), benzoylarginine amide (38 kat/mol enzyme) and carbobenzoxyglutaryltyrosine (110 kat/mol enzyme). Slight enzymatic activities towards benzoylarginine 2-naphthylamide and benzoylarginine p-nitroanilide could be measured. With the oxidized insulin B chain, lysosomal carboxypeptidase B exhibited only carboxypeptidase activity.
从牛脾脏中纯化至表观均一的溶酶体羧肽酶B（肽基-L-氨基酸水解酶，EC 3.4.18.1），通过凝胶过滤测定，在不存在尿素的情况下分子量为52000，在存在尿素的情况下分子量为25000，表明存在两个大小相同的亚基。经内切糖苷酶H切割后估计约15%的碳水化合物含量对酶活性无显著影响。等电聚焦将溶酶体羧肽酶B分离成几条蛋白带——每条都具有酶活性——等电点范围在4.6至5.2之间。用蛋白酶抑制剂E-64滴定酶活性位点中的巯基，每个分子产生一个巯基。在孵育溶液中加入硒代胱胺以及二硫苏糖醇和乙二胺四乙酸可实现最大激活。在这些条件下，羧肽酶可水解苯甲酰甘氨酰精氨酸（80 kat/mol酶）、苯甲酰精氨酸酰胺（38 kat/mol酶）和苄氧羰基谷氨酰酪氨酸（110 kat/mol酶）。可检测到对苯甲酰精氨酸2-萘酰胺和苯甲酰精氨酸对硝基苯胺的轻微酶活性。对于氧化胰岛素B链，溶酶体羧肽酶B仅表现出羧肽酶活性。
