Skidgel R A, Deddish P A, Davis R M
Department of Anesthesiology, University of Illinois College of Medicine, Chicago 60612.
Arch Biochem Biophys. 1988 Dec;267(2):660-7. doi: 10.1016/0003-9861(88)90074-4.
A carboxypeptidase which cleaves the C-terminal arginine or lysine from peptides was purified by a two-step procedure; gel filtration on Sephacryl S-300 and affinity chromatography on arginine-Sepharose. The activity increased 280% after the first step, indicating the removal of an inhibitor from the crude starting material. The activity in the crude seminal plasma eluted from the Sephacryl S-300 column with an apparent Mr 98,000 and after purification with an Mr 67,000, indicating that it binds to another protein in the crude seminal plasma. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single band at Mr 53,000 was seen which was converted to two smaller bands (Mr 32,000 and/or 26,000) after reduction. The seminal plasma carboxypeptidase has a neutral pH optimum, is inhibited by o-phenanthroline and by the inhibitor of carboxypeptidase B-type enzymes, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and can be activated by cobalt. The purified enzyme has a high specific activity (67.8 mumol/min/mg) with the ester substrate benzoyl (Bz)-Gly-argininic acid and readily cleaves Bz-Ala-Lys, Bz-Gly-Arg, and Bz-Gly-Lys. It also hydrolyzes biologically active peptides such as bradykinin (Km = 6 microM, kcat = 43 min-1), Arg6-Met5-enkephalin (Km = 103 microM, kcat = 438 min-1), and Lys6-Met5-enkephalin (Km = 848 microM, kcat = 449 min-1). The seminal plasma carboxypeptidase did not cross-react with antiserum to human plasma carboxypeptidase N; other properties distinguish it from the blood plasma enzyme as well as from pancreatic carboxypeptidase B and granular, acid carboxypeptidase H (enkephalin convertase). The carboxypeptidase could be involved in the control of fertility by activating or inactivating peptide hormones in the seminal plasma. In addition it could contribute to the degradation of basic proteins during semen liquefaction.
一种能从肽链上切割C末端精氨酸或赖氨酸的羧肽酶，通过两步法进行纯化；第一步是在Sephacryl S - 300上进行凝胶过滤，第二步是在精氨酸 - 琼脂糖上进行亲和层析。第一步后活性增加了280%，这表明从粗制起始材料中去除了一种抑制剂。从Sephacryl S - 300柱上洗脱的粗制精浆中的活性物质，其表观分子量为98,000，纯化后为67,000，这表明它在粗制精浆中与另一种蛋白质结合。当在十二烷基硫酸钠存在下通过聚丙烯酰胺凝胶电泳分析时，可见一条分子量为53,000的条带，还原后转变为两条较小的条带（分子量为32,000和/或26,000）。精浆羧肽酶的最适pH为中性，受邻菲罗啉以及羧肽酶B型酶的抑制剂2 - 巯基甲基 - 3 - 胍基乙基硫代丙酸抑制，并且可被钴激活。纯化后的酶对酯底物苯甲酰（Bz） - 甘氨酰 - 精氨酸具有高比活性（67.8 μmol/分钟/毫克），并且能轻易切割Bz - 丙氨酰 - 赖氨酸、Bz - 甘氨酰 - 精氨酸和Bz - 甘氨酰 - 赖氨酸。它还能水解生物活性肽，如缓激肽（Km = 6 μM，kcat = 43分钟-1）、精氨酸6 - 蛋氨酸5 - 脑啡肽（Km = 103 μM，kcat = 438分钟-1）和赖氨酸6 - 蛋氨酸5 - 脑啡肽（Km = 848 μM，kcat = 449分钟-1）。精浆羧肽酶与抗人血浆羧肽酶N的抗血清不发生交叉反应；它的其他特性也使其与血浆酶以及胰腺羧肽酶B和颗粒状酸性羧肽酶H（脑啡肽转化酶）有所区别。该羧肽酶可能通过激活或失活精浆中的肽激素参与生育控制。此外，它可能有助于精液液化过程中碱性蛋白质的降解。
