Yoshimoto T, Nishimura T, Kita T, Tsuru D
J Biochem. 1983 Oct;94(4):1179-90. doi: 10.1093/oxfordjournals.jbchem.a134463.
A post-proline cleaving enzyme [prolyl endopeptidase, EC 3.4.21.26] was purified about 3,700-fold from an extract of bovine brain by a series of column chromatographies on DEAE-Sephadex, hydroxyapatite and PCMB-T-Sepharose, and gel filtration on Sephadex G-200 using N-carbobenzoxy-Gly-Pro-beta-naphthylamide (Z-Gly-Pro-2-NNap), thyrotropin releasing hormone (TRH) and oxytocin as substrates. The purified enzyme appeared homogeneous as judged by disc gel and SDS gel electrophoreses. The enzyme was most active at pH 7.5 and 7.2 with Z-Gly-Pro-2-NNap and TRH, respectively, and hydrolyzed peptide bonds involving Pro-X (X=amino acid, peptide, ester and amide) bonds of synthetic substrates, oxytocin, vasopressin, neurotensin, substance P, tuftsin, bradykinin, and insulin B chain. However, the enzyme was inert toward collagen, gelatin, and casein. The enzyme was completely inactivated by diisopropylphosphorofluoridate (DFP), Z-Gly-Pro-chloromethyl ketone and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenylmethane sulfonylfluoride (PMSF) or metal chelators. Determination of the amino acid composition revealed that the enzyme contained 25 half-cystines. Modification of three cysteine residues of the enzyme by PCMB led to complete inactivation. The isoelectric point of the enzyme was 4.8, and the molecular weight was estimated to be 76,000 by ultracentrifugal analysis and 75,000-74,000 by both gel filtration and sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is present as a monomer. These results indicate that the post-proline cleaving enzyme from bovine brain is very similar to those previously purified from lamb brain and kidney in its enzymatic properties, substrate specificity and physicochemical properties, in sharp contrast with the results obtained by Tate, who reported that the bovine brain prolyl endopeptidase was inert toward oxytocin, vasopressin and bradykinin.
一种脯氨酸后切割酶[脯氨酰内肽酶，EC 3.4.21.26]通过在DEAE-葡聚糖、羟基磷灰石和对氯汞苯甲酸-对甲苯磺酰琼脂糖上进行一系列柱色谱，以及使用N-苄氧羰基-甘氨酰-脯氨酰-β-萘酰胺（Z-甘氨酰-脯氨酰-2-NNap）、促甲状腺激素释放激素（TRH）和催产素作为底物在葡聚糖G-200上进行凝胶过滤，从牛脑提取物中纯化了约3700倍。通过圆盘凝胶电泳和SDS凝胶电泳判断，纯化后的酶呈现均一性。该酶分别以Z-甘氨酰-脯氨酰-2-NNap和TRH为底物时，在pH 7.5和7.2时活性最高，能水解涉及合成底物、催产素、加压素、神经降压素、P物质、促吞噬素、缓激肽和胰岛素B链中Pro-X（X = 氨基酸、肽、酯和酰胺）键的肽键。然而，该酶对胶原蛋白、明胶和酪蛋白无活性。该酶能被二异丙基氟磷酸酯（DFP）、Z-甘氨酰-脯氨酰氯甲基酮和对氯汞苯甲酸（PCMB）完全灭活，而不受苯甲基磺酰氟（PMSF）或金属螯合剂的抑制。氨基酸组成测定表明该酶含有25个半胱氨酸。PCMB对该酶的三个半胱氨酸残基进行修饰导致其完全失活。该酶的等电点为4.8，通过超速离心分析估计分子量为76,000，通过凝胶过滤和十二烷基硫酸钠（SDS）凝胶电泳估计分子量为75,000 - 74,000，表明该酶以单体形式存在。这些结果表明，牛脑脯氨酸后切割酶在酶学性质、底物特异性和物理化学性质方面与先前从羊脑和肾中纯化的酶非常相似，这与泰特的结果形成鲜明对比，泰特报道牛脑脯氨酰内肽酶对催产素、加压素和缓激肽无活性。
