Kalwant S, Porter A G
Institute of Molecular and Cell Biology, National University of Singapore.
Biochem J. 1991 May 15;276 ( Pt 1)(Pt 1):237-44. doi: 10.1042/bj2760237.
Prolyl endopeptidase (EC 3.4.21.26) was purified from human brain by a series of column-chromatographic steps using DEAE-cellulose DE-52, hydroxyapatite, phenyl-Sepharose, Sephacryl S-200 and f.p.l.c. (Mono Q). The enzyme was purified by a factor of 943 and was homogeneous in a SDS/polyacrylamide gel as judged by Coomassie Blue staining. The Mr estimated by SDS/PAGE is 79,600, and under native conditions on Sephacryl S-200 it is 85,600. Therefore the enzyme exists as a monomer. With benzyloxycarbonylglycylproline p-nitroanilide as substrate, the optimum pH of the enzyme is 6.8, and with the substrate concentration between 0.059 mM and 0.37 mM the Km is 9.0 x 10(-4) M. The pI of the enzyme is 4.75. The enzyme is classified as a serine proteinase, as it is strongly inhibited by di-isopropyl fluorophosphate. However, other serine proteinase inhibitors do not inhibit the enzyme significantly, suggesting that the active site of prolyl endopeptidase differs from that of classical serine proteinases such as trypsin. Polyclonal antibodies were raised against purified human brain prolyl endopeptidase in rabbits. Western-blot analysis, enzyme-inhibition assays, antibody binding and immunoprecipitation experiments indicated that the polyclonal antibodies are both specific and inhibitory to the enzyme activity.
脯氨酰内肽酶（EC 3.4.21.26）通过一系列柱色谱步骤从人脑中纯化得到，这些步骤使用了DEAE - 纤维素DE - 52、羟基磷灰石、苯基琼脂糖、Sephacryl S - 200和快速蛋白质液相色谱（Mono Q）。该酶的纯化倍数为943，经考马斯亮蓝染色判断，在SDS / 聚丙烯酰胺凝胶中呈均一状态。通过SDS / PAGE估计的分子量为79,600，在Sephacryl S - 200上的天然条件下为85,600。因此，该酶以单体形式存在。以苄氧羰基甘氨酰脯氨酸对硝基苯胺为底物时，该酶的最适pH为6.8，当底物浓度在0.059 mM至0.37 mM之间时，Km为9.0×10⁻⁴ M。该酶的pI为4.75。该酶被归类为丝氨酸蛋白酶，因为它受到二异丙基氟磷酸的强烈抑制。然而，其他丝氨酸蛋白酶抑制剂对该酶的抑制作用不显著，这表明脯氨酰内肽酶的活性位点与胰蛋白酶等经典丝氨酸蛋白酶不同。用纯化的人脑脯氨酰内肽酶在兔体内制备了多克隆抗体。蛋白质免疫印迹分析、酶抑制试验、抗体结合和免疫沉淀实验表明，多克隆抗体对该酶活性具有特异性且具有抑制作用。
