Collins Patrick J, McMahon Gillian, O'Brien Pamela, O'Connor Brendan
School of Biotechnology and National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
Int J Biochem Cell Biol. 2004 Nov;36(11):2320-33. doi: 10.1016/j.biocel.2004.05.006.
The study and identification for the first time of a soluble form of a seprase activity from bovine serum is presented. To date, this activity has only been reported to be an integral membrane protease but has been known to shed from its membrane. The activity was purified 30,197-fold to homogeneity, using a combination of column chromatographies, from bovine serum. Inhibition by DFP, resulting in an IC(50) of 100:nM confirms classification as a serine protease. The protease after separation and visualisation by native PAGE was subjected to tryptic digestion and the subsequent peptides sequenced. Each peptide sequenced was found to be present in the primary structure of seprase/fibroblast activation protein (FAP), a serine gelatinase specific for proline-containing peptides and macromolecules. Substrate specificity studies using kinetic, RP-HPLC and LC-MS analysis of synthetic peptides suggest that this peptidase has an extended substrate-binding region in addition to the primary specificity site S(1). This analysis revealed at least five subsites to be involved in enzyme-substrate binding, with the smallest peptide cleaved being a tetrapeptide. A proline residue in position P(1) was absolutely necessary therefore showing high primary substrate specificity for the Pro-X bond, while a preference for a hydrophobic residue at the C-terminal end of the scissile bond (P'(1)) was evident. The enzyme also showed complete insensitivity to the prolyl oligopeptidase specific inhibitors, JTP-4819, Fmoc-Ala-pyrrCN and Z-Phe-Pro-BT. To date, no physiological substrate has clearly been defined for this protease but its ability to effectively degrade gelatin suggests a candidate protein substrate in vivo and a possible role in extracellular matrix protein degradation.
首次对牛血清中可溶性形式的丝氨酸蛋白酶活性进行了研究和鉴定。迄今为止，该活性仅被报道为一种整合膜蛋白酶，但已知它会从膜上脱落。使用多种柱色谱法从牛血清中纯化该活性，纯化倍数达30,197倍，使其达到同质。二异丙基氟磷酸（DFP）抑制作用导致IC(50)为100 nM，证实其属于丝氨酸蛋白酶。通过非变性聚丙烯酰胺凝胶电泳（native PAGE）分离并可视化后的蛋白酶进行胰蛋白酶消化，随后对所得肽段进行测序。发现所测序的每个肽段都存在于丝氨酸蛋白酶/成纤维细胞激活蛋白（FAP）的一级结构中，FAP是一种对含脯氨酸的肽和大分子具有特异性的丝氨酸明胶酶。使用合成肽的动力学、反相高效液相色谱（RP-HPLC）和液相色谱-质谱（LC-MS）分析进行的底物特异性研究表明，除了主要特异性位点S(1)外，该肽酶还有一个扩展的底物结合区域。该分析揭示至少有五个亚位点参与酶-底物结合，切割的最小肽段为四肽。因此，P(1)位的脯氨酸残基是绝对必需的，这表明对Pro-X键具有高一级底物特异性，同时在切割键的C末端（P'(1)）对疏水残基有明显偏好。该酶对脯氨酰寡肽酶特异性抑制剂JTP-4819、Fmoc-Ala-pyrrCN和Z-Phe-Pro-BT也完全不敏感。迄今为止，尚未明确确定该蛋白酶的生理底物，但其有效降解明胶的能力表明其在体内可能存在候选蛋白质底物，并可能在细胞外基质蛋白降解中发挥作用。
