Nyaruhucha C N, Kito M, Fukuoka S I
Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan.
J Biol Chem. 1997 Apr 18;272(16):10573-8. doi: 10.1074/jbc.272.16.10573.
The cDNA encoding a novel isoform of human trypsinogen was identified. The isoelectric points of the proenzyme and active forms calculated from the deduced amino acid sequence are consistent with those of mesotrypsin(ogen), known to be an inhibitor-resistant trypsin isoform. The cDNA attached with a bacterial signal peptide sequence was expressed in Escherichia coli. The recombinant proenzyme purified from periplasm showed enterokinase-dependent activation similar to a major isoform of human trypsinogen. The enzyme was far less inhibited by trypsin inhibitors such as soybean trypsin inhibitor, aprotinin, or pancreatic secretory trypsin inhibitor than the control trypsin. A gel filtration assay showed that the enzyme and aprotinin did not form a stable complex. It is noteworthy that the amino acid at position 198, which is in close vicinity to the active Ser, is Arg while those of other major trypsins are all Gly. It is concluded that the cloned cDNA encodes human mesotrypsinogen, a unique isoform of trypsinogen with inhibitor resistance.
一种编码人胰蛋白酶原新亚型的cDNA被鉴定出来。根据推导的氨基酸序列计算出的该酶原和活性形式的等电点，与中胰蛋白酶（原）的等电点一致，中胰蛋白酶（原）是一种已知的抗抑制剂胰蛋白酶亚型。连接有细菌信号肽序列的cDNA在大肠杆菌中表达。从周质中纯化得到的重组酶原表现出与人类胰蛋白酶原的主要亚型相似的肠激酶依赖性激活。与对照胰蛋白酶相比，该酶受大豆胰蛋白酶抑制剂、抑肽酶或胰腺分泌性胰蛋白酶抑制剂等胰蛋白酶抑制剂的抑制作用要小得多。凝胶过滤分析表明，该酶与抑肽酶未形成稳定复合物。值得注意的是，紧邻活性丝氨酸的第198位氨基酸是精氨酸，而其他主要胰蛋白酶的该氨基酸均为甘氨酸。结论是，克隆的cDNA编码人胰蛋白酶原中胰蛋白酶原，这是一种具有抑制剂抗性的独特胰蛋白酶原亚型。
