Aibe K, Yazawa H, Abe K, Teramura K, Kumegawa M, Kawashima H, Honda K
Drug Serendipity Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Ibaraki, Japan.
Biol Pharm Bull. 1996 Aug;19(8):1026-31. doi: 10.1248/bpb.19.1026.
A cDNA clone encoding the rabbit cysteine proteinase cathepsin K, which is predominantly expressed in osteoclasts and is closely related to cathepsins L (EC 3.4.22.15) and S (EC 3.4.22.27) [Tezuka K., Tezuka Y., Maejima A., Sato T., Nemoto K., Kamioka H., Hakeda Y., Kumegawa M., J. Biol. Chem., 269, 1106 (1994)], was expressed at high levels in Escherichia coli in a T7 expression system. The insoluble recombinant enzyme was solubilized in urea and refolded at an alkaline pH. Cathepsin K (37-kDa) was purified by gel filtration and its enzymatic characteristics were determined. The enzymatic activity of cathepsin K was strongly inhibited by cysteine proteinase inhibitors and its optimal pH was pH 5.5. Synthetic substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumaryl-amide, which is hydrolyzed by cathepsins L and S, was also cleaved by cathepsin K. On the other hand, benzyloxycarbonyl-Gly-Pro-Arg-7-(4-methyl)coumaryl-amide was the most suitable substrate for cathepsin K, but was hardly hydrolyzed by cathepsin L. The substrate specificity of cathepsin K, as determined using various chemogenic substrates, showed different characteristics from cathepsins L and S.
编码兔半胱氨酸蛋白酶组织蛋白酶K的cDNA克隆在T7表达系统中于大肠杆菌中高水平表达。组织蛋白酶K主要在破骨细胞中表达，且与组织蛋白酶L（EC 3.4.22.15）和组织蛋白酶S（EC 3.4.22.27）密切相关[Tezuka K., Tezuka Y., Maejima A., Sato T., Nemoto K., Kamioka H., Hakeda Y., Kumegawa M., J. Biol. Chem., 269, 1106 (1994)]。不溶性重组酶在尿素中溶解并在碱性pH下复性。通过凝胶过滤纯化组织蛋白酶K（37 kDa）并测定其酶学特性。组织蛋白酶K的酶活性受到半胱氨酸蛋白酶抑制剂的强烈抑制，其最适pH为5.5。被组织蛋白酶L和S水解的合成底物苄氧羰基-Phe-Arg-7-(4-甲基)香豆素酰胺也能被组织蛋白酶K切割。另一方面，苄氧羰基-Gly-Pro-Arg-7-(4-甲基)香豆素酰胺是组织蛋白酶K最合适的底物，但几乎不被组织蛋白酶L水解。使用各种化学产源底物测定的组织蛋白酶K的底物特异性显示出与组织蛋白酶L和S不同的特性。
