Liu Huijie, Wang Xuejuan, Yang Hanlin, Zhao Yan, Ji Shengping, Ma Hui, Zhou Yiting, Wang Yanjie, Zhang Hongan, Jiang Wenyan, Fang Chunyan, Feng Lingjun, Wang Xuejian
School of Pharmacy, Weifang Medical University.
Two Gland Division, Affiliated Hospital of Weifang Medical University.
Chem Pharm Bull (Tokyo). 2019;67(2):155-158. doi: 10.1248/cpb.c18-00667.
Aminopeptidase N, also known as CD13, is a transmembrance protease with many functions. CD13 is involved in inflammatory diseases and cancers. A convenient and reliable laboratory test method for detecting the suppressing effects of enzyme activity would be useful for study of CD13 inhibitors. Porcine CD13 (pCD13) was traditionally considered an enzyme source but has significant practical disadvantages. pCD13 is not a human source, and the accuracy and reliability of experimental results are greatly reduced. In this study, a modified detection method with K562-CD13 monoclonal cells, a human-derived cell line, was established to detect the suppressing effects of enzyme activity by the CD13 inhibitor. In this method, K562-CD13 monoclonal cells were used as enzyme source and L-leucine p-nitroaniline hydrochloride as substrate. Using CD13 enzyme activity analyses, we found that the ability of the catalytic substrate was weaker in K562 cells than in the other cell lines, and K562-CD13 cells expressed significantly higher levels of CD13 enzyme activity than parental K562 cells. The enzyme activity of CD13 was detected with the new method after ubenimex treatment. The enzyme activity was significantly inhibited by ubenimex in a dose-dependent manner. In summary, this study proposes a sensitive, stable, and objective laboratory method for detecting the inhibitory effect of the CD13 inhibitor.
氨肽酶N，也称为CD13，是一种具有多种功能的跨膜蛋白酶。CD13与炎症性疾病和癌症有关。一种方便可靠的检测酶活性抑制作用的实验室检测方法，对于CD13抑制剂的研究将是有用的。猪CD13（pCD13）传统上被认为是一种酶源，但存在明显的实际缺点。pCD13不是人源的，实验结果的准确性和可靠性大大降低。在本研究中，建立了一种使用人源细胞系K562-CD13单克隆细胞的改良检测方法，以检测CD13抑制剂对酶活性的抑制作用。在该方法中，K562-CD13单克隆细胞用作酶源，L-亮氨酸对硝基苯胺盐酸盐用作底物。通过CD13酶活性分析，我们发现K562细胞催化底物的能力比其他细胞系弱，并且K562-CD13细胞表达的CD13酶活性水平明显高于亲代K562细胞。用乌苯美司处理后，用新方法检测CD13的酶活性。乌苯美司以剂量依赖性方式显著抑制酶活性。总之，本研究提出了一种灵敏、稳定且客观的实验室方法来检测CD13抑制剂的抑制作用。
