Kodani Sean D, Barthélemy Morgane, Kamita Shizuo G, Hammock Bruce, Morisseau Christophe
Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616, USA.
Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, Davis, CA 95616, USA.
Anal Biochem. 2017 Dec 15;539:81-89. doi: 10.1016/j.ab.2017.10.014. Epub 2017 Oct 18.
Carboxylesterases are well known for their role in the metabolism of xenobiotics. However, recent studies have also implicated carboxylesterases in regulating a number of physiological processes including metabolic homeostasis and macrophage development, underlying the need to quantify them individually. Unfortunately, current methods for selectively measuring the catalytic activity of individual carboxylesterases are not sufficiently sensitive to support many biological studies. In order to develop a more sensitive and selective method to measure the activity of human carboxylesterase 1 (hCE1), we generated and tested novel substrates with a fluorescent aminopyridine leaving group. hCE1 showed at least a 10-fold higher preference for the optimized substrate 4-MOMMP than the 13 other esterases tested. Because of the high stability of 4-MOMMP and its hydrolysis product, this substrate can be used to measure esterase activity over extended incubation periods yielding a low picogram (femtomol) limit of detection. This sensitivity is comparable to current ELISA methods; however, the new assay quantifies only the catalytically active enzyme facilitating direct correlation to biological processes. The method described herein may allow hCE1 activity to be used as a biomarker for predicting drug pharmacokinetics, early detection of hepatocellular carcinoma, and other disease states where the activity of hCE1 is altered.
羧酸酯酶因其在外源化合物代谢中的作用而广为人知。然而，最近的研究也表明羧酸酯酶参与调节包括代谢稳态和巨噬细胞发育在内的许多生理过程，这凸显了单独对其进行定量的必要性。不幸的是，目前用于选择性测量单个羧酸酯酶催化活性的方法灵敏度不足以支持许多生物学研究。为了开发一种更灵敏、更具选择性的方法来测量人羧酸酯酶1（hCE1）的活性，我们制备并测试了带有荧光氨基吡啶离去基团的新型底物。与其他13种测试酯酶相比，hCE1对优化后的底物4-MOMMP的偏好至少高10倍。由于4-MOMMP及其水解产物具有很高的稳定性，该底物可用于在延长的孵育期内测量酯酶活性，检测限低至皮克（飞摩尔）级别。这种灵敏度与目前的ELISA方法相当；然而，新方法仅对具有催化活性的酶进行定量，便于与生物学过程直接关联。本文所述方法可能使hCE1活性用作预测药物药代动力学、早期检测肝细胞癌以及其他hCE1活性发生改变的疾病状态的生物标志物。
