期刊
ANALYTICAL BIOCHEMISTRY
卷 343, 期 1, 页码 143-151出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.04.032
关键词
fatty acid amide hydrolase; high-throughput screening; fluorescent assay; enzymatic assay; anandamide; amidase
A novel fluorescent assay to continuously monitor fatty acid amide hydrolase (FAAH) activity that is simple, sensitive, and amenable to high-throughput screening (HTS) of compound libraries is described in this article. Stable Chinese hamster ovary (CHO) cell lines expressing either human FAAH or an inactive mutant, FAAH-S241A, were established. Arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a novel fluorogenic substrate for FAAH, was designed and synthesized. FAAH catalyzes the hydrolysis of AAMCA to generate arachidonic acid and a highly fluorescent 7-amino, 4-methyl coumarin (AMC). The assay was done at 25 degrees C by incubating whole cell or microsomal preparations from FAAH-expressing cells with AAMCA. Release of AMC was monitored continuously using a fluorometer. Microsomal FAAH catalyzed the hydrolysis of AAMCA with an apparent K-m of 0.48 mu M and V-max of 58 pmol min(-1) mg protein(-1). The assay is specific for FAAH given that microsomes prepared from cells expressing FAAH-S241A or vector alone had no significant activity against AAMCA. Furthermore, the activity was inhibited by URB-597, an FAAH-specific inhibitor, in a concentration-dependent manner with an IC50 of 33.5 nM. The assay was optimized for HTS and had a Z' value ranging from 0.7 to 0.9. The assay is also compatible with ex vivo analysis of FAAH activity. (c) 2005 Elsevier Inc. All rights reserved.
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