A fluorescence-based assay for fatty acid amide hydrolase compatible with high-throughput screening.
Anal Biochem
; 343(1): 143-51, 2005 Aug 01.
Article
en En
| MEDLINE
| ID: mdl-16018870
ABSTRACT
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 0.48muM and V(max) of 58pmolmin(-1)mgprotein(-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 IC(50) of 33.5nM. 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.
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Banco de datos:
MEDLINE
Asunto principal:
Bioensayo
/
Ácidos Araquidónicos
/
Cumarinas
/
Colorantes Fluorescentes
/
Amidohidrolasas
/
Microsomas
Tipo de estudio:
Diagnostic_studies
/
Screening_studies
Límite:
Animals
/
Humans
Idioma:
En
Año:
2005
Tipo del documento:
Article