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A CE assay for the detection of agonist‐stimulated adenylyl cyclase activity
Author(s) -
Cunliffe Jennifer M.,
Whorton Matthew R.,
Sunahara Roger K.,
Kennedy Robert T.
Publication year - 2007
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200600571
Subject(s) - adenylyl cyclase , terbutaline , forskolin , chemistry , agonist , g protein coupled receptor , receptor , antagonist , g protein , microbiology and biotechnology , medicine , biochemistry , biology , asthma
A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC and G protein‐coupled receptor (GPCR) level. In the assay, cell membranes overexpressing GPCR and/or AC were incubated with modulators and substrate ATP to produce cAMP in a dose‐dependent manner. In both the CE‐UV and a radiochemical assay, the addition of forskolin (FSK) resulted in a two‐ to three‐fold maximum increase in AC activity with EC 50 s of 4.2 ± 0.7 and 2.4 ± 0.7 μM, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the β 2 ‐adrenergic receptor (β 2 AR) fused to the stimulatory G protein. Terbutaline (β 2 AR agonist) increased the basal rate of cAMP formation 1.7 ± 0.1‐fold resulting in an EC 50 of 62 ± 10 nM. The assay's ability to detect antagonists is demonstrated by the expected right‐shifted EC 50 of terbutaline by the β 2 AR antagonist propranolol. The CE‐UV assay offers advantages over the traditional radioactivity assay in terms of safety and labor.