A CE assay for the detection of agonist‐stimulated adenylyl cyclase activity

Abstract

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 EC50s of 4.2 +/- 0.7 and 2.4 +/- 0.7 microM, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the beta2-adrenergic receptor (beta2AR) fused to the stimulatory G protein. Terbutaline (beta2AR agonist) increased the basal rate of cAMP formation 1.7 +/- 0.1-fold resulting in an EC50 of 62 +/- 10 nM. The assay's ability to detect antagonists is demonstrated by the expected right-shifted EC50 of terbutaline by the beta2AR antagonist propranolol. The CE-UV assay offers advantages over the traditional radioactivity assay in terms of safety and labor.

Detection of G Protein Coupled Receptor Mediated Adenylyl Cyclase Activity by Capillary Electrophoresis Using Fluorescently Labeled ATP

“…Terbutaline increased AC activity by 56% with an EC 50 = 60 ± 9 nM ( n = 8, Figure B). This EC 50 agrees well with that obtained using unlabeled ATP as substrate (EC 50 = 62 ± 10 nM) …”

“…Furthermore, direct injection of membrane fragments did not adversely affect the separation in terms of extra peaks or capillary clogging, with migration time relative standard deviations (RSDs) being as low as 1% ( n = 6). The product BcAMP had a limit of detection of <1 nM, which is a ∼1000-fold increase over the CE-UV assay …”

“…The product BcAMP had a limit of detection of <1 nM, which is a ∼1000-fold increase over the CE-UV assay. 24 To determine appropriate reaction times for drug screening experiments, unfiltered sample was serially injected at 10-min intervals and product formation monitored for ∼50 min. Ideally, enzymatic rates are measured in the first 10-20% of substrate depletion to ensure the enzyme-substrate interaction is not affinity-limited (i.e., not limited by the dissociation constant) and that the forward reaction (BATP to BcAMP) dominates.…”

“…This EC 50 agrees well with that obtained using unlabeled ATP as substrate (EC 50 ) 62 ( 10 nM). 24 The quality of a high-throughput screening assay is measured by the Z′ factor 34 as defined by the following equation:…”

“…Capillary electrophoresis (CE) is well-suited for detecting enzyme activity due to its low sample volume requirements, high resolution, and rapid separations. , We have previously reported a CE assay with UV detection for the detection of GPCR-mediated AC activity using ATP as substrate, but the relatively high detection limit of 1 μM for cAMP limits the applicability of the method for physiological samples and prevents adoption to faster, miniaturized columns . Fluorescent substrates are often used in CE-enzyme assays to achieve high sensitivity. − We also previously demonstrated that BODIPY FL labeled ATP (BATP, Figure ) could be used as a substrate for purified AC cyotsolic domains in a CE with laser-induced fluorescence (LIF) detection enzyme assay .…”