Adenylyl cyclase (AC) activity assay

The effect of aptamers on β2AR-dependent stimulation of AC activity was assessed by 3’, 5’-cyclic AMP (cAMP) accumulation on HEK-293 membrane homogenates stably expressing β₂AR⁵³ (a clone developed in the laboratory that has an expression level of ~ 2 pmoles/mg), by measuring the conversion of [α-³²P]-ATP to [α-³²P]-cAMP as previously described⁵⁴. Typical assay setup contained a final total volume of 100-µL, performed in 3-steps. First, a premix sample (with or without ligand) of 60 µL consisting of final concentrations of 50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 1 mM ATP, 1 µM GTP, 1 mM cAMP, 2 µCi [α-³²P]-ATP, ATP-regenerating system [20 mM creatine phosphate and 13 units/100 µL of creatine-phosphokinase], and phosphodiesterase inhibitors [250 µM of Ro 20–1724 and 100 µM of 3-isobutyl-1-methylxanthine] with or without 100 nM isoproterenol were mixed. Second, HEK-293 membrane homogenates (150 µg) were incubated with individual aptamers (4 µM; heat denatured and refolded as described above) or assay buffer alone in a total volume of 40 µL for 20 min on ice. Then, to measure AC activity in response to isoproterenol (100 nM) or isoproterenol (100 nM) in combination with aptamers (4 µM), the premix samples (60 µL) and membrane mixtures (40 µL) were incubated at 37 °C for 10 min. Reactions were terminated with 0.8 mL cold trichloroacetic acid (6.25% wt/vol); and 100 µL of [³H]-cAMP (~25,000 cpm) was added as a recovery marker. Samples were pelleted by centrifugation at 1,500 × g for 20 min at 4°C. The [α-³²P]-cAMP formed was then isolated from the remaining ATP by applying the 1 mL reaction mixture to a sequential chromatography using a Dowex gel column followed by filtration on an aluminum oxide column and elution with 4 mL of 0.1 M imidazole, pH 7.5. The samples were counted for both ³H and ³²P, and the counts were converted to AC activity as picomole of cAMP/mg of protein/min as described previously⁵⁴.