Adenosine deaminase ec 3

Frozen tissue was homogenized in ice-cold preparation buffer using a sonicator (Soniprep 150). The buffer contained 50 mM Tris-HCl, pH 7.4, 7 mM MgCl₂, 1 mM EDTA, a cocktail of protease inhibitors (Roche Diagnostics, Mannheim, Germany), and 0.3 IU/mL adenosine deaminase (EC 3.5.4.4, Sigma-Aldrich). The membranes were precipitated by centrifugation at 4 °C for 40 min at 40,000× g (Thermo scientific, Sorvall Lynx 6000, Stockholm, Sweden) and washed through re-homogenization in the same buffer once more. The protein concentration was determined for the membrane homogenates by means of BCA Protein Assay (Pierce, Sweden) using as a standard bovine serum albumin. Pelleted membranes were resuspended to a concentration of 0.15 mg/mL, immediately used or stored at −80 °C until required.

[³H]-raclopride binding was displaced by quinpirole to determine the proportion of receptors in the high-affinity state (RH), the high-affinity value (Ki, High), and the low-affinity (Ki, Low) value. Ventral striatum membrane preparations (60 μg protein/ml) were incubated with increasing concentrations of quinpirole (0.01 nM to 1 mM) and 2 nM [³H]-raclopride (75 Ci/mmol, Novandi Chemistry AB, Sweden) in 250 μl of incubation buffer (50 mM Tris-HCl, 100 mM NaCl, 7 mM MgCl₂, 1 mM EDTA, 0.05% BSA, 1 mM DTT) and 0.3 IU/ml adenosine deaminase (EC 3.5.4.4, Sigma-Aldrich). The incubation took place for 90 min at 30°C in the presence or absence of 100 nM of the A2AR agonist CGS-21680. Non-specific binding was defined by radioligand binding in the presence of 100 μM (+)-butaclamol (Sigma-Aldrich, Sweden). The incubation was terminated by rapid filtration using Whatman GF/B filters (Millipore Corp, Sweden) and a MultiScreenTM Vacuum Manifold 96-well, followed by five washes (250 μl per wash) with ice-cold washing buffer (50 mM Tris-HCl pH 7.4). The filters were dried, 5 ml of scintillation cocktail was added, and the amount of bound ligand was determined after 12 h by liquid scintillation spectrometry.