Receptor binding studies

For competition binding assays, suspensions of nAChR-rich membranes from T. californica ray electric organ (1.25 nM α-Bgt binding sites) in 20 mM Tris-HCl buffer, pH 8.0, containing 1 mg/ml bovine serum albumin (BSA) (binding buffer), human α7 nAChR transfected GH₄C₁ cells (0.4 nM α-Bgt binding sites) in binding buffer, or a solution of heterologously expressed AChBP from L. stagnalis (2.4 nM in binding buffer) were incubated for 3 h with various amounts of the PLA₂s, followed by an additional 5 min incubation with 0.4 nM [¹²⁵I]α-Bgt. Nonspecific binding was determined by preliminary incubation of the preparations with 20 μM α-cobratoxin. The membrane and cell suspensions were applied to glass GF/C filters (Whatman, Little Chalfont, UK) presoaked in 0.25% polyethylenimine, and the unbound radioactivity was removed from the filter by washing (3 × 3 ml) with 20 mM Tris-HCl buffer, pH 8.0, containing 0.1 mg/ml BSA (washing buffer). The AChBP solutions were applied to two layers of DE-81 filters presoaked in PBS-T buffer, and washed (3 × 3 ml) with washing buffer. The bound radioactivity was determined using a Wizard 1470 Automatic Gamma Counter (Perkin Elmer). The binding results were analyzed using ORIGIN 7.5 (OriginLab Corporation, Northampton, MA, USA) fitting to a one-site or two-site dose-response competition curve.

For competition binding assays on the extracellular domain (ECD) of human α9 nAChR, PP PLA₂ in the concentration range 0.3–30 μM was incubated for 2 h at room temperature with the ECD (final concentrations of 30 μg/ml) in 50 μL of a 20 mM Tris–HCl buffer, pH 8.0, containing 1 mg/ml of the bovine serum albumin (binding buffer). Then [¹²⁵I]α-Bgt was added to the reaction mixtures to a final concentration of 0.2 nM. Simultaneously 15 μL Ni-NTA-agarose (QIAGEN) pre-washed in reaction buffer was added. After 6 min, the reaction was stopped by a rapid filtration on GF/C filters (Whatman) pre-soaked in 0.25% polyethylenimine and the unbound radioactivity was removed from the filters by washes (3×4 ml) with the 20 mM Tris–HCl buffer. Nonspecific binding was determined by preliminary incubation of the ECD with 10 μM α-cobratoxin. The bound radioactivity was determined using Wizard 1470 Automatic Gamma Counter (Perkin Elmer). The data were analyzed using ORIGIN 7.5 as a one-site dose-response curve.

SPR experiments were performed at 20°C, using a Biacore^® 2000 system (GE Healthcare, Biacore AB). AChBP was covalently immobilized to a CM5 sensor chip at acidic pH. For binding experiments, the running and dilution buffer was composed of 20 mM Tris (pH 7.4), 150 mM NaCl, and 0.005% Surfactant P20 (GE Healthcare, Biacore AB). The concentrations of Cro ranged from 5.5 to 46 μg/ml, and solutions were injected at a flow rate of 30 μl/min. Background signals were obtained by injection of samples to a blank-immobilized flow cell and these signals were subtracted from the sample signals. At the end of each run, a 10 s injection of 10 mM Gly/HCl pH 1.5 was performed to restore the complete binding capacity of the AСhBP coupled to the CM5 sensor chip. The kinetic constants, k_a (association rate constant), and k_d (dissociation rate constant), for the interaction between Cro and AChBP were calculated using Biacore BIAEVALUATION 3.1 software (Biacore AB). The curves were fitted according to the simple two-component model of interaction. The apparent dissociation constant (K_D^app) was obtained as the ratio of k_d and k_a (K_D^app = k_d/k_a).