Cytoscint es

Sodium-dependent transport of L-[³H]-glutamate was measured as previously described (Robinson et al. 1991 (link); Whitelaw and Robinson 2013 (link)). HKII or control treated synaptosomes (10 µL, containing ~40 µg of protein) were incubated with 0.5 µM L-[³H]-glutamate (PerkinElmer, Waltham, MA USA) in uptake buffer, containing 5 mM Tris base, 10 mM HEPES, 140 mM NaCl, 2.5 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM K₂HPO₄, 10 mM glucose (pH=7.2) at 37°C for 3 min. We have previously determined that ³H-glutamate uptake is linear until at least 5 min in this system (Robinson 1991 (link)). Uptake was measured in the absence and presence of sodium by substituting equimolar amounts of choline chloride for NaCl. The assay was terminated by the addition of 2 ml of ice-cold choline-containing buffer. Following termination, the suspensions were filtered onto glass filter paper (FP-100; Brandel, Gaithersburg, MD USA) using a Brandel cell harvester and rinsed three times with 2 ml of cold choline-containing buffer. Radioactivity was solubilized with 5 ml of Cytoscint ES (MP Biochemicals, Solon, OH USA) and measured using scintillation spectrometry (Beckman-Coulter Instruments LS 6500). Sodium dependent uptake was calculated as the difference between the signal in the sodium-containing buffer from the signal in the choline-containing buffer.

As previously reported,^{10 (link)} competition binding studies were performed in the presence of a fixed concentration of [³H]RTX and various concentrations of competing ligands. The binding assay mixtures were handled in borosilicate tubes and contained DPBS (with Ca²⁺ and Mg²⁺), bovine serum albumin (0.25 mg/mL), [³H]RTX (100 pM), and various concentrations of the ligands for a total volume of 350 μL. Non-specific binding was determined in the presence of non-radioactive RTX (100 nM). The tubes were incubated for 60 min in a 37 °C water bath. The mixtures were then cooled on ice for approximately 10 min, after which bovine glycoprotein fraction VI (100 μL; 2 mg/mL) was added to reduce nonspecific binding. The ice-cold contents were then transferred to 1.5 mL centrifuge tubes for centrifugation. Membrane-bound RTX was separated from free RTX as well as the glycoprotein-bound RTX by pelleting the membranes in a Beckman (Coulter) Allegra 21R centrifuge for 15 min at 12,000 rpm. Radioactivity in the pellets and in an aliquot of each supernatant was determined by scintillation counting in the presence of Cytoscint E.S. (MP Biomedicals). Equilibrium binding parameters (K_i, B_max, and cooperativity) were determined by fitting the Hill equation to the measured values.