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(0.2–0.3 mg protein/tube) for the binding assay. The
assay incubations were carried out at room temperature
for 120 min with 2.5 nM [3H]-(+)-MK-801 (22.5 Ci
mmol–1), 10 mM glutamic acid and 0.1 mM glycine in the
presence and absence of the test compound in a total
volume of 0.5 mL. Bound radioactivity was separated
by filtration through GF/C filters presoaked in 0.05%
polyethylenimmine and washed with ice-cold buffer (3×
5 mL). Non-specific binding was determined in the
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A 1 mM stock solution of the test compound was
prepared in 50% dimethylsulfoxide (DMSO). Subse-
quent dilutions were accomplished in buffer. The IC50
values were calculated from 3–4 displacement curves
based on 4–6 scalar concentrations of the test com-
pound in triplicate using the ALLFIT computer pro-
gram [24] and, in the case of tritiated glycine and
AMPA binding, converted to Ki values by application of
the Cheng-Prusoff equation [25]. In our experimental
conditions the dissociation constants (KD) for
[3H]glycine (10 nM) and [3H]AMPA (8 nM) were 75 6
and 28 3 nM, respectively.
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Valuable assistance in animal handling was provided
by M.G. Giovannini.
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