Determination of the Equilibrium Dissociation Constants and Number of Glycine Binding Sites in Several Areas of the Rat Central Nervous System, Using a Sodium‐Independent System

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Abstract
Parameters affecting the binding of [3H]glycine to membrane fractions isolated from the cerebral cortex, midbrain, cerebellum, medulla oblongata, and spinal cord of the rat were investigated in a Na+‐free medium. A [3H]glycine binding assay was established in which the binding was specific, saturable, pH‐sensitive, and reversible. Conditions were chosen in an effort to minimize binding to glycine uptake sites. From data on specific [3H]glycine binding, Scatchard plots were prepared and the KD and Bmax values were calculated. Two glycine binding sites (high and low affinity) were identified only in the medulla (KD: 44, 211 nM; Bmax: 361, 1076 fmol/mg protein) and spinal cord (KD: 19, 104nM;Bmax: 105, 486 fmol/mg protein). The ranges of the KD and Bmax values for the other three areas studied were 59 to 144 nM and 882 to 3401 fmol/mg protein, respectively. When the glycine content of each area, expressed as fmol/neuron, was plotted against the respective K0 (high affinity), a negative correlation was found (r=−0.90; p < 0.05). A similar negative correlation was found between the glycine content and Bmax (r=−0.88; p 3H]glycine binding.