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

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 K_D and B_max values were calculated. Two glycine binding sites (high and low affinity) were identified only in the medulla (K_D: 44, 211 nM; B_max: 361, 1076 fmol/mg protein) and spinal cord (K_D: 19, 104nM;B_max: 105, 486 fmol/mg protein). The ranges of the K_D and B_max 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 B_max (r=−0.88; p 3H]glycine binding.