Interaction of glycine with brain-cortex membrane fragments: kinetics, ionic requirements and amino acid specificity.

The binding of [14]glycine to rat brain-cortex membrane fragments, incubated in artificial cerebrospinal fluid, was studied in vitro by means of a nitrocellulose filter assay. The membranes were obtained from the large granule fraction (P2) of a brain-cortex homogenate, which was osmotically shocked and the larger membrane fractions isolated by centrifugation. Initial binding velocity lasts for about 2 min and equilibrium is reached in 10 min. The binding reaction is reversible, and [14C]glycine can de displaced by an excess of [12C]glycine or by dilution. Binding is strongly dependent on temperature and on sodium ions. The latter activate the binding process in a cooperative manner. Two binding components may be discerned: one with high affinity for glycine (Km = 40 +/- 8 muM) and one with lower affinity. Lowering the sodium concentration to 60 mM increases the Km of the high-affinity component to 59 muM, with no change in Vmax. The bound product is, after incubating the membranes at 37 degrees C for 10 min, 85% glycine. A large fraction of it may released by hypo-osmotic media.