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

Abstract

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 (P 2) 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 be 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 ( K m = 40 ± 8 μM) and one with lower affinity. Lowering the sodium concentration to 60 m M increases the K m of the high-affinity component to 59 μ M, with no change in V max. The bound product is, after incubating the membranes at 37 °C for 10 min, 85% glycine. A large fraction of it may be released by hypoosmotic media. The affinity of the receptor for different amino acids is in the order: glycine, alanine, serine, leucine. Negligible affinity is shown towards ß-alanine, lysine or arginine. γ-Aminobutyric acid (GABA), glutamate and aspartate inhibit glycine binding by a mechanism which is probably non-competitive. This affinity pattern is the same as that of the receptor involved in the transport of small neutral amino acids in brain slices. The sodium and temperature dependence are also characteristic of transport phenomena. The main conclusion of this work is that in brain-cortex only transport receptors, as opposed to synaptic receptors, can be demonstrated for glycine.