Strychnine-insensitive binding of [3H]glycine to synaptic membranes in rat brain, treated with Triton X-100

Binding of radiolabelled glycine, a putative inhibitory neurotransmitter in mammalian lower central structures, was examined by using the synaptic membranes of the brain of rat, treated with Triton X-100. This treatment with Triton markedly potentiated the binding of [3H]glycine detected at 2 degrees C and 30 degrees C. However, this binding was not affected by three different convulsants, strychnine, picrotoxin and bicuculline. The binding was saturable at 2 degrees C, with increasing concentrations of [3H]glycine up to 1 microM. Scatchard analysis revealed that the binding sites consisted of a single component with a Kd of 202 nM and a Bmax of 1.74 pmol/mg protein. The binding was inhibited, not only by various amino acids structurally related to glycine, including D- and L-serine and D-, L- and beta-alanine, but was also eliminated by some peptides containing glycine, such as gamma-D- and gamma-L-glutamylglycine, glycine methylester and N-methyl-glycine. In addition, the strychnine-insensitive binding of [3H]glycine was significantly abolished by numerous quinoxaline antagonists for excitatory amino acid receptors in the brain. These results suggest that synaptic membranes of brain, treated with Triton X-100, are useful to detect the strychnine-insensitive binding of [3H]glycine and superior to untreated membranes in terms of the freedom from the confounding effects of some endogenous amino acids.