A SYNAPTIC MEMBRANE GLYCINE-BINDING, GLUTAMATE-BINDING AND THIENYLCYCLOHEXYLPIPERIDINE-BINDING PROTEIN - ISOLATION AND IMMUNOCHEMICAL CHARACTERIZATION

Antibodies raised against a 43 kDa component of a complex of synaptic membrane proteins with ligand binding sites characteristic of glutamat e/N-methyl-D-aspartate (NMDA) receptors, were used previously to clone a cDNA For a glycine-, glutamate-, and thienylcyclohexylpirperidine ( TCP)-binding protein, pGlyBP (Kumar ei al., Biochem. Biophys. Res. Com mun. 216, 390-398, 1995). In the present studies, the antibodies were shown to label a 60 kDa protein, in synaptic membranes, that was relat ively hydrophilic as demonstrated by its predominant separation in the detergent-depleted phase of proteins solubilized with Triton X-114. A 55-60 kDa protein was purified From rat brain synaptic membranes by c hromatographic separation through matrices derivatized with 5,7-di-chl orokynurenic acid (5,7-DCK) followed by chromatography on a matrix der ivatized with 8-hydroxyquinoline (8-OHQ). The isolated fractions were highly enriched in strychnine-insensitive [H-3]glycine; NMDA- and glut amate-sensitive L-[H-3]glutamate, and MK-801-sensitive [H-3]TCP bindin g sites. The purified protein bound [H-3]glycine with a stoichiometry of 1.1-1.2 mol glycine per mol protein and exhibited both high (K-D = 280 nM) and low affinity (K-D = 30 mu M) glycine binding sites. Glycin e binding was inhibited by D-serine and R-(+)-3-amino-1-hydroxypyrroli din-2-one (R-(+)-HA-966). The K-D values for high and low affinity Sit es of glycine binding as well as those for the inhibition by R-(+)-HA- 966 were very similar to the K(D)s for glycine binding to the expresse d pGlyBP. Both L-glutamate and glycine activated [H-3]TCP binding to t he isolated proteins, but with relatively low affinity. The anti-43 kD a antibodies reacted strongly with the 55-60 kDa protein. Based on the se results, it appears that the 60 kDa glycoprotein in brain synaptic membranes described in the present study is the same protein as the cl oned pGlyBP. Copyright (C) 1996 Elsevier Science Ltd