PURIFICATION AND PHARMACOLOGICAL AND IMMUNOCHEMICAL CHARACTERIZATION OF SYNAPTIC MEMBRANE-PROTEINS WITH LIGAND-BINDING PROPERTIES OF N-METHYL-D-ASPARTATE RECEPTORS

A method was developed for the solubilization of approximately 50% of proteins in synaptic membranes that have ligand-binding characteristic s of N-methyl-D-aspartate (NMDA) receptors. Affinity chromatographic s eparation of the solubilized proteins through L-glutamate-derivatized matrices and subsequent elution by NMDA-containing buffers led to the purification of four predominant proteins with estimated sizes of 67-7 0, 53-62, 41-43, and 28-36 kDa. The co purification of NMDA-sensitive L-glutamate binding, dizocilpine-sensitive thienylcyclohexyl piperidin e (TCP)-binding, and strychnine-insensitive glycine-binding proteins w as achieved by this affinity chromatographic procedure. Glutamate, gly cine, and the polyamine spermidine increased both the ''on'' rate and the equilibrium level of [H-3]TCP binding to the isolated proteins. Th e group of proteins eluted by NMDA from the glutamate-derivatized matr ices could be further purified through size exclusion chromatography w ithout loss of ligand binding activity or separation of the NMDA-sensi tive glutamate-binding from the dizocilpine-sensitive TCP-binding prot eins. Polyclonal and monoclonal antibodies to the cloned NMDA receptor protein NMDAR1 did not react with any proteins in the solubilized mem brane proteins or the purified fractions. However, immunoreaction of a ntibodies raised against a glutamate binding protein and a phosphonoam inocarboxylic acid-binding protein indicated that these are two of the major proteins in the purified fractions. These studies indicate that these two proteins might be components of a complex that has some of the characteristics of NMDA receptors and that neuronal membranes may contain varieties of NMDA-like receptors composed of protein subunits that differ from the NMDAR1 and NMDAR2 receptor proteins.