Abstract
A method was developed for the solubilization of approximately 50% of proteins in synaptic membranes that have ligand-binding characteristics of N-methyl-D-aspartate (NMDA) receptors. Affinity chromatographic separation 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 67-70, 53-62, 41-43, and 28-36 kDa. The co-purification of NMDA-sensitive L-glutamate binding, dizocilpine-sensitive thienylcyclohexyl piperidine (TCP)-binding, and strychnine-insensitive glycine-binding proteins was achieved by this affinity chromatographic procedure. Glutamate, glycine, and the polyamine spermidine increased both the "on" rate and the equilibrium level of [3H]TCP binding to the isolated proteins. The group of proteins eluted by NMDA from the glutamate-derivatized matrices could be further purified through size exclusion chromatography of the NMDA-sensitive glutamate-binding from the dizocilpine-sensitive TCP-binding proteins. Polyclonal and monoclonal antibodies to the cloned NMDA receptor protein NMDAR1 did not react with any proteins in the solubilized membrane proteins or the purified fractions. However, immunoreaction of antibodies raised against a glutamate-binding protein and a phosphonoaminocarboxylic 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.
Highlights
L-Glutamic acid is theprincipal excitatory neurotransmitter in the mammalian central nervous system (Fonnum, 1984; Cotman and Monaghan, 1987)
The cDNAs for these receptor proteins were identified by functional expression cloning
A group of proteins previously purified from rat brain has recognition sites for NMDA receptor ligands such as L-glutamate, NMDA, glycine, 2-amino-5-phosphonopentanoate (2-AP5), thienylcyclohexyl piperidin(eTCP)p,hencyclidine (PCP), and dizocilpine (MK-801) (Ikin et al, 1990)
Summary
Immunochemical Characterization of Synaptic Membrane Proteins and Isolated Protein Fractions-The synaptic membrane proteins, solu-. Purification of Glutamate and NMDA-binding Proteins by Affznity bilized membrane proteins, and all protein fractions isolated through Chromatography-The isolation of synaptic membranes from 2 whole chromatographicprocedures weresubjectedto SDS-PAGE accordingto rat brain homogenateswas performed as described previouslyWhen et the procedures of Park and Labbe (1989). All protein samples were al., 1988).The purified synaptic membranes were solubilizedin 10 m~ dissolvedin sample buffer that contained 2% (w/v) SDS, 25m0M dithiopotassium phosphate buffer, pH 7.4, that contained four protease in- threitol, and 500 m~ iodoacetamide. The solubilizedmem- tively (see Park and Labb (1989)).Western blot analyses of the brane proteins were loaded onto a ReactiGel column that had been proteins isolated at various steps of the purification scheme used were reacted with L-glutamate (Wanget al., 1992),and fractions were eluted performed accordingto the procedures described previousl(yChenet al., by the successiveintroduction of 30 m~ KC1,5 m~ NMDA, and 1M KC1 1988; Eaton et al, 1990). Brane proteins (400-500 pg) or purified proteins (400-600 ng) were incubated in Microfuge tubes with 40 mM Tris/SO, buffer, pH 7.2,that
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