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 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.
Kumar
K N
KN
Department of Pharmacology and Toxicology, University of Kansas, Lawrence 66045.
Babcock
K K
KK
Johnson
P S
PS
Chen
X
X
Eggeman
K T
KT
Michaelis
E K
EK
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D016291:Dizocilpine Maleate / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D013570:Synaptic Membranes / Q000737:chemistry*
D005971:Glutamates; D008024:Ligands; D016194:Receptors, N-Methyl-D-Aspartate; D016291:Dizocilpine Maleate