J. Biol. Chem., Vol. 269, Issue 44, 27384-27393, Nov, 1994
Purification and pharmacological and immunochemical characterization of synaptic membrane proteins with ligand-binding properties of N-methyl-D- aspartate receptors
KN Kumar, KK Babcock, PS Johnson, X Chen, KT Eggeman and EK Michaelis
Department of Pharmacology and Toxicology, University of Kansas, Lawrence 66045.
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.
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