The purification of proteins having properties of an N-methyl-D-aspartate (NMDA) receptor from brain synaptic membranes and characterization of a glycine-binding protein expressed in Escherichia coli.

摘要

N-Methyl- scD-aspartate (NMDA) receptors are a subclass of glutamate receptors having strychnine-insensitive glycine and phencyclidine recognition sites. Proteins of {dollar}sim{dollar}40 kDa having binding sites for glycine were purified from rat brain synaptic membranes by affinity chromatography using 5,7-dichlorokynurenic acid (5,7-DCKA) PharmaLink{dollar}sp{lcub}rm TM{rcub}{dollar} matrices, followed by 8-hydroxyquinoline PharmaLink{dollar}sp{lcub}rm TM{rcub}{dollar} chromatography. These isolated proteins bound ({dollar}sp3{dollar}H) glycine in the presence of strychnine with a high (nM) and low ({dollar}mu{dollar}M) affinity (two sites). scD-Serine, HA-966 and 5,7-DCKA, inhibited ({dollar}sp3{dollar}H) glycine binding to these isolated proteins. Polyclonal antibodies developed against {dollar}sim{dollar}40 kDa proteins electroeluted from SDS-PAGE gels reacted specifically with an {dollar}sim{dollar}60 kDa protein in synaptic membranes following immunoblot analyses. Subsequent purifications led to the isolation of an {dollar}sim{dollar}60 protein which was shown to have MK-801 and ketamine specific ({dollar}sp3{dollar}H) TCP binding. The ({dollar}sp3{dollar}H) TCP binding could be activated by scL-glutamate and/or glycine. It is possible that these proteins are breakdown products of the NMDAR1/R2 proteins or distinct entities. The polyclonal antibodies raised against the {dollar}sim{dollar}40 kDa proteins were used to screen a rat hippocampal cDNA library and a cDNA clone expressing the antigenic protein was identified. The open reading frame of this clone describes a unique protein of 470 amino acids ({dollar}sim{dollar}53 kDa). E. coli transformed with the pBS vector plus p40 cDNA insert expressed a fusion protein ({dollar}sim{dollar}60 kDa) that was recognized by the antibodies to the 40 kDa proteins. The p40 expressed protein was purified from E. coli extracts using 5,7-DCKA PharmaLink{dollar}sp{lcub}rm TM{rcub}{dollar} affinity chromatography. The partially purified protein exhibited two binding sites for ({dollar}sp3{dollar}H) glycine with nM and {dollar}mu{dollar}M affinities. The binding of ({dollar}sp3{dollar}H) glycine was strychnine-insensitive and was displaceable by scD-serine, HA-966, and 5,7-DCKA. The p40 expressed protein exhibited activated MK-801 and ketamine specific ({dollar}sp3{dollar}H) TCP binding. These findings indicate that a synaptic membrane protein of {dollar}sim{dollar}60 kDa size has been isolated having a glycine and PCP recognition site characteristic of NMDA receptors. In addition, a cDNA clone has been identified of which the expressed protein exhibits nearly identical characteristics to the protein isolated from synaptic membranes. These proteins may be a subunit of a putative complex representing a different subtype of brain NMDA receptors.