PMID:9003035

Citation	Chazot, PL and Stephenson, FA (1997) Biochemical evidence for the existence of a pool of unassembled C2 exon-containing NR1 subunits of the mammalian forebrain NMDA receptor. J. Neurochem. 68:507-16
Abstract	Optimum conditions were determined for the solubilisation of native NMDA receptors of adult mammalian brain with the retention of [3H]MK-801 radioligand binding activity. The most efficient conditions were 1% Triton X-100/1 M NaCl. The efficiency of solubilisation was as follows: cloned NMDA receptors expressed in mammalian cells > forebrain receptors > cerebellar receptors. Triton X-100/1 M NaCl-solubilised forebrain NMDA receptors had a molecular size of 710,000 daltons, but significant NR1 immunoreactivity (41%) migrated as a monomer of 125,000 daltons. Immunoaffinity purification of NMDA receptors from forebrain by anti-NR1 911-920 antibody affinity chromatography from 1% Triton X-100/1 M NaCl solubilised extracts yielded purification of the NR1 Mr 120,000 immunoreactive species, but no detectable NR2A or NR2B immunoreactivity. Immunoprecipitation of NMDA receptors from Triton X-100/1 M NaCl extracts with anti-NR1 911-920 antibodies also resulted in precipitation of NR1 subunits, but with no detectable NR2A or NR2B subunits. In contrast, by immunoprecipitation with anti-NR1 17-35 antibodies, which recognise all forms of NR1, NR1, NR2A, and NR2B immunoreactivities were detected in the immune pellets. Similarly, a co-association of NR1, NR2A, and NR2B subunits was demonstrated following extraction of forebrain membranes with 1% sodium deoxycholate (pH 9) and purification by anti-NR1 911-920 antibody affinity chromatography. These results are consistent with the identification of a pool of unassembled C2 exon-containing NR1 subunits, i.e., NR1-1a, NR1-1b, NR1-2a, and NR1-2b, selectively solubilised by 1% Triton X-100/1 M NaCl.
Links	PubMed
Keywords	Age Factors; Alternative Splicing/physiology; Animals; Antibody Specificity; Cerebellum/chemistry; Chromatography, Affinity; Chromatography, Gel; Detergents; Exons/physiology; Gene Expression/physiology; Humans; Kidney/cytology; Mice; Molecular Weight; Precipitin Tests; Prosencephalon/chemistry; Receptors, N-Methyl-D-Aspartate/chemistry; Receptors, N-Methyl-D-Aspartate/genetics; Receptors, N-Methyl-D-Aspartate/immunology; Solubility; Transfection
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