Immunolocalization and biochemical characterization of N-methyl-D-aspartate receptor subunit NR1 from rat brain

The N-methyl-D-aspartate (NMDA) receptor subunit NR1 gene can produce eight isoforms in rat brain. A novel methodology for purifying NMDA receptor NR1 subunit from rat brain is reported here using chicken polyclonal antibodies (IgYs) against synthetic peptides corresponding to N1, C1 and C2' cassettes. The isolated protein was recognized by produced IgYs and commercial anti-NR1 IgGs, shown by MALDI-TOF MS a MW = 131,192 Da (glycosylated form); the enzymatically deglycosylated protein revealed a MW = 102,754 Da. The NMDA receptor NR1 subunit was characterized as being a heavily N-glycosylated protein. The isoelectric point was determined (6.3) as being different from that predicted for any of the isoforms (7.9-9.02). Attempts to separate the isoforms from the purified NR1 were unsuccessful, indicating the presence of just one isoform (NR1(111)). Immunohistochemistry on hippocampus regions CA1, CA3 and Dentate gyrus with anti-N1, anti-N2 and anti-C2' IgYs showed different staining intensity, depending upon the antibody assayed.