Expression of two synapse-enriched glycoproteins, gp65 and gp55, during rat brain development

479-492 and 1 : 100 against GABA, receptor purified from adult bovine'cerebral cortex. The titre is defined as that dilution of serum which would produce an absorbance equal to half of the maximum response. The immune response against purified GABA, receptor, measured by ELISA was blocked by preincubating the serum with the free Cys-a3 479-492 peptide (Fig. la) . A Western blot of purified GABA, from adult bovine cerebral cortex is shown in Fig. 1( b). Antisera raised against Cys-a3 479-492 reacted with a polypeptide with M , 59 000-60 000. This is in contrast with the antiserum raised against the a 1 isoform-specific peptide, a 1 1-1 5, which reacted predominantly with a polypeptide of M , 53 000. Both a 1 and a 3 immunoreactivity were found in GABA, receptor preparations purified from adult bovine cerebral cortex, cerebellum, hippocampus and 12-day-old calf cerebral cortex (Stephenson et al. (1989)). Thus, we have demonstrated the presence of a polypeptide which was predicted from the identification of a cDNA which encodes the a3 subunit. This polypeptide has M , 59 000-60 000 compared with a molecular mass of 52 000 Da calculated from the deduced primary structure; the difference is attributed to N-glycosylation (Levitan et al., 1988). The a 3 subunit was not found previously because in one-dimensional SDS/PAGE it comigrates with the Psubunit ( M , 57 000-58 000). Kirkness & Turner (1988) have recently shown that the P-band can be resolved using antibodies which can be predicted to recognize all three a subunit isoforms. In several species, photoaffinity labelling of the GABA, receptor with the benzodiazepine, [ 3H]flunitrazepam has shown that bands other than the a-subunit incorporated radiolabel (reviewed in Stephenson, 1988). The discovery of isoforms of the a-subunit with different M , may explain these results. The isoform-specific antibodies described here may serve as important tools in the elucidation of their functional importance.