Immunological identification of multiple alpha-like subunits of the gamma-aminobutyric acidA receptor complex purified from neonatal rat cortex.
Antibodies were prepared against a synthetic peptide corresponding to amino acid sequences 174-203 of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1-subunit. The antibodies recognized this synthetic alpha 1-peptide, but failed to react with the homologous peptide sequence, 170-199, of the bovine beta 1-subunit. On Western blots, anti-alpha 1-subunit antibody recognized a 50-kilodalton (kDa) protein in affinity-purified receptor preparations from adult rat cortex and cerebellum. In receptor purified from neonatal cortex, the anti-alpha 1-antibody reacted with 50-kDa, 53-54-kDa, and 59-kDa proteins. After digestion with endoglycosidase F, these three protein bands retained differing electrophoretic mobilities. The 50-kDa and 59-kDa subunits of affinity-purified neonatal receptor, which were photoaffinity-labeled with [3H]flunitrazepam, were immunoprecipitated to different extents by alpha-subunit antibody. These data suggest the existence in GABAA receptor from neonatal cortex of three proteins (50 kDa, 53 kDa, and 59 kDa) which have immunological homology to alpha 1-subunit of bovine GABAA receptor. The presence of an alpha- and a beta-like subunit with similar mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis may account for the relatively high concentration of protein in the 53-54-kDa band which has been observed in receptor purified from neonatal cortex. The presence of multiple alpha-like subunits may be related to the presence of a relatively high concentration of type II GABA receptor in this tissue.
Sato
T N
TN
Department of Biology, Georgetown University, Washington, District of Columbia 20057.
Neale
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D015153:Blotting, Western; D002540:Cerebral Cortex / Q000378:metabolism*; D006026:Glycoside Hydrolases; D015151:Immunoblotting; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D051381:Rats; D011963:Receptors, GABA-A / Q000276:immunology / Q000302:isolation & purification*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D011963:Receptors, GABA-A; D006026:Glycoside Hydrolases