Evidence for the existence of differential O-glycosylated alpha 5-subunits of the gamma-aminobutyric acidA receptor in the rat brain.
Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the gamma-aminobutyric acidA (GABAA) receptor alpha 5-subunit. These anti-peptide alpha 5(2-10) or anti-peptide alpha 5(427-433) antibodies reacted specifically with GABAA receptors purified from the brains of 5-10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABAA receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABAA receptors with N-Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABAA receptors with neuraminidase and O-Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide alpha 5(2-10) and the anti-peptide alpha 5(427-433) antibody. These results indicate the existence of at least two different alpha 5-subunits of the GABAA receptor that differ in their carbohydrate content. In contrast to other alpha- or beta-subunits of GABAA receptors so far investigated, at least one of these two alpha 5-subunits contains O-linked carbohydrates.
Department of Biochemical Psychiatry, University Clinic for Psychiatry, Vienna, Austria.
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Sieghart
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Buchstaller
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Fuchs
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Höger
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Adamiker
D000818:Animals; D001921:Brain / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000494:pharmacology; D006031:Glycosylation; D007536:Isomerism; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D011963:Receptors, GABA-A; D006026:Glycoside Hydrolases; C020668:glycanase