Glycoprotein as a constituent of purified gamma-aminobutyric acid/benzodiazepine receptor complex: structures and physiological roles of its carbohydrate chain.
The effect of treatments with various enzymes and chemically modifying agents on [3H]muscimol binding to a purified gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex from the bovine cerebral cortex was examined. Treatments with pronase, trypsin, guanidine hydrochloride, and urea significantly decreased the binding of [3H]muscimol, but dithiothreitol, N-ethylmaleimide, reduced glutathione, oxidized glutathione, cysteine, and cystine had no significant effect. These results indicate that the GABA receptor indeed consists of protein, but -SH and -S-S- groups in the protein are not involved in the exhibition of the binding activity. On the other hand, column chromatography using concanavalin A-Sepharose eluted protein having [3H]muscimol binding activity and staining of glycoprotein using an electrophoresed slab gel indicated the existence of two bands originating from the subunits of the GABA/benzodiazepine receptor complex. Furthermore, treatments with various glycosidases such as glycopeptidase A, beta-galactosidase, and alpha-mannosidase significantly increased the binding of [3H]muscimol. These results strongly suggest that GABA/benzodiazepine receptor complex is a glycoprotein and that its carbohydrate chain may be a hybrid type. Treatment with beta-galactosidase resulted in the disappearance of the low-affinity site for [3H]muscimol binding and in an increase of Bmax of the high-affinity site, without changing the KD value. These results suggest that the carbohydrate chain in the receptor complex may have a role in exhibiting the low-affinity binding site for GABA. The observation that the enhancement of [3H]muscimol binding by treatments with beta-galactosidase and glycopeptidase A were much higher than that with alpha-mannosidase may also indicate a special importance of the beta-galactosyl residue in the inhibition of GABA receptor binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000494:pharmacology; D006023:Glycoproteins / Q000032:analysis*; D006026:Glycoside Hydrolases / Q000494:pharmacology; D008970:Molecular Weight; D009118:Muscimol / Q000378:metabolism; D011963:Receptors, GABA-A / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism
D006023:Glycoproteins; D011963:Receptors, GABA-A; D009118:Muscimol; D006026:Glycoside Hydrolases; D010450:Endopeptidases