Glycoprotein as a Constituent of Purified γ‐Aminobutyric Acid/Benzodiazepine Receptor Complex: Structures and Physiological Roles of Its Carbohydrate Chain

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Abstract
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.sbd.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. Furthermore, the observation that the activation of high-affinity [3H]muscimol binding by benzodiazepines disappeared following .beta.-galactosidase treatment suggests that the carbohydrate chain in the receptor complex may also be involved in the functional coupling between the GABA receptor and the benzodiazepine receptor.