Article date: 1986/3/1
PubMed ID: 3005837
Journal name: Molecular pharmacology (ISSN: 0026-895X)
The ability to photolabel benzodiazepine receptors from various regions of the rat brain with 3H-flunitrazepam has allowed for the structural examination of these receptors by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. Results for all regions studied revealed the labeled receptor to consist of a single major band of radioactivity with the apparent molecular weight of approximately 50,000. Under our conditions of labeling we do not significantly label any higher molecular weight forms of the receptor. Exposure of the benzo-diazepine receptors to either of the glycosidases neuraminidase (N) and endoglycosidase-H (E) results in the specific removal of sialic acids and complete asparagine-linked carbohydrate moieties, respectively. This type of structural modification of the receptor resulted in an apparent decrease in the molecular weight, as determined by increased mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis, for all regions examined (cortex + N + E, 8,000-10,000; hippocampus + N, 7,000, + E, 12,000; cerebellum + N, none, + E, 4,000). These results point to a heterogeneity in the posttranslational glycosylation of the benzodiazepine receptor that may be due to brain region-specific differences in glycosylation. The removal of these carbohydrate moieties alters the binding of agonists and antagonists to the benzodiazepine receptor. Cortical agonist binding following either glycosidase treatment resulted in no apparent shift in the Kd but a significant decrease in the Bmax. The Bmax change may be the result of a large decrease in affinity or denaturation of a subpopulation of benzodiazepine receptors. Antagonist binding also showed no apparent Kd shift but a significant increase in the Bmax. The increase may have resulted from the activation of “hidden” benzodiazepine receptors or a shift of low affinity sites to sites of higher affinity. Cerebellar agonist or antagonist binding was not altered, in terms of either Kd or Bmax, by either enzyme treatment, correlating well with the small amount of carbohydrate removal seen following such treatments. The ability of these enzymes to modify the apparent molecular weight of the benzodiazepine receptors and the strong correlation to altered ligand binding, in a regional specific manner, generally parallel the description given of type 1 and type 2 benzodiazepine receptors.
Author List: Sweetnam P M, Tallman J F
Publication Types: Journal Article
Substances mentioned in the article: Benzodiazepinones; Carbohydrates; Receptors, GABA-A; Sialic Acids; Tritium; Muscimol; Flumazenil; gamma-Aminobutyric Acid; Flunitrazepam; Asparagine; Neuraminidase; Acetylglucosaminidase; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Pentobarbital;
Mesh terms: Acetylglucosaminidase/pharmacology; Animals; Asparagine/analysis; Benzodiazepinones/metabolism; Brain Chemistry; Carbohydrates/analysis; Electrophoresis, Polyacrylamide Gel; Flumazenil; Flunitrazepam/metabolism; Kinetics; Male; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Molecular Weight; Muscimol/pharmacology; Neuraminidase/pharmacology; Pentobarbital/pharmacology; Rats; Rats, Inbred Strains; Receptors, GABA-A/analysis; Sialic Acids/analysis; Tritium; gamma-Aminobutyric Acid/pharmacology;