Role of sulfhydryl groups in Y2 neuropeptide Y receptor binding activity.
Benextramine, a tetramine disulfide, irreversibly inhibits neuropeptide Y (NPY) binding to the 50-kDa Y2 NPY receptor in bovine hippocampus (Li, W., MacDonald, R. G., and Hexum, T. D. (1991) Eur. J. Pharmacol. 207, 89-91). Evidence is presented that this inhibition occurs through a thiol-disulfide exchange. Treatment of bovine hippocampal membranes with benextramine inhibited NPY affinity cross-linking to the 50-kDa receptor. This inhibition of labeling was not affected by washing the membranes, but could be completely reversed by the addition of several thiol reducing reagents, including reduced glutathione, beta-mercaptoethanol, and cysteine. Benextramine inhibited 70% of NPY-specific labeling and was much more effective than other sulfhydryl reactive agents, such as oxidized glutathione, cystamine, and 5,5'-dithio-bis(2-nitrobenzoic acid). Furthermore, the sulfhydryl-modifying agents N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid specifically decreased NPY affinity labeling. Finally, NPY labeling of the 50-kDa receptor was reduced by the heavy metal ions Zn2+, Cu2+, and Hg2+. Preincubation with NPY prevented Y2 receptors from being inactivated by either 400 microM N-ethylmaleimide or 1 mM benextramine. These results suggest that one or more benextramine-sensitive sulfhydryl groups on the Y2 receptor are important for NPY binding activity.
Department of Pharmacology, University of Nebraska Medical Center, Omaha 68198.
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Li
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MacDonald
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Hexum
D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D002417:Cattle; D003538:Cystamine / Q000031:analogs & derivatives / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006624:Hippocampus / Q000378:metabolism; D066298:In Vitro Techniques; D008566:Membranes / Q000378:metabolism; D009478:Neuropeptide Y / Q000378:metabolism*; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D000317:Adrenergic alpha-Antagonists; D000345:Affinity Labels; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter; D013438:Sulfhydryl Compounds; C029998:benextramine; D003538:Cystamine