Specific [3H]quinuclidinyl benzilate binding activity in Digitonin-solubilized preparations from bovine brain |
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Authors: | Orest Hurko |
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Institution: | Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014 USA |
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Abstract: | Receptors for the specific muscarinic radioligand 3H]quinuclidinyl benzilate (3H]QNB) were solubilized by digitonin from a particulate preparation of bovine brain without significant alteration in binding affinities for muscarinic antagonists. Electron microscopy and sucrose density gradient sedimentation analysis confirmed the solubility of these receptors in aqueous solutions of digitonin. Equilibrium and kinetic studies of 3H]QNB binding to solubilized receptors indicated that binding was stereoselective and was blocked by muscarinic compounds. These tests permit tentative identification of digitonin-solubilized 3H]QNB binding sites as muscarinic acetylcholine receptors. Digitonin-solubilized receptors were homogeneous with respect to sedimentation behavior and binding affinities for agonist and antagonist drugs, unlike membrane-bound receptors. Enzyme digestion studies and treatment with group-specific reagents indicated that muscarinic receptors are proteins whose binding activity could be disrupted by reduction with dithiothreitol or by modification of sulfhydryl residues. |
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