Graduate Thesis Or Dissertation
 

Structural characterization of the cardiac muscarinic acetylcholine receptor

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  • The physical properties of the purified cardiac muscarinic acetylcholine receptor have been examined by D₂O/H₂O sucrose gradient sedimentation and sephacryl S-300 gel filtration in Triton X-405 and dodecyl-β-maltoside. From the sedimentation experiments, the partial specific volume of the receptor-Triton X-405 complex were 0.813 cm³/g and 5.30 S, respectively and the receptor-dodecyl-β-maltoside were 0.783 cm³/g and 9.30 S, respectively. An estimate of the molecular weight of the mAcChR in Triton x-405 and dodecyl-β-maltoside was 143,000 and 255,000, respectively. Gel filtration in Triton X-405 and dodecyl-β-maltoside gave an estimate of Stokes radii 4.29 nm and 5.26, respectively with an estimated molecular weight of 137,000 and 256,000, respectively. Frictional ratios (f/f₀) of 1.21 and 1.22 were calculated in Triton X-405 and dodecyl-β-maltoside, respectively. The amount of bound Triton X-405 was estimated at 1.021 g/g mAcChR and bound dodecyl-β-maltoside at 2.538 g/g mAcChR giving apparent molecular weights of 70,900 and 72,032, respectively for the uncomplexed mAcChR. After correction for carbohydrate, these values are within 1.5% and 3.0%, respectively of the molecular weight of the mAcChR calculated from sequence data. Biochemical characterization of detergent solubilized and purified mAcChR was attempted using sucrose gradient sedimentation and ligand binding studies. Sucrose gradients in the absence of ligands with both detergent solubilized and purified receptor show a relatively homogenous population of mAcChRs, while in the presence of saturating agonist (carbachol) concentration about 25% of the mAcChR peak migrates to a higher apparent molecular weight. This high molecular weight peak was reduced when the detergent-solubilized receptor is extracted in the presence of GppNHp or when the carbachol is removed by dialysis, suggesting an interaction between mAcChR and inhibitory guanine nucleotide binding protein (Gi). The high molecular weight peak in purified receptor preparation was eliminated when agonist treated receptor was reduced with dithiothreitol, suggesting a dimerization of mAcChR via intra or intermolecular disulfide bonds. In ligand binding studies, antagonists showed about the same affinity for the purified mAcChR as was found for the membrane bound and detergent solubilized preparations, while agonist binding is somewhat variable possibly due to receptor dimerization or aging. Purified mAcChR was phosphorylated with the catalytic subunit of cAMP-dependent kinase. Phosphorylation of the mAcChR in digitonin/cholate showed a stoichiometry of about 0.70 mol Pi/mol mAcChR and resulted in a loss of ligand binding sites that was reversed by treatment with the phosphatase calcineurin in the presence of calcium and calmodulin. Upon reconstitution into lipids, the apparent stoichiometry was increased by about fifteen fold to 10 mol Pi/mol of ligand binding sites. Carbachol-stimulated covalent incorporation of phosphate was found only in the reconstituted system in the presence of G[subscript i], suggesting that the agonist-stimulated phosphorylation may result in part from a unique receptor conformation that occurs in other association with this protein. Ligand binding studies indicated that phosphorylation of the mAcChR in the detergent-solubilized or reconstituted state did not affect its interaction with carbachol or L-quinuclidinyl benzilate in vitro.
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