Solubilization and sedimentation analysis of muscarinic acetylcholine receptors.
To investigate if G-protein-receptor interactions can be characterized using sucrose density gradients (SDG) we have determined the experimental conditions for muscarinic acetylcholine receptor (mAChR) solubilization and analysis on SDG. Solubilization of 65-80% of [3H]QNB bound mAChR was accomplished with 1% of detergent. Analysis of solubilized receptors on SDG containing 0.4 M KCl and 0.1% detergent demonstrated that the physical properties of the receptor-detergent complexes are influenced by the solubilizing detergent as well as detergents included in the SDG. Neither GTP gamma S nor NaF and AlCl3 altered the sedimentation properties of mAChR, suggesting that the solubilized mAChR is no longer associated with G-protein under these conditions. Receptors bound to [3H]oxotremorine and [3H]QNB had similar sedimentation properties, suggesting that, once solubilized, mAChRs do not remain associated with G-proteins. Covalent labeling with [3H]PrBCM followed by solubilization and analysis on SDS-gel electrophoresis demonstrated the presence of intact receptor molecule. These observations suggest that the changes in the sedimentation properties of detergent-receptor complexes are independent of G-protein interactions and are influenced by the nature of the detergent associated with the mAChR during analysis.
Traish
A
A
Department of Biochemistry, Boston University, School of Medicine, MA 02118.
Moran
E
E
D000818:Animals; D002793:Cholic Acids / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D005374:Filtration; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008297:Male; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013552:Swine
D002793:Cholic Acids; D011976:Receptors, Muscarinic; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011813:Quinuclidinyl Benzilate; D019204:GTP-Binding Proteins; D004072:Digitonin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate