Rinken A
Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
J Biochem. 1996 Jul;120(1):193-200. doi: 10.1093/oxfordjournals.jbchem.a021385.
Sixteen different detergents were studied for solubilization of functional complexes between m2 muscarinic acetylcholine receptors (mAChR) and guanine nucleotide-binding regulatory proteins (G proteins). More than 40% of solubilized mAChR retained their GTP-dependent high affinity for agonist binding after solubilization with sucrose monolaurate, whereas all other detergents studied gave considerably lower solubilization yields or caused the loss of the high affinity for agonist binding. The preformation of mAChR-G protein complexes in membranes revealed that a large excess of G proteins did not increase the portion of high-affinity binding sites, but caused GTP- and Mg2(+)-dependent inhibition of the binding of radioactive antagonists to mAChR. The optimization of detergent concentration and other experimental conditions revealed that up to 47% of the solubilized receptors indicated the GTP-dependent high affinity for agonist binding after mixing solubilized mAChR with purified G proteins in sucrose monolaurate in the presence of Mg2+ and carbachol. These results give the first clear proof of the formation of functional complexes between mAChR and G proteins in solution and indicate that GTP-dependent high-affinity agonist binding is connected to the direct interactions between mAChR and G proteins and that other membrane components are not necessary.
研究了16种不同的去污剂对毒蕈碱型乙酰胆碱受体（mAChR）与鸟嘌呤核苷酸结合调节蛋白（G蛋白）之间功能复合物的增溶作用。在用月桂酸蔗糖酯增溶后，超过40%的增溶mAChR保留了其对激动剂结合的GTP依赖性高亲和力，而所研究的所有其他去污剂的增溶产率都低得多，或导致对激动剂结合的高亲和力丧失。膜中mAChR - G蛋白复合物的预形成表明，大量过量的G蛋白并不会增加高亲和力结合位点的比例，但会导致GTP和Mg2 +依赖性地抑制放射性拮抗剂与mAChR的结合。去污剂浓度和其他实验条件的优化表明，在Mg2 +和卡巴胆碱存在的情况下，将增溶的mAChR与纯化的G蛋白在月桂酸蔗糖酯中混合后，高达47%的增溶受体显示出对激动剂结合的GTP依赖性高亲和力。这些结果首次明确证明了溶液中mAChR与G蛋白之间形成了功能复合物，并表明GTP依赖性高亲和力激动剂结合与mAChR和G蛋白之间的直接相互作用有关，且其他膜成分并非必需。
