caa a22 4500 397555644 CHVBK 20180308164801.0 cr unu---uuuuu 161202e199612 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021546 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021546 Purification and Functional Reconstitution with GTP-Binding Regulatory Proteins of Hexahistidine-Tagged Muscarinic Acetyicholine Receptors (m2 Subtype) [Elektronische Daten] [Mariko Kato Hayashi, Tatsuya Haga] We have expressed human m2 muscarinic acetylcholine receptors tagged with six histidine residues at the carboxy-terminal region in insect cells (Sf9) and purified them using metal-immobilized Chelating Sepharose gels. Co2+-immobilized gels were found to be much more efficient for purification of m2 receptors than gels containing Ni2+ or other metal ions. Twenty-fold purification was attained by a simple, single-step procedure, and approximately 40% of solubilized receptors were recovered as a partially purified preparation with a specific activity of 1.6 nmol/mg of protein. Purified receptors were functionally active in that carbamylcholine stimulated binding of [35S]GTPγS to the G-protein G12 reconstituted in lipid vesicles with purified m2 receptors. The extent of stimulation of [35S]GTPγS binding to G12 by hexahistidine-tagged m2 receptors was essentially the same as that observed for m2 receptors that lack histidine tags. In addition, palmitoylation at the carboxy-terminal region was not impaired by the hexahistidine-tag fusion. The method described in this study should be applicable to the purification of other G-protein-coupled receptors in functionally active form. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence G-protein nationallicence histidine-tag nationallicence muscarinic receptor nationallicence purification nationallicence Hayashi Mariko Kato Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113 aut Haga Tatsuya Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113 aut The Journal of Biochemistry Oxford University Press 120/6(1996-12), 1232-1238 0021-924X 120:6<1232 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021546 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021546 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hayashi Mariko Kato Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113 aut NATIONALLICENCE 700 1- Haga Tatsuya Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/6(1996-12), 1232-1238 0021-924X 120:6<1232 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford