PURIFICATION OF RECOMBINANT PORCINE M2 MUSCARINIC ACETYLCHOLINE-RECEPTOR FROM CHINESE-HAMSTER OVARY CELLS - CIRCULAR-DICHROISM SPECTRA AND LIGAND-BINDING PROPERTIES

The recombinant porcine m2 muscarinic acetylcholine receptor (rPm2R) f rom Chinese hamster ovary cells has been purified to homogeneity. Two mg of purified rPm2R, with a specific activity of 12 nmol of R-(-)-qui nuclidinyl benzilate/mg of protein, were obtained from 30 mi of packed Chinese hamster ovary cells. The apparent molecular mass (78.5 kDa) a nd specific activity for the rPm2R preparation were the same as that f or the Pm2R purified from atrial tissue, but the yield was 100 times g reater. Purified rPm2R bound agonist and antagonist with the same affi nities and coupled to the inhibitory guanine nucleotide-binding protei n with the same efficiency as the purified native atrial Pm2R. Ligand binding studies were consistant with a single class of antagonist bind ing sites but two subclasses of agonist binding sites. The fraction of rPm2R having high affinity for agonists was increased by mM Mg2+, low detergent concentration, and low temperature. Circular dichroism spec tra obtained for the purified rPm2R with and without agonists were ind istinguishable, but spectra for the antagonist-occupied receptor showe d reproducibly deeper characteristic negative deflections at 208 and 2 20 nm. Secondary structure analysis of the CD spectra predicted 53% al pha-helix for the free receptor and 49% alpha-helix for the R-(-)-quin uclidinyl benzilate-receptor complex.