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) from Chinese hamster ovary cells has been purified to homogeneity. Two mg of purified rPm2R, with a specific activity of 12 nmol of R-(-)-quinuclidinyl benzilate/mg of protein, were obtained from 30 ml of packed Chinese hamster ovary cells. The apparent molecular mass (78.5 kDa) and specific activity for the rPm2R preparation were the same as that for the Pm2R purified from atrial tissue, but the yield was 100 times greater. Purified rPm2R bound agonist and antagonist with the same affinities and coupled to the inhibitory guanine nucleotide-binding protein with the same efficiency as the purified native atrial Pm2R. Ligand binding studies were consistent with a single class of antagonist binding 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 spectra obtained for the purified rPm2R with and without agonists were indistinguishable, but spectra for the antagonist-occupied receptor showed reproducibly deeper characteristic negative deflections at 208 and 220 nm. Secondary structure analysis of the CD spectra predicted 53% alpha-helix for the free receptor and 49% alpha-helix for the R-(-)-quinuclidinyl benzilate-receptor complex.
Peterson
G L
GL
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331, USA.
Toumadje
A
A
Johnson
W C
WC
Schimerlik
M I
MI
D000818:Animals; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D003902:Detergents; D006801:Humans; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D017433:Protein Structure, Secondary; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine; D013696:Temperature
D003902:Detergents; D008024:Ligands; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D011813:Quinuclidinyl Benzilate; D008274:Magnesium