Solubilization and hydrodynamic properties of pig atrial muscarinic acetylcholine receptor in dodecyl beta-D-maltoside.
The pig atrial muscarinic acetylcholine receptor (mAcChR) has been solubilized from the membrane-bound state in high yield and in stable conformation by the non-ionic detergent dodecyl beta-D-maltoside (DBM). The yield and selectivity for receptor solubilization is dependent on the detergent/protein ratio during extraction. Extraction at 2 mg of DBM/mg of protein gave a 75% yield of solubilized receptor with a 1.5-fold enrichment. A double-extraction procedure, in which non-receptor protein was first extracted at 0.4 mg of DBM/mg of protein and mAcChR was selectively solubilized by a second extraction at 0.35 mg of DBM/mg of protein, gave a 50% overall yield and a 2.8-fold enrichment. Both preparations had a half-life of about 20 days on ice without addition of muscarinic ligands. Receptor stability was decreased by the presence of cations, particularly bivalent cations, and enhanced by the agonist carbachol. Dissociation constants for the interaction of the DBM-solubilized receptor with the antagonist L-quinuclidinyl benzilate (Kd = 223 pM) and the agonist carbachol (Kd = 100 microM) were similar to those for the digitonin/cholate-solubilized receptor. Pig atrial mAcChR purified in digitonin/cholate and exchanged into DBM displayed reliable hydrodynamic behaviour during sucrose density sedimentation in gradients of 2H2O and H2O and during gel filtration in Sephacryl S-300. DBM is thus the first detergent which will solubilize a stable form of the ligand-free mAcChR in yields similar to those with digitonin, and is the only stabilizing detergent thus far suitable for hydrodynamic studies. DBM is also likely to be similarly useful in studying other membrane proteins for which digitonin has been the solubilizing detergent of choice.
Peterson
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GL
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331.
Rosenbaum
L C
LC
Schimerlik
M I
MI
D000818:Animals; D002217:Carbachol / Q000378:metabolism; D002462:Cell Membrane / Q000032:analysis; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D005960:Glucosides / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism*; D008024:Ligands; D008274:Magnesium; D015636:Magnesium Chloride; D008565:Membrane Proteins / Q000032:analysis; D008970:Molecular Weight; D009206:Myocardium / Q000032:analysis*; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013552:Swine
D005960:Glucosides; D006027:Glycosides; D008024:Ligands; D008565:Membrane Proteins; D011976:Receptors, Muscarinic; D015636:Magnesium Chloride; D011813:Quinuclidinyl Benzilate; C040358:dodecyl maltoside; D002217:Carbachol; D008274:Magnesium