Glycerol, sodium phosphate, and sodium chloride permit the solubilization and partial purification of rat hepatic alpha 1-receptors by 3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate.
CHAPS [3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate], a zwitterionic detergent, has been used to solubilize the rat hepatic alpha 1-adrenergic receptor. Although the use of this detergent alone permitted a poor receptor solubilization, the inclusion of sodium phosphate, sodium chloride, and glycerol to the medium allowed 30% of the binding activity observed in plasma membranes to be recovered. Binding of the selective alpha 1-adrenergic antagonist, [3H]prazosin, by the solubilized preparation was saturable and of high affinity. In addition, binding of the radioligand was inhibited by a variety of adrenergic agents with affinity, specificity, and stereoselectivity comparable to that observed in plasma membranes. The use of glycerol in the solubilization medium permitted recovery of the solubilized receptor in a stable form (T1/2 = 72 h at 4 degrees C). Sequential affinity and size-exclusion gel chromatography allowed a 1000-fold purification of the solubilized receptor. The Stokes' radius and the apparent molecular mass of the purified receptor-Chaps complex (48.4 A and 160,000 Da, respectively), determined by gel filtration chromatography, were similar to those previously obtained for the rat hepatic alpha 1-receptor purified after solubilization with the nonionic detergent digitonin. These data indicate that the combination of Chaps, sodium phosphate, sodium chloride, and glycerol permitted the solubilization and partial purification of hepatic alpha 1-receptor in an active and stable form. The use of this technique might be useful for the solubilization of other membrane-bound proteins by Chaps whose biophysical characteristics make it an ideal detergent for reconstitution experiments.
Parini
A
A
Cellular and Molecular Research Laboratory, Cardiac Unit, Massachusetts General Hospital, Boston 02114.
Graham
R M
RM
D000818:Animals; D002462:Cell Membrane / Q000032:analysis; D002793:Cholic Acids / Q000494:pharmacology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005260:Female; D005990:Glycerol / Q000494:pharmacology*; D008099:Liver / Q000032:analysis*; D010710:Phosphates / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D011942:Receptors, Adrenergic, alpha / Q000302:isolation & purification*; D012965:Sodium Chloride / Q000494:pharmacology*; D012995:Solubility
D002793:Cholic Acids; D010710:Phosphates; D011942:Receptors, Adrenergic, alpha; D012965:Sodium Chloride; D005990:Glycerol; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C018279:sodium phosphate