Solubilization and reconstitution of alpha 2-adrenergic receptors from rat and calf brain.
Sodium cholate and digitonin were used to solubilize alpha2-adrenergic receptors from rat and calf brain. Sodium cholate extracted 40-50% of the membrane protein and 25-30% of the binding capacity. Digitonin extracted only 20-30% of the membrane protein and only 10-15% of the binding capacity of the native membranes. Both detergents were removed by dialysis in the presence of phospholipids, and the solubilized protein was precipitated upon addition of poly(ethyleneglycol) and magnesium. In the solubilization/reconstitution process no purification of the alpha2-adrenergic receptor was obtained, most probably due to its inactivation by the solubilization conditions. The reconstituted protein(s) tested for binding properties, using p-[3H]aminoclonidine and/or [3H]clonidine, maintained the pharmacological profile of the native alpha2-adrenergic receptor. The potency order of various alpha2-agonists and alpha2-antagonists as well as their stereoselectivity were identical to those of the native alpha2-receptor. Specific receptor binding decreases in the presence of the guanyl nucleotides GTP or guanosine 5'-[beta, gamma-imido]-triphosphate but not ATP, thus indicating a co-solubilization of GTP regulatory components (stimulatory protein Ns or inhibitory protein Ni or both). Adenylate cyclase activity of the reconstituted preparation is stimulated threefold by sodium fluoride, suggesting the presence of both Ns-protein and the catalytic unit (C) in the reconstituted protein(s).
D000262:Adenylyl Cyclases / Q000302:isolation & purification; D000818:Animals; D001923:Brain Chemistry; D002384:Catalysis; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D019826:Cholic Acid; D002793:Cholic Acids; D004072:Digitonin; D005260:Female; D008297:Male; D011485:Protein Binding; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000302:isolation & purification*; D012995:Solubility
D002793:Cholic Acids; D011942:Receptors, Adrenergic, alpha; D000262:Adenylyl Cyclases; D019826:Cholic Acid; D004072:Digitonin