Solubilization of active opiate receptors.

Abstract

Receptors that reversibly bind opiates and opioid peptides have been solubilized from brain and neuroblastoma-glioma hybrid cell NG108-15 membranes. Active receptors are specifically solubilized with a new type of detergent 3-[(3-cholamidopropyl)dimethylammonio}1-propanesulfonate, which is a zwitterionic derivative of cholic acid. The solubilized receptor complexes behave as large molecules with a Stokes radius of 70 A and contain protein as an essential constituent.A necessary first step in the study of the structure and mechanism of action of opiate receptors is their separation, in an active form, from membranes. was from Calbiochem; f3-endorphin (camel) was from Boehringer Mannheim; etorphine-HCI was a gift from Robert Willette (National Institute of Drug Abuse) and naloxone-HCI was donated by Endo Laboratories (New York). Other opiates were gifts from Arthur Jacobson (National Institute of Arthritis, Metabolism, and Digestive Diseases). Sodium cholate and Zwittergent were from Calbiochem; CHAPS was synthesized as described (7). Dithiothreitol and sucrose (ultra-pure reagent) were from Bethesda Research Laboratories (Rockville, MD).Cell Growth. Neuroblastoma-glioma hybrid NG108-15 cells were grown as described (3), except that 850-cm2 tissue culture roller bottles (Corning) were used. The bottles were gassed at the time of planting with 5% C02/95% air, but not upon subsequent feeding.Brain Membrane Preparation. Crude mitochondrial fractions (P2) from rat brain were prepared as described (8) and stored at -800C. Bovine brain membranes were prepared from whole brain, without the cerebellum and medulla, and homogenized in 10 vol of 50 mM Tris-HCl (pH 7.5). After filtration through cheesecloth, the pellet from i centrifugation at 40,000 X g for 30 min was resuspended in a total of 2 vol of 50 mM Tris-HCl (pH 7.5) and frozen at -80'C until use.Solubilization. NG108-15 cell homogenate was centrifuged at 50,000 rpm (160,000 X g) for 30 min at 40C. The pellet was resuspended in 50 mM Tris-HCl (pH 7.5) to a protein concentration of 8-15 mg/ml. The suspension was homogenized on ice in the presence of 10 mM CHAPS with 25 strokes of a ground-glass homogenizer, then centrifuged for 60 min at 160,000 X g in cellulose nitrate tubes. The clear supernatant was separated from a floating cloudy layer and the pellet by puncturing the tube with a 23-gauge needle at a point 1 cm above the pellet and gently withdrawing the liquid into a 10-ml syringe.Aliquots of brain membrane suspensions were diluted with 2 vol of 50 mM Tris-HCl (pH 7.5), 50 ,g of pancreatic trypsin inhibitor per ml (Worthington), 0.1 mM phenylmethylsulfonyl fluoride (Calbiochem), and 0.01% dimethyl sulfoxide (Eastman). The suspension was centrifuged at 165,000 X g for 15 min, and the pellet was resuspended in 2 vol of the above buffer with 10 mM CHAPS to a protein concentration of approximately 12 mg/ml. This suspension was agitated at 40C for 1 hr, then centrifuged at 105,000 X g for 60 min. The clear, slightly yellow supernatant fraction was carefully...

Solubilization and purification of rat liver 5′-nucleotidase by use of a zwitterionic detergent and a monoclonal-antibody immunoadsorbent

“…A similar value of Mr is obtained by gel-exclusion chromatography (results not shown). It is noteworthy that Evans & Gurd (1973) have previously suggested that the mouse liver enzyme may be a dimer of two identical-size subunits (Mr 70000-75 000) in the native state, and that both Naito & Lowenstein (1981) (Hjelmeland, 1980;Simonds et al, 1980)}, suggesting that different zwitterionic detergents will be useful for the purification of different membrane proteins. Similarly, the use of a monoclonal-antibody immunoaffinity step will be specific to the protein of interest.…”

Bimodal regulation of adenylate cyclase

“…For instance, opiate receptors have been solubilized with full retention of their agonist specificity [47]. Another solubilised opiate receptor preparation retained the ability of Na ÷ to modify binding [48], although no effects of GTP were detected.…”

Agonist regulation of the expression of the delta opioid receptor in NG108‐15 cells

“…Delta opiate receptor numbers were measured in membrane fractions by equilibrium radioligand binding analysis with 2 nM [3H]diprenorphine as previously described [20]. Specific binding is defined as the difference between the amount of radioactive ligand bound in the presence and absence of 10/.tM non-radioactive ligand after 10 min incubation at 37°C.…”