Glycoprotein Properties of Muscarinic Acetylcholine Receptors from Bovine Cerebral Cortex

Muscarinic acetylcholine receptors from bovine cerebral cortex were solubilized in digitonin for the subsequent determination of several biochemical properties. The digitonin-solubilized receptors were representative of the entire membrane-bound population of muscarinic receptors with respect to carbohydrate content, isoelectric point, and molecular weight. The glycoprotein nature of the solubilized receptors was demonstrated by their quantitative binding to wheat germ agglutinin-agarose. The presence of a bound antagonist did not decrease the extent of receptor binding to this lectin. Treatment of receptors with neuraminidase to remove N-acetylneuraminic acid residues reduced binding to wheat germ agglutinin-agarose by 40%; further treatment with endoglycosidases D and H, to remove all N-linked carbohydrate, decreased binding by a total of 67%. Removal of N-acetylneuraminic acid residues had no effect on agonist binding properties of the membrane-bound receptors. The carbohydrate-specific enzymes were further used to assess the contribution of carbohydrate to the isoelectric point and molecular weight of the receptor. Muscarinic receptors solubilized in either digitonin or Triton X-100 focused as one major species with a pI of 4.3. Neuraminidase treatment resulted in an increase of 0.17 units in the pI of the receptor. Muscarinic receptors labeled with the covalent muscarinic antagonist propylbenzilylcholine mustard migrated as a single major polypeptide with a molecular weight of 73,000 on sodium dodecyl sulfate-urea-polyacrylamide gels. The exclusion of urea from these gels severely retarded receptor mobility, indicating a strong tendency for aggregation of receptors in SDS. Removal of N-linked carbohydrate by endoglycosidase treatment reduced the molecular weight of the antagonist binding polypeptide by no more than 5%. These results demonstrate the glycoprotein nature of muscarinic receptors from mammalian cerebral cortex and provide evidence for their heterogeneity with respect to carbohydrate content.     

Glycoprotein Properties of the Solubilized Atrial Muscarinic Acetylcholine Receptor

Abstract: The muscarinic acetylcholine receptor from porcine atria exhibits sialoglycoprotein characteristics based on its sensitivity to neuraminidase digestion and its ability to interact specifically with lectin affinity resins when solubilized with a digitonin/cholate mixed detergent system. Differential lectin binding properties of the neuraminidase-treated and untreated receptor suggest that high-affinity binding to immobilized wheat germ agglutinin is accomplished through the presence of both terminal sialic acid and internal N -acetylglucosamine or its β(1→4)-linked oligomers.     

Presence of Various Carbohydrate Moieties Including β-Galactose and N-Acetylglucosamine Residues on Solubilized Porcine Brain Neurokinin-1/Substance P Receptors

Neurokinin-1 (NK-1)/substance P (SP) receptors were solubilized using 10 mM 3-[( cholamidopropyl)-dimethylammonio]-1- propanesulfate from porcine striatal membranes (solubilization yield, 80%). In solubilized preparations, [3H]SP apparently bound to a single class of high-affinity sites (KD = 0.82 +/- 0.13 nM) as in membrane homogenates. The ligand selectivity pattern observed in both membrane and solubilized receptor preparations indicated that [Sar9,Met(O2)11]SP = SP much greater than senktide = [Nle10]neurokinin A. This suggests the selective labeling of the NK-1 receptor class in both assays. Solubilized receptors were retained on agarose-coupled lectins that bind N-acetylglucosamine-galactose and beta-galactose (Ricinus communis I and Ricinus communis II), mannose (concanavalin A and lentil), and N-acetylglucosamine (wheat germ agglutinin) but not on lectins binding fucose (Lotus A) and N-acetylgalactosamine (Doli-chos biflorus A). Thus, it appears that porcine brain NK-1/SP receptors are enriched with various carbohydrate moieties, beta-galactose and N-acetylglucosamine-galactose residues being especially abundant. This situation is rather different from that in various other members of the rhodopsin seven-transmembrane receptor superfamily.     

Solubilization in good yield of active opiate binding sites from mammalian brain

Active opiate binding sites have been solubilized from mammalian brain cell membranes. The presence of 0.5-0.1 M NaCl during treatment of membranes from rat brain, human frontal cortex, and bovine corpus striatum with glycodeoxycholate or digitonin resulted in the extraction of active opiate binding sites in yields ranging up to 43%. The criteria for solubility of the sites were their inability to sediment at 10(5) x g after 2 hr and their apparent molecular weight of 3- 4 x 10(5) as determined by gel filtration. The receptors in solution resemble the membrane-bound sites with respect to saturability, stereo-specificity, sensitivity to heat and reagents, and high affinity for opioid ligands. The interaction of solubilized sites with immobilized lectins was used to demonstrate the glycoprotein nature of the opiate receptor. Soluble receptors from all species studied were retained by wheat germ agglutinin(WGA)-agarose and could be specifically eluted with N-acetylglucosamine. No retention of solubilized material was observed with eight other lectins examined, including horseshoe crab lectin, a sialic acid specific agglutinin. The receptor protein eluted from WGA columns was enriched 25-50-fold over the crude soluble fraction.     

Characterization of a Digitonin-Solubilized Bovine Brain H3 Histamine Receptor Coupled to a Guanine Nucleotide-Binding Protein

The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD = 4.6 nM; Bmax = 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15-30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD = 6.1 nM; Bmax = 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size-exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide-binding protein.     

Characterization of covalently cross-linked pancreatic somatostatin receptors

The receptor for somatostatin present in rat pancreatic plasma membranes was characterized by affinity labeling with [125I-Tyr11]somatostatin utilizing three different heterobifunctional cross-linking agents: N-5-azido-2-nitrobenzoyloxy-succinimide, N-succinimidyl 6-(4-azido 2'-nitrophenylamine)hexanoate, and N-hydroxysuccinimidyl 4-azido-benzoate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a broad band of Mr = 92,000 when any of the three cross-linkers was used; N-succinimidyl 6-(4-azido 2'-nitrophenylamine), however, was most efficient. Labeling of the Mr = 92,000 protein band was not affected by reducing agents but was sensitive to somatostatin and guanine nucleotides, particularly GTP gamma S, at concentrations which reduced binding to the receptor. The affinity-labeled protein could be solubilized completely with Zwittergent 3-12, partially with Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and poorly with Zwittergent 3-08 and digitonin. When exposed to agarose-coupled lectins, the detergent solubilized, labeled Mr = 92,000 protein was completely adsorbed to wheat germ agglutinin, partially to ricin communis II, and not at all to concanavalin A or lotus or lentil lectin. The Mr = 92,000 protein bound to wheat germ agglutinin-agarose was not eluted by N-acetylglucosamine but was by triacetylchitotriose, providing a considerable purification of the somatostatin receptor. These data allow us to conclude that the somatostatin receptor is a monomeric glycoprotein with an Mr = 90,000 binding subunit which probably contains a polymeric arrangement of N-acetylglucosamine residues.     

Interactions of brain muscarinic acetylcholine receptors with plant lectins

We previously reported that muscarinic acetylcholine receptors (mAChRs) from porcine brains are glycoproteins. When porcine brain membranes were solubilized with digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), approximately 20% of the receptors were solubilized, most (90% or more) of which bound to Sepharose 4B conjugated with wheat germ agglutinin (WGA). In contrast, when membranes were solubilized with Lubrol PX, a much larger fraction (approximately 60%) of the receptors were solubilized. However, about a third of this solubilized receptor population remained unbound to WGA-Sepharose even in the presence of an excess amount of the lectin-Sepharose. These results suggested a structural heterogeneity of the mAChR in terms of its carbohydrate moiety. The effects of lectins on the ligand binding properties of mAChRs were also studied. WGA or concanavalin A (ConA) was found to cause a 2- to 3-fold increase in the affinity of membrane-bound receptors to an antagonist [3H]quinuclidinyl benzylate [( 3H]QNB) without affecting the maximum number of sites, whereas the lectins had no significant effects on the binding of the agonist [3H]cis-methyldioxolane. When the membranes were dissolved with detergents, lectin did not increase the [3H]QNB affinity: These lectins caused an approximately 2 fold decrease in the affinity of digitonin-solubilized receptors for [3H]QNB. Thus the lectins exert differential effects on agonist and antagonist binding to the brain membrane mAChRs, most likely by modulating some intermolecular interactions.     

Large scale preparation and characterization of membrane-bound and detergent-solubilized muscarinic acetylcholine receptor from pig atria

The muscarinic acetylcholine receptor (mAcChR) has been prepared from pig atrial membranes by new large scale procedures which result in 30-40 fold enrichment of the receptor in the membrane-bound state and a further three fold enrichment during solubilization. The membrane-bound receptor was prepared by differential and sucrose density gradient centrifugation in 25 mM imidazole, 1 mM EDTA, pH 7.4. A double extraction procedure using a mixed digitonin/cholate detergent was used to solubilize the receptor at a 60-70% yield. The membrane and solubilized preparations had specific activities of 3.5-5 and 8-12 pmol [3H]L-quinuclidinyl benzilate (QNB) binding sites per mg of protein, respectively. The presence of imidazole, which behaved as a weak muscarinic ligand, stabilized the receptor during solubilization and storage. Both the membrane-bound and detergent-solubilized mAcChR bound antagonists at a single class of sites and agonists at two subclasses of QNB sites. The proportion of high affinity agonist sites in the solubilized receptor was about 1/3 that in the membrane receptor. [3H]Propylbenzilylcholine mustard covalently labeled a single prominent atropine-sensitive component with an apparent molecular weight of 70-74,000 on SDS-polyacrylamide gels for both the membrane and solubilized receptor.     

Solubilization, Characterization, and Partial Purification of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-, Quisqualate-, Kainate-Sensitive l-Glutamate Binding Sites from Porcine Brain Synaptic Junctions

L-[3H]Glutamate binding sites were solubilized from porcine brain synaptic junctions by Triton X-114 in the presence of KCl. The solubilized binding sites bound L-[3H]glutamate reversibly with KD and Bmax values of 1.48 +/- 0.18 microM and 178.2 +/- 15.9 pmol/mg of protein, respectively. These binding sites appeared to be integral membrane glycoproteins, with sugar moieties recognized by wheat germ agglutinin. A 49.3-fold purification of these binding sites was achieved by Triton X-114 solubilization, anion-exchange chromatography, and affinity chromatography using wheat germ agglutinin-Sepharose. The apparent molecular mass of the partially purified binding sites was 620 +/- 50 kDa. L-[3H]Glutamate bound to the solubilized preparation could be effectively displaced by agonists of non-N-methyl-D-aspartate (NMDA) L-glutamate receptors but not by NMDA or alpha-amino-4-phosphonobutyrate. The rank order for the competitive ligands in displacing L-[3H]glutamate was: quisqualate greater than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid greater than L-glutamate greater than kainate.     

Solubilization of muscarinic acetylcholine receptors from mammalian brain: An analytical approach

Muscarinic receptor sites were extracted from microsomal membranes of mammalian brain using digitonin. These detergent preparations were submitted to the control of unambiguous solubilization criteria, and were found to correspond to molecularly dispersed material. In contrast, the corresponding salt preparations did not fulfill these criteria. After comparing various assay methods, we describe a simple and rapid charcoal adsorption binding technique, which was selected for measuring digitonin-extracted muscarinic receptors. Under these conditions the digitonin preparations, from four different species revealed identical biochemical properties in a stereospecific labeling system using [³H]dexetimide and [³H]levetimide.     

Electrophoretic characterization of muscarinic receptors under denaturating and nondenaturating conditions: computer-assisted Ferguson plot analysis

The physical properties of the covalently labeled [( 3H]propylbenzilycholine mustard) muscarinic acetylcholine receptor from rat brain were studied by sodium dodecyl sulfate-polyacrylamide electrophoresis and computer-assisted Ferguson plot analysis. No proteolytic degradation or dimerization of the ligand binding subunit was found. No clues for different molecular weight forms or anomalous migration of the muscarinic receptor were detected. The weighted regression analysis of Ferguson plots gave an apparent molecular mass of 64-65 kDa. A new method for the electrophoretic separation of native (quinuclidinyl[3H]benzilate labeled) muscarinic receptor-detergent complexes was used for the comparison of wheat germ agglutinin binding, and not lectin binding receptors which were obtained by selective solubilization from porcine striatum. For this purpose, the computer-assisted Ferguson plot analysis is particularly suitable, since it renders possible the statistical assessment of both size and charge differences. Both receptor-detergent complexes were found to differ; statistically significant in their net charge but not in their size. The data support the view that muscarinic receptors from different sources may differ considerably in their glycosylation and that the receptor from porcine striatum can reversibly associate with a low-molecular-mass component which contains sialic acid.     

Alpha 1- and beta 2-adrenergic receptors co-expressed on cloned MDCK cells are distinct glycoproteins

We have explored the molecular differences between alpha 1- and beta 2-adrenergic receptors that are co-expressed by a clonally-derived cell line, Madin-Darby canine kidney clone D (MDCK-D). MDCK-D membranes were pre-labeled with selective alpha 1- and beta-adrenergic radioligands and were then solubilized with the non-ionic detergent digitonin. Solubilized alpha 1- and beta 2-adrenergic receptors were retained by immobilized wheat germ agglutinin and were eluted following addition of N-acetyl-D-glucosamine or sialic acid. Both receptors were also retained by immobilized Limax flavus lectin, a sialic acid-binding lectin. Lectins that were specific for N-acetyl-D-glucosamine residues did not bind to these receptors. These results indicate that both alpha 1 and beta 2 receptors are sialylated glycoproteins. The solubilized alpha 1- and beta 2-adrenergic receptors migrated with different elution profiles from an Ultragel AcA 34 column. The apparent molecular sizes of the digitonin-receptor complexes were 68A for the alpha 1 receptor and 55A for the beta 2 receptor. These results show that alpha 1- and beta 2-adrenergic receptors can be present on the same cell as distinct sialic acid-containing glycoproteins.     

The heterogeneity and nucleotide modulation of cholinergic muscarinic receptors are restored by poly (ethylene) glycol-6000 precipitation of solubilized atrial receptors

The membrane bound muscarinic receptor exhibits heterogeneity of binding sites for agonists that are modulated by guanine nucleotides. These properties are generally lost upon receptor solubilization by several detergents, but may be recovered after rapid detergent removal by poly (ethylene) glycol-6000 precipitation of the digitonin plus cholate solubilized atrial muscarinic receptor. The results are discussed in terms of muscarinic receptor interactions in the intact membrane.     

Purification of opioid receptor in the presence of sodium ions.

Opioid receptor was solubilized from rat brain membranes with a mixture of the detergents CHAPS and digitonin in the presence of protease inhibitors and 1 M NaCl. The solubilized receptor bound mu-opioid agonists and antagonists with affinities similar to those of native membrane receptor. The affinity of solubilized receptor for the agonist PL017 was greatly reduced by GTP gamma S, suggesting the receptor is still associated with G-protein. The solubilized material was passed through an opioid antagonist (10cd) affinity column and a wheat germ agglutinin column, set up in series, to obtain a partially purified receptor preparation. This partially purified material bound mu-agonist with low affinity and the binding affinity was no longer affected by GTP gamma S. The partially purified receptor was further purified by repeating the affinity and lectin chromatography with smaller size column. Binding of opioid antagonist [3H]diprenorphine to the partially or purified receptors was dependent upon the presence of sodium ions. The purified receptor showed saturable and stereospecific binding for opioid ligands, was predominantly of the mu-type, and exhibited as a diffuse band with a medium molecular mass of 62 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The average specific binding activity of the purified receptor was 18.8 +/- 2.3 pmol/micrograms protein, a value close to the theoretical estimation.     

Heterogeneity of solubilized muscarinic cholinergic receptors: binding and hydrodynamic properties

Previous studies have described the conversion, after detergent solubilization, of the multiple populations of membrane-bound muscarinic agonist binding sites to a population of uniform affinity. This paper describes the solubilization of at least two receptor species, distinct in their agonist binding characteristics, which are capable of interconversion by transition metal ions. This finding enabled a more detailed examination of the molecular properties and regional differences of brain muscarinic receptors than was previously possible. Muscarinic receptors (mAChR) obtained from the rat cerebral cortex or medulla pons were solubilized using digitonin or the zwitterion detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). The equilibrium binding of the antagonist [3H]-4-N-methylpiperidyl benzilate ([3H]4NMPB) to detergent-solubilized receptors resembled binding to neural membranes and exhibited subnanomolar affinity, saturability, and simple mass action kinetics. Agonist binding to soluble preparations was measured by competition of [3H]4NMPB binding sites. Saturation isotherms for agonist binding to digitonin- and Chaps-solubilized mAChR obtained from various brain regions appear flattened and have Hill coefficients in the range 0.52-0.78. Computerized modelling techniques indicate that the best fit to the experimental data is provided by a model specifying two soluble muscarinic agonist binding sites with differing dissociation constants, KH and KL, respectively. Solubilization of cerebral cortex membranes with Chaps or digitonin resulted in a population with a composition of high- and low-affinity sites similar to that found in the membrane-bound state. In contrast, solubilization of the medulla pons resulted in an approximately 40% loss of high-affinity sites. Solubilized receptors retained the sensitivity to transition metals ions, but were insensitive to guanine nucleotides. Density gradient centrifugation indicated that Chaps-solubilized mAChR are composed of two molecular forms with S20,W equal to 9.9 S and 14.9 S. The 14.9 S species comprises approximately 30% of the total binding activity in the cortex and approximately 40% in the medulla. We identify the 14.9 S species as being associated with a guanylnucleotide binding protein because treatment of medulla membranes with guanylylimidodiphosphate prior to solubilization results in disappearance of 14.9 S with 9.9 S unchanged. Sedimentation of cortical mAChR in the presence of Cu+2 leads to an increase in 14.9 S to almost 50% of the total binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nonionic detergents increase the stoichiometry of ligand binding to the rat hepatic galactosyl receptor

When digitonin is used to expose intracellular galactosyl (Gal) receptors in isolated rat hepatocytes, only about half of the binding activity for 125I-asialoorosomucoid (ASOR) is found as compared to cells solubilized with Triton X-100. The increased ligand binding in the presence of detergent is not due to a decrease in Kd but could be due either to an increase in the number of ASORs bound per receptor or to exposure of additional receptors. Several experiments support the former explanation. No additional activity is exposed even when 80% of the total cell protein is solubilized with 0.4% digitonin. It is, therefore, unlikely that receptors are in intracellular compartments not permeabilized by digitonin and inaccessible to 125I-ASOR. Digitonin-treated cells are not solubilized by Triton X-100 if they are first treated with glutaraldehyde under conditions that retain specific binding activity. 125I-ASOR binding to these permeabilized/fixed cells increases about 2-fold in the presence of Triton X-100 and a variety of other detergents (e.g., Triton X-114, Nonidet P-40, Brij-58, and octyl glucoside) but not with the Tween series, saponin, or other detergents. When these fixed cells are washed to remove detergent, 125I-ASOR binding decreases almost to the initial level. Affinity-purified Gal receptor linked to Sepharose 4B binds approximately twice as much 125I-ASOR in the presence of Triton X-100 as in its absence. The results suggest that the increase in Gal receptor activity in the presence of nonionic detergents is due to an increase in the valency of the receptor rather than to exposure of additional receptors.     

Reconstitution of the rat liver vasopressin receptor coupled to guanine nucleotide-binding proteins

The V1 vasopressin receptor has been solubilized from rat liver membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate (CHAPS) and reconstituted into phospholipid vesicles. There is essentially complete solubilization of the receptor by 3% CHAPS at a protein concentration of 15 mg/ml. Reconstitution into soybean phospholipid vesicles is readily achieved either by gel filtration chromatography or by membrane dialysis. The binding of [3H]vasopressin to proteoliposomes is specific, saturable, reversible, and magnesium-dependent. In contrast, the detergent-soluble vasopressin receptor does not display specific binding. The apparent affinity of the reconstituted receptor for [3H]vasopressin is approximately 4-fold lower than that of the receptor in native membranes. In addition, the binding of [3H]vasopressin to reconstituted vesicles is not sensitive to 100 microM guanosine 5'-O-thiotriphosphate (GTP gamma S) as it is in native membranes. However, the apparent affinity of the reconstituted receptor for ligand approximates that of native membranes when membranes are prebound with vasopressin prior to solubilization and reconstitution into vesicles. Furthermore, vesicles reconstituted from membranes prebound with vasopressin show GTP gamma S sensitivity of [3H] vasopressin binding. This finding strongly suggests that vasopressin stabilizes a receptor-G-protein complex during solubilization. The rat liver vasopressin receptor is a glycoprotein, as shown by its specific binding to the lectin "wheat germ agglutinin." The vasopressin receptor can be reconstituted from the N-acetylglucosamine-eluted peak of a wheat germ agglutinin-Sepharose column, and [3H] vasopressin binding activity is purified 5-6-fold from membranes by this chromatographic procedure. The functionality of the partially purified receptor is indicated by its ability to bind ligand with high affinity and by its ability to functionally interact with a G-protein when vasopressin is bound prior to solubilization.     

Dopamine D1 receptors characterized with [3H]SCH 23390. Solubilization of a guanine nucleotide-sensitive form of the receptor

Dopamine D1 receptors were solubilized from canine and bovine striatal membranes with the detergent digitonin. The receptors retained the pharmacological characteristics of membrane-bound D1 receptors, as assessed by the binding of the selective antagonist [3H]SCH 23390. The binding of [3H]SCH 23390 to solubilized receptor preparations was specific, saturable, and reversible, with a dissociation constant of 5 nM. Dopaminergic antagonists and agonists inhibited [3H]SCH 23390 binding in a stereoselective and concentration-dependent manner with an appropriate rank order of potency for D1 receptors. Moreover, agonist high affinity binding to D1 receptors and its sensitivity to guanine nucleotides was preserved following solubilization, with agonist dissociation constants virtually identical to those observed with membrane-bound receptors. To ascertain the molecular basis for the existence of an agonist-high affinity receptor complex, D1 receptors labeled with [3H] dopamine (agonist) or [3H]SCH 23390 (antagonist) prior to, or following, solubilization were subjected to high pressure liquid steric-exclusion chromatography. All agonist- and antagonist-labeled receptor species elute as the same apparent molecular size. Treatment of brain membranes with the guanine nucleotide guanyl-5'-yl imidodiphosphate prior to solubilization prevented the retention of [3H]dopamine but not [3H]SCH 23390-labeled soluble receptors. This suggests that the same guanine nucleotide-dopamine D1 receptor complex formed in membranes is stable to solubilization and confers agonist high affinity binding in soluble preparations. These results contrast with those reported on the digitonin-solubilized dopamine D2 receptor, and the molecular mechanism responsible for this difference remains to be elucidated.     

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.     

Solubilization in high yield of opioid receptors retaining high-affinity delta, mu and kappa binding sites

The binding sites for opiates (agonist and antagonist) and opioid peptides can be solubilized from rat brain membranes with digitonin in the presence of Mg2+ (10 mM). High affinity and high capacity binding to the soluble delta, mu, and kappa receptors is obtainable when the membranes are treated in Mg2+ (30 degrees C, 60 min) prior to solubilization. The yields of solubilized binding sites extracted with digitonin, 40-90%, are higher than those obtained from Mg2+-pretreated membranes with other detergents commonly used for receptor solubilization. The stability of the digitonin-soluble opioid receptor at room temperature makes it useful for purification and characterization.