Digitonin-solubilized Receptors Research Articles

3H]-(R)-N6-Phenylisopropyladenosine Agonist Binding to the Solubilized A1Adenosine Receptor from Bovine Epidydimal Spermatozoa

The high-affinity agonist radioligand [3H]-(R)-N6-phenylisopropyladenosine ([3H]-R-PIA) was used to investigate agonist A1adenosine receptors interactions in soluble preparations from bovine spermatozoa. The digitonin-solubilized receptor shows a high-affinity state with aKdof 5.32 ± 1.17 nMand aBmaxof 460 ± 33 fmol/mg protein. The binding capacity, higher than that of the membrane-bound form, indicates that the soluble preparation is likely enriched with binding sites. In the presence of guanylyl-5′-imidodiphosphate (Gpp(NH)p), [3H]-R-PIA binds to the soluble receptor with aKdof 7.97 ± 1.44 nMand aBmaxof 400.8 ± 27 fmol/mg protein. The radioligand rapidly dissociates with aK−1of 0.125 min−1although specific [3H]-R-PIA is still found in solubilized A1receptor. The A1agonistN6-cyclopentyladenosine differentiates two affinity states, whereas Gpp(NH)p shifts the agonist curve to the right and all the receptors are in the low-affinity state. In the presence of NaCl, the agonist still recognizes two affinity states with a lower affinity than that observed in the absence of NaCl. Analyses of sedimentation profiles show the existence of a population of A1receptors tightly coupled to Gi, the pertussin toxin-sensitive component of the G protein family.

Extracti-Gel TM D chromatography is a simple, efficient method for removing digitonin during receptor purification: application to the kl opioid receptor

Digitonin is widely used for extracting active neurotransmitter receptors from membranes. However, its low critical micellar concentration has made its removal from samples problematic. Here we report that digitonin can be efficiently removed (> 90%) from solution using Extracti-Gel TM D, a detergent-absorbing matrix. Active K l opioid receptors solubilized from brain survive Extracti-Gel TM D chromatography with a recovery of 50–55% and 25% dilution by added volume. The loss of receptor and the dilution, however, are compensated for to a large extent by the disinhibition of binding that results from the removal of digitonin. Extracti-Gel TM D chromatography had little or no effect on the apparent equilibrium dissociation constant for [ 3H]U-69,593 binding to the K l receptor. We conclude that Extracti-Gel TM D column chromatography is a simple, highly efficient and practical method for markedly reducing the concentration of digitonin in biological samples. Application of the procedure should allow characterization of digitonin-solubilized receptors with minimal complications from bound digitonin and extend the usefulness of digitonin to studies going beyond the initial stages of receptor purification.

Solubilization of high affinity peptide-YY receptors from porcine brain.

We have previously characterized peptide-YY (PYY) receptors in porcine hippocampal membranes. We demonstrate here that brain PYY receptors can be extracted in the active state using digitonin. Among several detergents tested for their suitability to extract active PYY receptors, digitonin gave the most favorable results, as judged by specific binding of [125I]PYY to the solubilized receptors. The binding of [125I]PYY to digitonin extract was dependent on incubation time, temperature, and protein and magnesium ion concentrations and had a pH optimum of 6-7. Solubilized PYY receptors maintained the rank order of potencies for various related peptides and PYY fragments characteristic of the membrane PYY receptor: PYY greater than neuropeptide Y (NPY) much much greater than avian and porcine pancreatic polypeptide, and PYY greater than PYY-(22-36) much much greater than PYY-(1-22) and PYY-(22-28), respectively. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the digitonin extract; the high affinity component had affinities and binding capacities similar to those of the membrane PYY receptor. Solubilized PYY receptors also retained their sensitivity to guanine nucleotides. PYY was cross-linked to its receptors with disuccinimidyl suberate, solubilized with digitonin, and cross-linked to digitonin-solubilized receptors. The resulting complexes were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. Using these procedures, we identified a PYY receptor species with a molecular size of 50,000, which was the same size as the labeled protein in native membrane homogenates. Solubilized NPY receptors were also the same size. The solubilized cross-linked PYY receptor was adsorbed by wheat germ agglutinin-agarose and Concanavalin-A, suggesting its glycoprotein nature. These data suggest that the specific binding properties of the PYY receptor are inherent in the solubilized glycoprotein molecules. The solubilization in digitonin of PYY receptors from membranes should allow a more complete molecular and functional characterization of PYY-mediated events and purification of the receptor.

A rapid procedure for the purification of cardiac 1,4-dihydropyridine receptors from porcine heart

Highly purified porcine cardiac sarcolemma was used as a source for purification of mammalian cardiac 1,4-dihydropyridine receptors associated with the voltage-dependent Ca2+ channel. The cardiac digitonin-solubilized receptor prelabeled with (+)-[3H]PN 200-110 was enriched at least 236-fold using an improved, rapid three-step purification protocol which could be completed within 12 h. The purity of the preparation was at least 22%, the yield of the receptors 24%. Photoaffinity labeling experiments with (-)-[3H]azidopine allowed the identification of the cardiac alpha 1 subunit. In contrast to the purified rabbit or guinea-pig skeletal muscle Ca2+ channel complex, none of the purified polypeptides underwent rapid and substantial phosphorylation by the catalytic subunit of the cyclic AMP-dependent protein kinase in vitro.

Sphingosine Interacts Directly with the Receptor Complex to Inhibit Thyrotropin-Releasing Hormone Binding

Sphingosine inhibition of [3H] [N3-Me-His] TRH (MeTRH) binding, previously shown to be independent of its effects on protein kinase-C, has been further characterized in GH3 cell membranes and in a partially purified, digitonin-solubilized receptor preparation. In membranes, as in intact cells, sphingosine inhibited [3H]MeTRH binding by decreasing receptor affinity, but, in contrast to its effect in intact cells, did not affect the number of available binding sites. The inhibition of binding was linear up to 75 microM sphingosine (in the presence of 100 microM BSA at 0.1 mg membrane protein/ml), yielding an apparent Ki of 51 microM. Since GTP decreases the affinity for MeTRH binding in GH3 cell membranes, we studied interactions between GTP and sphingosine. While the effects of low concentrations of GTP gamma S and sphingosine were additive, sphingosine inhibition of MeTRH binding surpassed and was not affected by the addition of maximally inhibitory concentrations of GTP gamma S. Also, sphingosine (75 microM) did not affect the ability of a maximally effective dose of TRH to stimulate the low Km GTPase (vehicle, +35 +/- 5%; sphingosine, +32 +/- 10%); there was a 25% decrease in total GTPase activity in the presence of sphingosine. MeTRH binding to digitonin-solubilized receptors, which had properties similar to those described previously by others, including no effect of GTP on binding, was inhibited by sphingosine. In solubilized receptors, as in membranes, sphingosine caused a decrease in apparent affinity without changes in the number of binding sites. These data suggest that sphingosine interacts directly with the TRH receptor [or an associated factor(s) in the receptor complex] to decrease affinity by a mechanism that does not involve uncoupling of G-proteins.

Limited proteolysis of the vasoactive intestinal peptide receptor: comparison of its folded structure in the membrane-bound and detergent-solubilized states

Limited proteolysis was used to probe and compare the conformation of the rat lung vasoactive intestinal peptide (VIP) receptor in membrane-bound and detergent-solubilized states. It had been shown previously that the activity of the detergent-solubilized VIP receptor is sensitive to the nature of the detergent used for extraction (Patthi, S., Simerson S. and Velicelebi, G. (1988) J. Biol. Chem., 263, 19363–19369). Receptors that were extracted from the membrane using digitonin retained the ability to bind 125I-VIP, while those solubilized in Triton X-100 displayed little or no detectable activity. In order to correlate the differences observed in the activity of the receptor with its folded state, membrane-bound and detergent-solubilized receptors were covalently labeled with 125I-VIP and subjected to limited proteolysis using trypsin, chymotrypsin or carboxypeptidase Y. Digitonin-solubilized receptors most closely resembled the membrane-bound protein in terms of protease sensitivity and proteolytic cleavage products. By contrast, receptors solubilized in Triton X-100 displayed increased sensitivity to proteases and produced distinctly different proteolytic patterns. Thus, the differences observed in the activities of receptors solubilized in digitonin and those solubilized in Triton X-100 could be correlated with detectable differences in the conformation of the protein in each respective detergent solution. These results suggest that digitonin provides an environment that is more compatible with the native folded state of the receptor, similar to its conformation in the membrane.

Solubilization of High Affinity Corticotropin-Releasing Factor Receptors from Rat Brain: Characterization of an Active Digitonin-Solubilized Receptor Complex

The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of [125I]Tyro-ovine CRF ([125I]oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for [125I] oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, [125I]oCRF labeled the same size receptor complex. These data suggest that either the guanine nucleotide-binding protein (Ns) is tightly associated with the CRF receptor after solubilization and is insensitive to guanine nucleotides, or that high affinity binding for soluble CRF receptors is not dependent on the coupling of a guanine nucleotide-binding protein. The solubilization of CRF receptors from membranes in digitonin should allow for the more complete molecular and functional characterization of CRF-mediated events and purification of the receptor.

Pituitary Thyrotropin-Releasing Hormone Receptors: Local Anesthetic Effects on Binding and Responses

Pharmacological agents are widely used to probe the mechanism of action of TRH. A number of these drugs behave as local anesthetics at high concentrations. The effect of local anesthetics on the binding of [3H]Me-TRH to specific receptors was studied using the GH4C1 line of rat pituitary tumor cells. [3H]Me-TRH binding was inhibited by classical local anesthetics with the order of potency (IC50 values): dibucaine (0.37 mM) greater than tetracaine (1.2 mM) greater than lidocaine (3.3 mM) greater than procaine and benzocaine (greater than 10 mM). IC50 values for other drugs with local anesthetic properties that inhibited [3H]Me-TRH were: 100 microM trifluoperazine, 100 microM imipramine, 170 microM chlorpromazine, 300 microM verapamil, and 700 microM propranolol. Inhibition by tetracaine and verapamil increased as the pH was raised from 6 to 8.5, indicating that the free base form of the amine drugs was the inhibitory species, and the local anesthetic effect was greater at 37 C than at 24 C or 0 C. [3H]Me-TRH binding to receptors in isolated membranes was inhibited to the same extent as binding to receptors on intact cells. Local anesthetics were 3- to 20-fold less potent at inhibiting [3H]Me-TRH to digitonin-solubilized receptors than binding to intact cells. In contrast, the potency of chlordiazepoxide, a putative TRH antagonist, to inhibit [3H]Me-TRH binding was equal using cells and solubilized receptors (IC50 = 10 microM). Local anesthetics inhibited TRH-stimulated PRL release and also inhibited basal PRL secretion and secretion stimulated by two nonhormonal secretagogues, (Bu)2cAMP and a phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization and characterization of the κ-opioid receptor type from guinea-pig cerebellum

The κ-opioid receptor (κOp) from guinea-pig cerebellum membranes has been solubilized in an active form and in good yield (30–40%) with digitonin in 10 mM Tes-KOH buffer, pH 7.4 [ 3H]Bremazocine (K D = 1.1 nM in the soluble extract) was used to monitor the binding and hydrodynamic characteristics of the digitonin-solubilized receptor, s-κ Op. Opioid agonists and antagonists bind s-κ Op with a generally lower (yet still high) apparent affinity than they do its membrane-bound counterpart, m-κ Op, but with the same rank order of potency. In particular, U50488, a κ selective agonist, binds s-κ Op with a considerably higher apparent affinity than do [D-Ala 2,MePhe 4,Glyol 5]enkephalin, a μ selective agonist, and [D-Thr 2,Leu 5]enkephalyl-Thr, a δ selective agonist. s-κ Op has also retained substantial stereospecificity since levorphanol was nearly 200-fold as potent as dextrorphan in competing with binding of [ 3H]bremazocine in the soluble extract. However, s-κ Op appeared to be desensitized to regulation by Na + ions: the selective inhibition by the allosteric effector of binding of agonists at the m-κ Op was no longer observed in digitonin extracts from guinea-pig cerebellum membranes. The s-κ Op behaved as a unique macromolecular entity both in molecular exclusion chromatography (app. r s = 6.7 nm) and in sedimentation of linear density gradients (app. S 20,w = 11.8 S).

Regulation of porcine brain alpha 2-adrenergic receptors by Na+,H+ and inhibitors of Na+/H+ exchange.

Previous reports from this laboratory have demonstrated that alpha 2-adrenergic receptors accelerate Na+/H+ exchange in NG108-15 neuroblastoma X glioma cells and evoke platelet secretion via a pathway involving Na+/H+ exchange. The present studies were designed to examine whether agents that interact with Na+/H+ antiporters also might influence alpha 2-adrenergic receptor-ligand interactions. We observed that Na+ decreases receptor affinity for the agonists epinephrine, norepinephrine, and UK14304 and slightly increases receptor affinity for the antagonists yohimbine and idazoxan in digitonin-solubilized preparations from porcine brain cortex. Increases in [H+] also decrease receptor affinity for agonists and cause either a slight increase or no change in receptor affinity for antagonists. Amiloride analogs accelerate the rate of [3H] yohimbine dissociation from digitonin-solubilized receptors with a relative effectiveness that parallels their ability to block Na+/H+ exchange in other systems. Interestingly, these modulatory effects of Na+,H+ and 5-amino-substituted analogs of amiloride are retained in homogeneous preparations of the alpha 2-adrenergic receptor, suggesting that the allosteric-binding sites for these agents are on the receptor-binding protein itself.

Characterization of [formula omitted] binding to solubilized cholecystokinin (CCK) receptors of rat pancreas

The binding of [ 3 H](±) L-364,718 (3 S(−)- N-(2,3-dihydro-l-methyl-2-oxo-5-phenyl-1 H-1,4-benzodiazepine-3- yl)-1 H-indole-2-carboxamide],an extremely potent nonpeptide cholecystokinin (CCK) receptor antagonist, to digitonin-solubilized CCK receptors from rat pancreas was characterized. [ 3 H](±) L-364,718 binding to digitonin-solubilized receptors was assayed using polyethylene glycol precipitation followed by rapid filtration to separate free and bound [ 3 H(±) L-364,718 . Specific [ 3 H](±) L-364,718 binding to solubilized receptors was dependent on the digitonin and receptor concentration and, under optimal conditions, represented greater than 90% of the total binding. Scatchard analysis indicated a single class of binding sites with a K d of 0.53 nM and a B max of 3.1 pmol/mg protein. Specific [ 3 H](±) L-364,718 binding to solubilized CCK receptors was inhibited by both CCK receptor agonists and antagonists in a stereospecific manner. After solubilization, the affinities of various antagonists to displace specific [ 3 H](±) L-364,718 binding were similar to those obtained with membrane-bound receptors; however, the affinities of CCK agonists were reduced 10–100 times. Collectively, the data presented indicate that [ 3 H](±) L-364,718 represents a new antagonist ligand which has apparent advantages over the agonist ligand [ 125I]CCK in assaying digitonin-solubilized receptors. Gel filtration of the digitonin-solubilized CCK receptors followed by [ 3 H](±) L-364,718 binding determinations revealed an estimated molecular weight of 400,000 dartons.

Monoclonal antibodies that coimmunoprecipitate the 1,4-dihydropyridine and phenylalkylamine receptors and reveal the calcium channel structure

Monoclonal hybridoma cell lines secreting antibodies against the (+)-PN 200-110 and the (-)-demethoxyverapamil binding components of the voltage-dependent calcium channel from rabbit transverse-tubule membranes have been isolated. The specificity of these monoclonal antibodies was established by their ability to coimmunoprecipitate (+)-[3H]PN 200-110 and (-)-[3H]demethoxyverapamil receptors. Monoclonal antibodies described in this work cross-reacted with rat, mouse, chicken, and frog skeletal muscle Ca2+ channels but not with crayfish muscle Ca2+ channels. Cross-reactivity was also detected with membranes prepared from rabbit heart, brain, and intestinal smooth muscle. These antibodies were used in immunoprecipitation experiments with 125I-labeled detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin] solubilized membranes. They revealed a single immunoprecipitating component of molecular weight (Mr) 170,000 in nonreducing conditions. After disulfide bridge reduction the CHAPS-solubilized (+)-PN 200-110-(-)-demethoxyverapamil binding component gave rise to a large peptide of Mr 140,000 and to smaller polypeptides of Mr 30,000 and 26,000 whereas the digitonin-solubilized receptor appeared with subunits at Mr 170,000, 140,000, 30,000, and 26,000. All these results taken together are interpreted as showing that both the 1,4-dihydropyridine and the phenylalkylamine receptors are part of a single polypeptide chain of Mr 170,000.

Immunochemical studies of the muscarinic acetylcholine receptor.

Muscarinic receptors have been purified from calf forebrain plasma cell membranes by affinity chromatography on a dexetimide-agarose gel. SDS-PAGE analysis showed a single 70 kDa band. Monoclonal antibodies have been prepared against these affinity purified 70 kDa protein(s). One antibody, M-35, immunoprecipitated up to 80% of digitonin-solubilized muscarinic receptors. M-35 had agonist-like effects on guinea-pig myometrium: it increased the intracellular cyclic GMP content, decreased prostaglandin-induced cyclic AMP accumulation and caused muscle contractions. The two first effects were inhibited by atropine. M-35 was used to visualize muscarinic receptors at the surface of human fibroblastic cells. In the particular cell line used, the receptors have a low affinity for pirenzepine, were negatively coupled to adenylate cyclase and mediated increase in the phosphatidyl-inositol breakdown.

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.

Interactions of Brain Musearinic Acetylcholine Receptors with Plant Lectins1

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.

Drug Potencies on Partially Purified Brain D2 Dopamine Receptors

To examine the sensitivities of partially purified dopamine receptors to various dopaminergic agonists and antagonists, canine brain striatum dopamine receptors were enriched by isoelectric focusing. The digitonin-solubilized receptors were prelabelled with [3H]spiperone and focused for two time periods. After 5 h (incomplete focusing), radioactive peaks were detected at pH 6 and 9-11. Only the pH 6 peak revealed drug sensitivities expected of D2 receptors. Receptor recovery of the pH 6 peak was 79% with purification being sevenfold. After focusing overnight to equilibrium, the pH 6 peak further separated into peaks at pH 4.6 and 6.8. The receptor was identified only in the pH 4.6 fraction. The recovery of receptors in the pH 4.6 peak was low (10%), indicating little enrichment of the receptor. The rank order of binding of neuroleptics and dopamine agonists to the purified material was similar to that of the original preparation of soluble receptors. Dopamine did not bind to the purified pH 4.6 fraction unless the phosphate buffer (used during focusing) was replaced with Tris buffer. The absence of receptors in the pH 6.8 and pH 10 fractions, although both contained prelabeled [3H]spiperone, indicates the importance of testing agonists and antagonists on each fraction at each step in purification.

Solubilization and characterization of thyrotropin-releasing hormone receptors from rat brain.

The thyrotropin-releasing hormone receptor from rat brain was solubilized in a stable unbound form. The natural glycoside digitonin was the only detergent of a variety tested capable of solubilizing the active receptor. The digitonin-solubilized receptor exhibited binding kinetics for 3-[3H]methylhistidine thyrotropin-releasing hormone virtually identical to membrane preparations and responded with a similar order of potency to a series of thyrotropin-releasing hormone analogues that inhibit binding of the labeled ligand. Gel filtration analysis and sucrose density gradient centrifugation indicated a Stokes radius of 5.2 nm, a sedimentation coefficient of 11.2 s, and a corresponding calculated molecular weight of 244,000 for the detergent-receptor complex. The soluble receptor shows a gradual loss of binding activity over 48 to 72 hr when kept at 4 degrees C. However, the preparation may be frozen at -20 degrees C with no significant loss of activity.

Monoclonal antibodies against the native or denatured forms of muscarinic acetylcholine receptors.

BALB/c mice were immunized with affinity-purified muscarinic acetylcholine receptors from calf brain and their splenocytes fused with NS1 myeloma cells. Hybrid cultures were grown and selected for production of antibodies on the basis of enzyme immunoassays on calf and rat forebrain membrane preparations. Thirty-four clones were retained and six of them further subcloned. Two of these subclones produced antibodies that selectively recognized muscarinic acetylcholine receptor-bearing membranes. The M-35b antibodies interacted only with native digitonin-solubilized receptors, and not with denatured receptors. The M-23c antibodies did not react with active digitonin-solubilized receptors but recognized the denatured form. The M-23c antibodies should thus be useful in the purification of the receptor and its precursor translation products, while the M-35b antibodies could be used for the immunocytochemical localization of the receptor in cells and tissues of different species.

Enrichment, solubilization, and partial characterization of digitonin-solubilized muscarinic receptors derived from canine ventricular myocardium.

The subcellular distribution of cardiac muscarinic receptors was defined in canine ventricular myocardium, and receptors were solubilized from subcellular fractions enriched in muscarinic receptor content. The subcellular location of muscarinic receptors in cardiac tissue was determined by measurement of the distribution of [3H](+/-)quinuclidinyl benzilate-binding activity in particulate fractions isolated from canine ventricular myocardium. Based upon excellent correlation between [3H](+/-)quinuclidinyl benzilate binding and activity of the sarcolemmal Na+,K+-ATPase throughout the subcellular fractions, muscarinic receptors appeared to be localized to sarcolemma in canine ventricular myocardium. Therefore, membrane fractions enriched in sarcolemma were used as a source of cardiac muscarinic receptors for solubilization. Treatment of membrane vesicle fractions with digitonin (0.6%) resulted in solubilization of [3H](+/-)quinuclidinyl benzilate-binding activity with an extraction yield of 25-35%. Criteria of pharmacological specificity and stereospecificity established the identity of the solubilized binding activity of muscarinic receptors. Solubilization of muscarinic receptors was documented by demonstration of hydrodynamic behavior consistent with molecularly dispersed material. Upon glycerol gradient centrifugation, digitonin-solubilized muscarinic receptors from cardiac tissue sedimented with an apparent sedimentation coefficient of 9S. Pharmacological characterization of the digitonin-solubilized receptors revealed 8- to 39-fold reductions in affinities for muscarinic antagonists compared to the affinities exhibited by receptors in the membrane-bound state. Substantially greater reductions in agonist affinities (reduction of at least 700-fold for all agonists studied) suggested selective loss of ability of the digitonin-solubilized receptors to exhibit high affinity agonist interactions. In contrast to membrane-bound receptors, digitonin-solubilized receptors also demonstrated a loss of guanine nucleotide regulation, as well as steep agonist:radioligand competition curves with slope factors of 1.0, suggesting a homogeneous population of agonist-binding sites. Interpreted within the context of a model of state interconversion for membrane-bound receptors, the results suggested that either muscarinic receptors of a single state were selectively solubilized, or that solubilization induced conversion of all receptors to a single low affinity state, possibly by removal of constituents necessary for assumption of a high affinity agonist conformation.

Characterization of the mammalian beta-2 receptor in 8 M urea

Treatment of canine pulmonary membranes with detergent-free 8 M urea yielded a single class of large MW high-affinity binding sites exhibiting the stereospecificity and order of potency for beta-ligands characteristic of the beta receptor. This urea-solubilized receptor, demonstrating a 3–4 fold decreased affinity for beta-ligands as compared to membrane-bound and digitonin-solubilized receptor, exhibited a marked loss of activity when concomitantly exposed to solubilizing concentrations of detergent.