Subunit structure and peptide mapping of junctional and extrajunctional acetylcholine receptors from rat muscle.

We have purified the junctional acetylcholine receptor from normal rat skeletal muscle and compared its structure with that of the extrajunctional receptor from denervated muscle. The two receptors from leg muscle were distinguished by isoelectric focusing and by reaction with sera from patients with myasthenia gravis. The junctional form of the acetylcholine receptor was purified from normal leg muscle by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose followed by sucrose gradient centrifugation. Analysis of radioiodinated receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the subunit structure of the junctional receptor was similar to that previously determined for the extra-junctional form (Froehner, S. C., et al. (1977) J. Biol. Chem. 252, 8589-8596), with major polypeptides, whose apparent molecular weights in 9% polyacrylamide gels were 45 000 and 51 000. In addition, several minor polypeptides were found. When the two receptors were labeled with different isotopes of iodine and run together on a sodium dodecyl sulfate gel, the subunits of one receptor could not be resolved from those of the other. As seen earlier with the extrajunctional form, the affinity alkylating reagent [3H]MBTA labeled the 45 000- and 49 000-dalton polypeptides of the junctional receptor. Peptide mapping showed that the two MBTA binding subunits are structurally related, although they are unrelated to the other polypeptides, and that the 45 000- and 51 000-dalton polypeptides of the junctional receptor were indistinguishable from those of the extrajunctional receptor. In addition, peptide mapping of the four subunits of acetylcholine receptor isolated from Torpedo californica electric organ showed that these four polypeptides appear to be structurally unrelated.     

Selective photoaffinity labeling of acetylcholine receptor using a cholinergic analogue

A bisazido derivative was synthesized from bis(3-aminopyridinium)-1,10-decane diiodide and it was shown that it was bound (KD congruent to 2.2 muM) specifically to purified acetylcholine receptor and fulfilled the requirements for a photoaffinity label. Like the parent compound the derivative could transform membrane-bound receptor from a low ligand affinity conformation(s) to a high ligand affinity form (s), a transition which is thought to resemble desensitization processes observed in vivo. Photolysis of 3H-labeled bisazido reagent was carried out in the presence of the receptor. After dodecyl sulfate-polyacrylamide gel electrophoresis of labeled purified receptor two of the four subunits (mol wt 40 000 and 60 000) contained 90% of the bound radioactivity while for membrane-bound receptor the subunits of mol wt 40 000 and 50 000 were labeled. The results favor the assumption that the specific ligand binding sites are located on mol wt 40 000 subunits and labeling of the other subunits reflects (a) their proximity to the ligand-binding site and (b) alterations in subunit topography between membrane-bound and solubilized states.     

Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding

The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[¹²⁵I]bungarotoxin but not [³H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [³H]acetylcholine, [³H]decamethonium, [³H]nicotine, [¹⁴C]dimethyl-d-tubocurarine, and α-[¹²⁵I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing.The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (K_D = 0.02 μM) and 5.1 nmoles a lower affinity (K_D = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.     

Purification and characterization of a nicotinic acetylcholine receptor from chick brain

Immunohistochemical studies have previously shown that both the chick brain and chick ciliary ganglion neurons contain a component which shares antigenic determinants with the main immunogenic region of the nicotinic acetylcholine receptor from electric organ and skeletal muscle. Here we describe the purification and initial characterization of this putative neuronal acetylcholine receptor. The component was purified by monoclonal antibody affinity chromatography. The solubilized component sediments on sucrose gradients as a species slightly larger than Torpedo acetylcholine receptor monomers. It was affinity labeled with bromo[3H]acetylcholine. Labeling was prevented by carbachol, but not by alpha-bungarotoxin. Two subunits could be detected in the affinity-purified component, apparent molecular weights 48 000 and 59 000. The 48 000 molecular weight subunit was bound both by a monoclonal antibody directed against the main immunogenic region of electric organ and skeletal muscle acetylcholine receptor and by antisera raised against the alpha subunit of Torpedo receptor. Evidence suggests that there are two alpha subunits in the brain component. Antisera from rats immunized with the purified brain component exhibited little or no cross-reactivity with Torpedo electric organ or chick muscle acetylcholine receptor. One antiserum did, however, specifically bind to all four subunits of Torpedo receptor. Experiments to be described elsewhere (J. Stollberg et al., unpublished results) show that antisera to the purified brain component specifically inhibit the electrophysiological function of acetylcholine receptors in chick ciliary ganglion neurons without inhibiting the function of acetylcholine receptors in chick muscle cells. All of these properties suggest that this component is a neuronal nicotinic acetylcholine receptor with limited structural homology to muscle nicotinic acetylcholine receptor.     

Separate sites of low and high affinity for agonists on Torpedo californica acetylcholine receptor

We have studied alkylation of the membrane-bound acetylcholine receptor (AcChR) from Torpedo californica electric organ by the cholinergic agonist bromo-acetylcholine (BrAcCh). Following reduction of the AcChR with dithiothreitol (DTT) under strictly controlled conditions, a single class of binding sites was covalently labeled by BrAcCh. The extent of alkylation was dependent on the concentration of both DTT and BrAcCh and reached a maximum when a number of sites equivalent to the number of alpha-bungarotoxin (alpha-BTx) binding sites were labeled. The reaction with BrAcCh was completely inhibited by saturating concentrations of alpha-BTx. On the contrary, complete alkylation of the AcChR with [3H]BrAcCh consistently inhibited only approximately 50% of alpha-BTx binding. The effects of DTT reduction and subsequent BrAcCh alkylation on the cation-gating properties of the AcChR were investigated in rapid kinetic experiments. DTT reduction resulted in a slight decrease in the maximum cation flux and a small shift in the effective dissociation constant to higher acetylcholine (AcCh) concentration. The flux response was completely inhibited by maximal alkylation of the membrane vesicles by BrAcCh. A low-affinity binding site for AcCh, which is likely to be important in AcChR activation, has been revealed for T. californica AcChR by studying the effects of cholinergic ligands on the fluorescence of a probe, 4-[(iodoacetoxy)ethylmethylamino]-7-nitro-2,1,3-benzoxadiazole (IANBD), covalently bound to the AcChR protein. Maximal labeling by BrAcCh did not affect the binding of AcCh to the low-affinity binding site, as monitored by changes in the fluorescence of this probe. This low-affinity binding site must therefore be distinct from the site labeled by BrAcCh. The results strongly support the notion that the nicotinic AcChR contains multiple binding sites for cholinergic ligands.     

Purification and characterization of the glycine receptor of pig spinal cord

A large-scale purification procedure was developed to isolate the glycine receptor of pig spinal cord by affinity chromatography on aminostrychnine agarose. After an overall purification of about 10 000-fold, the glycine receptor preparations contained three major polypeptides of Mr 48 000, 58 000, and 93 000. Photoaffinity labeling with [3H]strychnine showed that the [3H]strychnine binding site is associated with the Mr 48 000 and, to a much lesser extent, the Mr 58 000 polypeptides. [3H]Strychnine binding to the purified receptor exhibited a dissociation constant KD of 13.8 nM and was inhibited by the agonists glycine, taurine, and beta-alanine. Gel filtration and sucrose gradient centrifugation gave a Stokes radius of 7.1 nm and an apparent sedimentation coefficient of 9.6 S. Peptide mapping of the [3H]strychnine-labeled Mr 48 000 polypeptides of purified pig and rat glycine receptor preparations showed that the strychnine binding region of this receptor subunit is highly conserved between these species. Also, three out of six monoclonal antibodies against the glycine receptor of rat spinal cord significantly cross-reacted with their corresponding polypeptides of the pig glycine receptor. These results show that the glycine receptor of pig spinal cord is very similar to the well-characterized rat receptor protein and can be purified in quantities sufficient for protein chemical analysis.     

Purification of the dihydropyridine receptor of the voltage-dependent Ca2+ channel from skeletal muscle transverse tubules using (+) [3H]PN 200-110

The rabbit skeletal muscle T-tubule membranes preparation is the richest source of organic Ca2+ blocker receptor associated with the voltage-dependent Ca2+ channel. Solubilization by 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) in the presence of glycerol leads to a 52% recovery of active receptors as determined by (+)[3H]PN 200-110 binding experiments. The dissociation constant of the (+) [3H]PN 200-110 solubilized-receptor complex was 0.4 +/- 0.2 nM by equilibrium binding and 0.13 nM from the rate constants of association (k1 = 0.116 nM-1 min-1) and dissociation (k-1 = 1.5 10(-2) min-1). The (+) [3H]PN 200-110 receptor has been substantially purified by a combination of filtration of Ultrogel A2 column and lectin affinity chromatography in the presence of trace amount of specifically bound (+) [3H]PN 200-110. The purified material contained polypeptides of apparent molecular weights of 142 000, 32 000 and 33 000. These three components copurified with (+)[3H]PN 200-110 binding activity.     

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.     

Characterization of the transient agonist-triggered state of the acetylcholine receptor rapidly labeled by the noncompetitive blocker [3H]chlorpromazine: additional evidence for the open channel conformation

The kinetics of covalent labeling of the alpha, beta, gamma, and delta chains of the acetylcholine receptor (AcChR) from Torpedo marmorata by the noncompetitive blocker [3H]chlorpromazine ([3H]CPZ) are investigated by using rapid mixing photolabeling techniques. In an initial study [Heidmann, T., & Changeux, J. P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1897-1901], it was shown that the rate of [3H]CPZ labeling increases 100-1000-fold upon simultaneous addition of nicotinic agonists to the AcChR and that prior addition of these agonists abolishes the effect. The data were interpreted in terms of the rapid labeling of the transient active state of the AcChR where the ion channel is in its open configuration. This interpretation was recently challenged [Cox, R. N., Kaldany, R. R. J., Di Paola, M., & Karlin, A. (1985) J. Biol. Chem. 260, 7186-7193] on the ground of studies with a different noncompetitive blocker, [3H]quinacrine azide, and the suggestion was made that this compound labels the rapidly desensitized closed channel conformation of the AcChR. In this paper it is shown that the rate of rapid labeling of the AcChR by [3H]CPZ decreases to negligible values upon exposure of the AcChR to nicotinic agonists, in the 100-500-ms time range. The absolute values of the rate constants of this decrease (10-15 s-1 for saturating concentrations of acetylcholine and carbamoylcholine) and their variation with agonist concentration (apparent dissociation constants of 40 microM and 0.4 mM for acetylcholine and carbamoylcholine, respectively) are those expected for the rapid desensitization of the AcChR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity chromatography of the bovine cerebral cortex A1 adenosine receptor

An approximate 140-fold purification of the A1 adenosine receptor of bovine cerebral cortex has been obtained via affinity chromatography. The affinity column consists of Affi-Gel 10 coupled through an amide linkage to XAC, a high-affinity A1 adenosine receptor antagonist. As assessed by [3H]XAC binding, bovine brain membranes solubilized with the detergent CHAPS had a specific binding activity of 1.1 pmol/mg protein. Interaction of solubilized A1 adenosine receptors with the XAC-Affi-Gel was biospecific and 30% of the receptor activity was bound by the gel. Demonstration of [3H]XAC binding in the material eluted from the column with R-PIA required insertion of receptor into phospholipid vesicles. The specific activity of the affinity column purified receptor was 146 +/- 22 pmol/mg protein with typically 5-15% of the bound receptor recovered. The purified receptor displayed high-affinity antagonist binding and bound agonists with the potency order expected of the bovine brain A1 adenosine receptor: R-PIA greater than S-PIA greater than NECA. In purified preparations, the photoaffinity probe [125I]PAPAXAC-SANPAH specifically labelled a protein of molecular mass 38,000 which has previously been shown to be the A1 adenosine receptor binding subunit.     

Evidence that the acetylcholine binding site is not formed by the sequence alpha 127-143 of the acetylcholine receptor

The sequence alpha 127-143 of the alpha subunit of the acetylcholine receptor has been proposed to contain several important features: (1) the acetylcholine binding site, (2) the only N-glycosylation site of the alpha subunit, at asparagine-alpha 141, and (3) two cysteine residues, at alpha 128 and alpha 142, that may participate in a disulfide bond known to be near the binding site. We tested these hypotheses by using antisera to receptor and its subunits and monoclonal antibodies to the synthetic peptide alpha 127-143 cyclized by a disulfide bond between alpha 128 and alpha 142. Antisera to receptor and its alpha subunit were able to immunoprecipitate the iodinated peptide, and this reaction was inhibited by soluble receptor, but not by membrane-bound receptor. alpha-Bungarotoxin did not inhibit antiserum binding to solubilized receptor. Similarly, cholinergic ligands had little or no effect on binding to immobilized receptors of anti-peptide monoclonal antibodies. In addition, these monoclonal antibodies, when bound to the receptor, did not affect toxin binding kinetics. By contrast, preincubation with concanavalin A did inhibit monoclonal antibody binding. Reduction of the receptor significantly decreased the binding of three of the monoclonal antibodies, but subsequent alkylation with N-ethylmaleimide or the affinity labeling reagent bromoacetylcholine had no additional effect on binding. A dithiothreitol concentration about 100-fold higher that the one needed to reduce the disulfide near the acetylcholine binding site was necessary to inhibit monoclonal antibody binding. We conclude that the sequence alpha 127-143 is not fully exposed on the surface when the receptor is in the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biophysical characterization of the purified alpha 1-adrenergic receptor and identification of the hormone binding subunit

The alpha 1-adrenergic receptor has been solubilized in active form from rat hepatic membranes with the nonionic detergent, digitonin, and purified by affinity and gel filtration chromatography to homogeneity with a specific activity of 14,400 pmol/mg of protein. The affinity chromatographic steps of the purification procedure were achieved by the use of a newly synthesized analog (2-[4(2-succinoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, CP-57,609) of the highly selective alpha 1-adrenergic antagonist, prazosin, immobilized via an amide linkage to agarose. The resulting purified receptor bound [3H]prazosin and a variety of adrenergic agents with the specificity, stereoselectivity, and affinities equivalent to those observed with membrane-bound and solubilized receptor preparations. The purified receptor.digitonin complex had a Stokes radius of 49 A and a sedimentation coefficient (s20w) of 7.1, as determined by AcA-34 gel filtration chromatography and sucrose gradient density centrifugation, respectively. Based on these hydrodynamic parameters, the calculated molecular weight of the receptor.digitonin complex was estimated at 147,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis following the final purification step revealed a single band of protein at 59,000 daltons from which [3H]prazosin binding activity could be recovered after renaturation of the receptor protein. These findings indicate that the protein purified from rat hepatic membranes is the hormone binding component of the alpha 1-adrenergic receptor and that the receptor molecule most likely contains more than one Mr = 59,000 subunit.     

Solubilisation and Characterisation of a Putative Quisqualate-Type Glutamate Receptor from Chick Brain

The brains of 1-day-old chicks were shown to be a rich source of binding sites with the pharmacological characteristics expected of a quisqualate-type glutamate receptor. alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) bound with KD and Bmax values, measured at 0 degree C in the presence of the chaotrope potassium thiocyanate, of 55 nM and 2.6 pmol/mg protein. The regional localisations of [3H]AMPA and [3H]kainate binding sites were manifestly different. The membrane-bound [3H]AMPA binding sites were efficiently solubilised by N-octyl-beta-D-glucopyranoside (1%) in the presence of 0.2 M thiocyanate. In the detergent extract the affinity was 69 nM and there was an apparent increase in the number of sites (Bmax, 4.6 pmol/mg protein). The rank order of potency for competitive ligands in displacing [3H]AMPA binding was quisqualate approximately AMPA greater than 6-cyano-7-nitroquinoxaline-2,3-dione greater than L-glutamate greater than kainate and was identical for the membrane-bound and solubilised sites. Dissociation was biphasic with rate constants of 0.117 min-1 and 0.015 min-1. The association rate constants for [3H]AMPA at the solubilised sites were 1.45 x 10(6) M-1 min-1 and 6.55 x 10(6) M-1 min-1. The kinetically derived KD values were 80.7 nM and 2.3 nM. The detection of higher affinity binding sites by kinetic analysis but not by equilibrium binding may be explained by the greater sensitivity of dissociation data to small populations of high-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

A monoclonal antibody capable of modulating opioid binding to rat neural membranes

A monoclonal antibody capable of inhibiting opioid binding to rat neural membranes has been produced. Spleen cells from a BALB/c mouse, immunized with a partially purified opioid receptor complex, were fused with P3-X63.Ag8.653.3 myeloma cells. The cell line OR-689.2.4 secreted an IgM that was capable of partially inhibiting opioid binding to rat neural membranes under equilibrium binding conditions, while not affecting the binding of nonopioid ligands. Control mouse immunoglobulins and heat-denatured OR-689.2.4 did not inhibit opioid binding to membranes. The purified immunoglobulin inhibited the binding of [3H]dihydromorphine in a titrable, saturable, and reversible manner, as well as the binding of the delta-ligand [3H][D-Ala2,D-Leu5]enkephalin, the kappa-ligand [3H] ethylketocyclazocine, and 3H-labeled antagonists. In addition to blocking the binding of opioids to membranes, the immunoglobulin could also displace bound [3H]dihydromorphine from neural membranes. The 125I-labeled immunoglobulin specifically bound to neural membranes with a Kd of 1.3 nM and a maximal number of binding sites of 41.8 fmol/0.25 mg of membrane protein. In a titrable manner, the immunoglobulin precipitated opioid binding sites from a solubilized preparation of neural membranes. When OR-689.2.4 conjugated to Sepharose was incubated with the partially purified opioid receptor complex, labeled with 125I, a 35,000-dalton protein was specifically bound by the immunoglobulin. This antibody provides a tool for probing the multiple opioid binding sites.     

The mammalian beta 2-adrenergic receptor: purification and characterization

The beta 2-adrenergic receptors from hamster, guinea pig, and rat lungs have been solubilized with digitonin and purified by sequential Sepharose-alprenolol affinity and high-performance steric-exclusion liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveal a peptide with an apparent Mr of 64 000 in all three systems that coincides with the peptide labeled by the specific beta-adrenergic photoaffinity probe (p-azido-m-[125I]iodobenzyl)carazolol. A single polypeptide was observed in all three systems, suggesting that lower molecular weight peptides identified previously by affinity labeling or purification in mammalian systems may represent proteolyzed forms of the receptor. Purification of the beta-adrenergic receptor has also been assessed by silver staining, iodinated lectin binding, and measurement of the specific activity (approximately 15 000 pmol of [3H]dihydroalprenolol bound/mg of protein). Overall yields approximate 10% of the initial crude particulate binding, with 1-3 pmol of purified receptor obtained/g of tissue. The purified receptor preparations bind agonist and antagonist ligands with the expected beta 2-adrenergic specificity and stereoselectivity. Peptide mapping and lectin binding studies of the hamster, guinea pig, and rat lung beta 2-adrenergic receptors reveal significant similarities suggestive of evolutionary homology.     

The gamma-aminobutyric-acid/benzodiazepine-receptor protein from rat brain. Large-scale purification and preparation of antibodies

The gamma-aminobutyric-acid-receptor protein complex from rat brain was solubilized in high yield, purified in milligram amounts by benzodiazepine affinity chromatography and used to generate a high-titer rabbit antiserum. High concentrations of Triton X-100 detergent plus KCl solubilized about 90% of the membrane-bound gamma-aminobutyric acid receptor (assayed by [3H]muscimol binding) and benzodiazepine receptor (assayed by [3H]flunitrazepam binding) activities. Both activities were retained on an affinity column using an immobilized benzodiazepine ligand, and most of the column-absorbed receptor could be eluted by a solution of free benzodiazepine plus 4 M urea. The purified protein bound [3H]muscimol and [3H]flunitrazepam with receptor-like pharmacological specificity and specific activities of about 1700 pmol and 700 pmol bound/mg protein, respectively, for the two ligands. This corresponds to a purification of over 600-fold and a near theoretical purity, with a yield of milligram quantities from 100 g brain. Four peptide bands were observed on gel electrophoresis in sodium dodecyl sulfate, with molecular mass values of 31, 47, 52 and 57 kDa. The latter two were most significantly stained, and identified as receptor subunits by photolabeling with [3H]flunitrazepam (52 kDa) and [3H]muscimol (57 kDa), and by reaction on Western blots with monoclonal antibodies to this protein produced by Schoch et al. [(1985) Nature (Lond.) 314, 168-171]. Rabbit antiserum was raised to the purified protein and could, at high dilutions, both coprecipitate soluble gamma-aminobutyric-acid/benzodiazepine-receptor-binding activities and stain the receptor subunits (principally 52-kDa band) on Western blots.     

The interaction of fibronectin fragments with fibroblastic cells

We have examined the interaction of the purified cell-binding domain of fibronectin with fibroblastic baby hamster kidney cells. When the cell-binding region of fibronectin is part of a large 75,000-dalton fragment, the direct binding of the tritium-labeled fragment to cells in suspension can be observed. There is a single class of 10(5) sites/cell with an apparent dissociation constant of 4 X 10(-7) M. When the cell-binding region is part of a smaller 11,500-dalton fragment, an interaction with cells can only be observed indirectly via inhibition assays. The apparent affinity of this fragment for the cell surface fibronectin receptor is low. This 11,500-dalton fragment competitively inhibits both the direct binding of soluble [3H]fibronectin to cells in suspension and the spreading of cells on fibronectin-coated substrates, suggesting that the fragment binds to the same receptor site as intact fibronectin. Possible models describing the mechanism of the interaction of fibronectin with its receptor are proposed.     

The nicotinic acetylcholine receptor from Discopyge tschudii: purification, characterization and reconstitution into liposomes

The nicotinic acetylcholine receptor from Discopyge tschudii electroplax was purified by affinity chromatography on Affi-Gel 401 using bromoacetylcholine as the ligand. Its specific activity was about 4000 pmol 125I-alpha-bungarotoxin/mg protein. SDS-polyacrylamide gel electrophoresis revealed four bands of apparent molecular weights: 41,200, 49,500, 60,000 and 66,300. The amino acid composition of each individual subunit was determined. Native membranes, rich in nicotinic receptor exhibited carbamylcholine-catalysed cation transport (blocked by curare and desensitized by prior incubation with the cholinergic agonist). The functional activity of the purified material could be reconstituted into soybean lecithin liposomes. Our data show that the Discopyge tschudii nicotinic receptor is similar to that from Torpedo californica.     

Guanine nucleotide regulation of [3H]vasopressin binding to liver plasma membranes and solubilized receptors. Evidence for the involvement of a guanine nucleotide regulatory protein.

A guanine nucleotide regulatory protein may be involved in vasopressin-receptor-mediated polyphosphoinositide breakdown in rat liver. Therefore we examined the effects of the non-hydrolysable guanine nucleotide guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) on [3H]vasopressin ([3H]AVP) binding to hepatic plasma membranes and detergent extracts. [3H]AVP bound to a single set of high-affinity binding sites in membranes. Addition of p[NH]ppG decreased the affinity of receptor binding without altering the maximal binding capacity. The rate of dissociation of [3H]AVP from membrane-bound receptors was also enhanced by p[NH]ppG. Solubilization of [3H]AVP-prelabelled membranes with dodecyl beta-D-maltoside resulted in a [3H]AVP-receptor complex that was unstable in solution. Incubation of these extracts for 5 min at 30 degrees C resulted in a 40% loss of bound [3H]AVP, whereas in the presence of p[NH]ppG there was a 54% loss. However, when membranes were prelabelled with [3H]AVP and p[NH]ppG and then solubilized, the resulting hormone-receptor complex was still temperature-labile but insensitive to the further addition of p[NH]ppG. The molecular size of soluble vasopressin receptors was estimated by gel filtration. The [3H]AVP-receptor complex was eluted as a single peak with an apparent molecular size of 258 kDa. However, no peak was detected when solubilized extract was made from membranes prelabelled with [3H]AVP and p[NH]ppG, suggesting that this receptor complex had dissociated during chromatography. It is possible therefore that the high-Mr complex contains the hormone, its receptor and a guanine nucleotide binding protein.