Histrionicotoxin and alkylguanidine interactions with the solubilized and membrane-bound muscarinic acetylcholine receptor from porcine atria

The interaction of alkylguanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkylguanidines with alkyl chain lengths from one to ten carbons displaced l-[³H]quinuclidinyl benzilate (l-[³H]QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of ?ln K_i versus alkyl carbon chain number, a value of ?(473 ± 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The binding of alkylguanidines to the digitonin/cholate solubilized mAcChR was complex in nature resulting in titration curves that did not obey the law of mass action for simple competitive inhibition at higher alkyl carbon numbers and a sigmoidal plot of ?ln K_i versus carbon number. Decahydrohistrionicotoxin bound in a competitive manner versus l-[³H]QNB to both the membrane-bound (K_i = (6.9 ± 1.4) × 10^?6 M) and the solubilized (K_i = (1.5 ± 0.3) × 10^?5 M) preparations.     

Photoaffinity labeling of the solubilized, partially purified muscarinic acetylcholine receptor from porcine atria by p-azidoatropine methyl iodide

The synthesis of a tritiated photoaffinity analogue of the muscarinic antagonist atropine, [3H]-p-azidoatropine methyl iodide is described. The compound appeared to bind to a single class of sites in membrane-bound, solubilized, and partially purified preparations of muscarinic receptor from porcine atria with a dissociation constant (determined by competition vs. [3H]-L-quinuclidinyl benzilate) of about 1.0 X 10(-7) M. This value was in agreement with the apparent dissociation constant (8.5 X 10(-8)M) determined by measuring the concentration dependence of covalent incorporation into a partially purified receptor preparation. Competition experiments indicated that the specific covalent labeling could be blocked by the muscarinic agonist carbamylcholine and the antagonists L-quinuclidinyl benzilate and atropine. An apparent molecular weight of 75 000 +/- 5000 was found for specifically labeled peptide(s) in a solubilized, partially purified receptor preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Preparation and characterization of muscarinic-acetylcholine-receptor-enriched membranes from pig atria.

A procedure was developed for the large scale preparation of membranes from pig atria which are enriched 10-13 fold in the muscarinic acetylcholine receptor. The procedure involved differential centrifugation and sucrose-gradient centrifugation in solutions containing 150 mM-NaClO4 and 5 mM-EDTA to minimize membrane aggregation. The final membrane preparation bound about 1.1 pmol of L-quinuclidinyl benzilate/mg of protein. Comparable results were obtained with either fresh or frozen tissue. About the same yield (120 pmol of L-quinuclidinyl benzilate sites/100 g of tissue) and specific activity of membranes were obtained from different regions of the atria. The final preparation was stable at -80 degrees C in buffered sucrose solutions. The membranes appeared mostly as sheets or fragments and partly as closed vesicles in the electron microscope and were heterogeneous in isopycnic Percoll gradients. Marker enzyme studies showed that the receptor was enriched in parallel with the plasma membrane markers guanylate cyclase (particulate form) and (Na+ + K+)-activated ATPase. Some contamination by mitochondrial outer and endoplasmic reticulum membranes was evident from the distribution of monoamine oxidase and glucose-6-phosphatase activity, but the preparation was largely free of sarcoplasmic reticulum, mitochondrial inner, and lysosomal membranes.     

Heterooligomers of the muscarinic receptor and G proteins purified from porcine atria

Muscarinic receptor extracted from porcine atria in digitonin-cholate copurified with Gα_o, Gα_i1-3, and caveolins. The presence of complexes was confirmed by coimmunoprecipitation of the receptor, α-subunits, and caveolins in various combinations. Homooligomers of α_i2 were detected on Western blots, and heterooligomers of α_i2 and α_o were identified by coimmunoprecipitation; thus, a complex may contain at least two α-subunits. Other combinations of α-subunit were not detected. The ratio of total α-subunit to receptor was near 1, as measured by [³⁵S]GTPγS and the antagonist [³H]quinuclidinylbenzilate, and the binding of [³⁵S]GTPγS was manifestly biphasic. The ratio of α_o to α_i1,2 also was near 1, as determined from the intensity of Western blots. Cardiac muscarinic receptors therefore can be purified as a mixture of complexes that contain caveolins and oligomers of α-subunit, some of which are heteromeric. Each complex would appear to contain equal numbers of α-subunit and the receptor.     

Purification of the muscarinic acetylcholine receptor from porcine brain

The muscarinic acetylcholine receptor of porcine cerebrum has been purified to apparent homogeneity by affinity chromatography, with conjugated 3-(2'-aminobenzhydryloxy)tropane (ABT) as described previously (Haga, K., and Haga, T. (1983) J. Biol. Chem. 258, 13575-13579). In a single step purification using 900 ml of digitonin/cholate-solubilized preparations and 300 ml of the ABT-agarose gel, we obtained, in a yield of 10-15%, more than 250 pmol of muscarinic receptors which bind [3H]N-methylscopolamine with a specific activity of 1,000-5,000 pmol/mg of protein (1,000-5,000-fold purification). The muscarinic receptors eluted from the ABT-agarose gel with 0.1 mM atropine were adsorbed to hydroxylapatite and then recovered as a concentrated solution. Muscarinic receptors were further purified by rechromatography with the same gel or by gel permeation high pressure liquid chromatography. The amino acid composition of the purified receptor was determined, and the specific activity of the purified preparation was estimated to be 13,100 pmol/mg of protein on the basis of amino acid composition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptors with or without radioiodination revealed a single, major band with an apparent Mr of 70,000 either by silver staining or radioautogram. The major band corresponded to the band which specifically bound [3H]propylbenzylcholine mustard (irreversible muscarinic ligand). The purified receptor showed essentially the same specificity for muscarinic ligands as unpurified receptors.     

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.     

Purification and characterization of a membrane-bound sialidase from pig liver

A membrane-bound sialidase in pig liver microsomes was solubilized with a nonionic detergent, IGEPAL CA630, and purified to homogeneity by sequential chromatographies on SP-Toyopearl, Butyl-Toyopearl (1st), SuperQ-Toyopearl, Hydroxyapatite, Butyl-Toyopearl (2nd), GM1-Cellulofine affinity, and sialic acid-Cellulofine affinity columns. The molecular weight of the purified enzyme was estimated to be 57 kDa on SDS-PAGE. The pH optimum was 4.8 for the activity measured using 4-methylumbelliferyl-alpha-N-acetylneuraminic acid (4MU-Neu5Ac) as the substrate. The enzyme activity was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, iodoacetamide and p-chloromercuribenzoic acid. While the enzyme could effectively hydrolyze 4MU-Neu5Ac, it failed to significantly cleave a sialic acid residue(s) from sialyllactose, glycoproteins or gangliosides at pH 4.8. These results suggest that the purified enzyme is a novel sialidase with a substrate specificity distinct from those of known membrane-bound sialidases in mammalian tissues.     

Structural studies of a membrane-bound acetylcholine receptor from Torpedo californica

We investigated the differential repair of DNA lesions induced by bifunctional mitomycin C, monofunctional decarbamoyl mitomycin C and ultraviolet irradiation in normal human, Xeroderma pigmentosum and Fanconi's anemia cells using assays for the survival of clone-forming ability, alkaline sucrose sedimentation and hydroxyapatite chromatography of DNA. Four FA cell lines exhibited about 5 to 15 times higher sensitivity to MC killing, despite normal resistance to u.v. and DMC, than did normal human cells. The XP cells, however, were highly sensitive to u.v. and DMC killings due to their deficiency in excision repair, but the cells unexpectedly had an almost normal capacity for surviving MC and repairing the MC interstrand cross-links.In experiments to determine the sedimentation velocity of the DNA in alkaline sucrose gradients, normal and XP cells showed evidence for single-strand cutting following MC treatment. The sedimentation velocity of the DNA covalently cross-linked by MC in an FA strain was 2.5 times faster than that of the untreated control, and remained unaltered during post-incubation due to the lack of half-excision⁴ of cross-links. However, FA cells, but not XP cells, had the normal ability to incise DNA with the DMC monoadducts. Hydroxyapatite chromatography revealed the reversibly bihelical property of MC cross-linked DNA after denaturation. Normal and XP cells lost such reversibility during post-MC incubation as the result of cross-link removal with first-order kinetics (half-life = 2 h). The three FA lines studied exhibited two- to eightfold reduced rates of cross-link removal than normal and XP cells, indicating a difference in the repair deficiency of the FA strain. Thus we have been led to conclude that FA cells may have different levels of deficiency in half-excision repair of interstrand cross-links induced by MC, despite having normal mechanisms for repair of u.v.-induced pyrimidine dimers and DMC monoadducts, and vice versa in XP cells.     

Isolation and functional characterization of the chick M5 muscarinic acetylcholine receptor gene

The chick is a widely used system for study of the actions of muscarinic acetylcholine receptors in the cardiovascular, visual, and nervous systems. We report the isolation and functional analysis of the gene encoding the chick M5 muscarinic receptor. RT-PCR analysis indicates that the M5 receptor is expressed at low levels in embryonic chick brain and heart. When expressed in stably transfected Chinese hamster ovary cells, the M5 receptor exhibits high-affinity binding to muscarinic antagonists and mediates robust activation of phospholipase C activity.     

Isolation and characterization of monoclonal antibodies specific for the cardiac muscarinic acetylcholine receptor

A number of monoclonal antibodies were raised against the purified porcine atrial muscarinic acetylcholine receptor. The antibodies were shown to exhibit a high degree of specificity for the receptor by their ability to recognize the purified receptor but not other porcine atrial glycoproteins in enzyme-linked solid-phase immunosorptive assays and by immunoblot analyses. Several of the antibodies were able to quantitatively precipitate the muscarinic receptor in both pig and rat heart and a portion of the receptor from rat cerebellum but little if any receptor from rat cerebral cortex. Thus, these monoclonal antibodies not only exhibit specificity for the muscarinic receptor but also are specific for the cardiac receptor subtype.     

Phosphorylation of membrane-bound acetylcholine receptor by protein kinase C: characterization and subunit specificity

Acetylcholine receptor (AChR) from Torpedo electric organ in its membrane-bound or solubilized form is phosphorylated by the Ca2+/phospholipid-dependent protein kinase (PKC). The subunit specificity for PKC is different from that observed for cAMP-dependent protein kinase (PKA). Whereas PKC phosphorylates predominantly the delta subunit and the phosphorylation of the gamma subunit by this enzyme is very low, PKA phosphorylates both subunits to a similar high extent. We have extended our phosphorylation studies to a synthetic peptide from the gamma subunit, corresponding to residues 346-359, which contains a consensus PKA phosphorylation site. This synthetic peptide is phosphorylated by both PKA and PKC, suggesting that in the intact receptor both kinases may phosphorylate the gamma subunit at a similar site, as has been previously demonstrated by us for the delta subunit [Safran, A., et al. (1987) J. Biol. Chem. 262, 10506-10510]. The diverse pattern of phosphorylation of AChR by PKA and PKC may play a role in the regulation of its function.     

Pharmacological and biochemical characterization of complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein

Complexes of agonist-bound muscarinic acetylcholine receptor (mAChR) and guanine nucleotide-binding protein (G protein) were solubilized and isolated from rat heart. Heart membranes were incubated with mAChR agonists or antagonists, solubilized using digitonin and cholate, and subjected to chromatography over wheat germ agglutinin-Affi-Gel. Eluted fractions were precipitated using a cardiac-selective anti-mAChR antibody (Luetje, C. W., Brumwell, C., Norman, M. G., Peterson, G. L., Schimerlik, M. I., and Nathanson, N. M. (1987) Biochemistry 26, 6892-6898). Using samples obtained from membranes initially incubated with carbachol (10 nM, 100 nM, or 1 mM), G alpha immunoreactivity was detected on Western blots probed using antibodies with specificity for G alpha subunits. The G alpha immunoreactivity was not detected when atropine alone (10 nM or 1 microM) or when excess atropine (1 microM) plus carbachol (100 nM) was used during the membrane preincubation. G beta immunoreactivity, when detectable on Western blots, was present in substoichiometric amounts relative to that of G alpha. The G alpha immunoreactivity was not present if GTP was included during incubation of membranes with agonist and following membrane solubilization. Further results indicate that although agonist binding to receptors is rapidly reversed by GTP or GDP (t1/2 less than 10 min), the mAChR-G protein complex is reversed more slowly or not at all. It was also shown that at high agonist concentrations, the cardiac mAChR interacts with both Go and Gi-like proteins. Together, these results demonstrate the utility of an immunoaffinity approach to the purification and biochemical study of receptor-G protein interactions.     

Properties of the membrane-bound acetylcholine receptor from cow brain nucleus caudatus

A new procedure for isolation of the membrane-bound acetylcholine receptor from cow brain N. caudatus is proposed. The procedure described includes tissue disruption by homogenization and ultrasonication, isolation of a crude membrane fraction and subsequent washing of the membranes with 1 M NaCl and 0,6 M KCl. The receptor yield is 40 nmoles per 1 g of tissue. The receptor is of a muscarine type. The K alpha values for the complexes with acetylcholine (3,8.10(-5) M), D,L-muscarine (3,7.10(-5) M), pilocarpine (3,7.10(-5) M), methylfurmethide (3,7.10(-5) M), atropine (2,5.10(-8) M and 1,2.10(-9) M) and platyphyllin (2,3.10(-7) M) were determined. The atropine binding within the pH range of 5 to 8 is weakly dependent on concentrations of Ca2+, Mg2+ and EDTA and EGTA sodium salts as well as on pH. Atropine binding is inhibited by high concentrations of NaCl. It is concluded that N. caudatus from cow brain is a convenient source for isolation of the muscarine acetylcholine receptor.     

A microtechnique for quantification of detergent-solubilized muscarinic and nicotinic acetylcholine receptors using a semi-automated cell harvestor

A specific method for the rapid assay of muscarinic acetylcholine receptors (mAChR), either detergent-solubilized or in neuroblastoma cells, is described. This method is also applicable to the assay of nicotinic acetylcholine receptors. The procedure employs a cell harvestor and microtiter plates, and has the advantage of requiring small quantities of radioligand, microgram quantities of detergent-solubilized cholinergic receptor or less cells. The binding parameters such as the equilibrium dissociation constants (Kd) of mAChR and nicotinic acetylcholine receptor (nAChR) and inhibition constants (Ki) for antagonists determined by the present method are in excellent agreement with values determined by other methods. This assay procedure for mAChR and nAChR should facilitate the rapid screening of cholinergic agonists/antagonists and also the further purification and characterization of mAChR.     

Affinity chromatography of the muscarinic acetylcholine receptor

A novel compound, 3-(2'-aminobenzhydryloxy)-tropane (ABT), and an ABT-agarose gel were synthesized and used for the purification of solubilized muscarinic receptors. ABT had a high affinity with an apparent dissociation constant (Kd) of 7 nM for the muscarinic receptors solubilized from the porcine brain by digitonin. An ABT-agarose gel was prepared by coupling ABT with epoxy-activated Sepharose 6B, and the degree of substitution to the gel was determined to be 4-5 mumol/ml of the gel by UV absorption spectrum. During affinity chromatography using 10 ml of the ABT-agarose gel and 100 ml of the digitonin-solubilized preparation, 70% of muscarinic receptors were adsorbed to the gel, in marked contrast with the adsorption of only 2% of proteins. Approximately 25% of muscarinic receptors applied to the gel were eluted biospecifically with 1 mM muscarinic ligands. The purified fraction showed a high affinity for [3H]quinuclidinyl benzylate with a Kd of 0.4 nM and similar specificity for muscarinic ligands to that of unpurified soluble receptors. The protein concentration of the purified fraction was too low to be determined accurately, but very approximately a purification of 10(3)-fold was indicated.