Characterization of acetylcholine receptor-rich and acetylcholinesterase-rich membrane particles from Torpedo californica electroplax

Large-scale purification of acetylcholinesterase-rich and acetylcholine receptor-rich membrane fragments from Torpedo californica electroplax is described. Electron microscopy studies reveal structural differences in the two types of particles and the results are discussed in terms of structural aspects of the postsynaptic cleft. Polyacrylamide gel electrophoresis of receptor-rich fragments reveals that the fragments contain the same polypeptide components observed in receptor preparations purified from the same electroplax membranes, indicating that purified Torpedo receptor is not composed of species degraded by proteolysis. Results obtained from fluorescence studies of a cholinergic analog allow conclusions to be reached regarding species differences in electroplax acetylcholine receptor preparations.     

Some molecular properties of an isolated acetylcholine receptor ion-translocation protein

An improved procedure for isolation and purification of acetylcholine receptor from Torpedo californica electroplax membranes is described. The purified material contains the neurotransmitter recognition site and a second binding subsite which complexes inorganic cations and bis-quaternary cholinergic analogs. In addition to the transmitter recognition site the isolated macromolecule contains the molecular features necessary for ion-translocation during postsynaptic depolarization, since a chemically excitable membrane can be formed from purified acetylcholine receptor and Torpedo phospholipids.     

cAMP,not Ca2+/calmodulin,regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Conductance Properties of Artificial Lipidic Membranes Containing a Proteolipid from Electrophorus : Response to cholinergic agents

Previous studies have shown that a special proteolipid extract from the electric organ of Electrophorus showed high affinity binding for acetylcholine and other cholinergic agents. This proteolipid has now been incorporated into ultrathin lipidic membranes, and the membrane resistance was studied. The resistance decreased from 7.27 ± 0.82 x 10⁵ ohm cm² in the control membrane to 1.83 x 10⁵ ohm cm² with addition of 72 µg/ml proteolipid. The decrease in resistance followed a potential function of order four with the proteolipid concentration in the membrane-forming solution. The presence of this proteolipid determined some type of cationic selectivity which was not observed in the control. At a critical point of proteolipid concentration the conductance spontaneously fluctuated between two levels. The membrane current jumped from one state to another by way of single discrete steps, reminiscent of those obtained with the excitatory inducing material or the macrocyclic antibiotics. In membranes containing another proteolipid having no cholinergic binding properties, the increase in conductance was smaller, and had a linear function with the concentration. In this case the "flip flop" fluctuation and the cationic selectivity were not observed. The membranes containing the cholinergic proteolipid reacted to the addition of acetylcholine by a rapid and transient increase in conductance that was considerably reduced or abolished by a previous application of d-tubocurarine. These membranes also interacted with other cholinergic agents, such as gallamine triethiodide, hexamethonium, and α-bungarotoxin. These results suggest that this special proteolipid, when added to the artificial membranes, induces a "chemical excitability" toward cholinergic ligands.     

Effect of cholinergic ligands and local anesthetics on acetylcholine receptor enriched membrane preparations from Torpedo californica electroplax.

Pyrene was introduced in acetylcholine receptor (AcChR)-rich membrane preparations of Torpedo californica electroplax. The lifetime of the singlet excited state of pyrene was used to probe the properties of the hydrocarbon regions of the lipid bilayer as well as the possible perturbing effects of cholinomimetic agents on this region. After excitation with a single 15-ns pulse with a Q-switched ruby laser, the lifetime of the pyrene singlet excited state in the membranes was 200 ns. In desensitized membranes the pyrene fluorescence lifetimes remained unchanged when the cholinergic ligands carbamylcholine, d-tubocurarine, decamethonium, and hexamethonium, as well as α-bungarotoxin, were present. By contrast, the lifetime was shortened when local anesthetics were present. In sensitized membranes no changes in the pyrene lifetimes were detected when the membranes were converted from their resting state to a carbamylcholine-induced “desensitized state.” Water-soluble fluorescence quenchers affected the lifetime of pyrene in membranes. The second order rate constants for the pyrene-quencher interaction were used to detect changes in fluidity and/or membrane lipid accessibility to quenchers induced by ligands or anesthetics. No changes were detected in the quenching constants of nitromethane or Tl⁺ in the presence of cholinergic agents (with the exception of d-tubocurarine); on the other hand, a marked decrease in Tl⁺ accessibility was induced by the anesthetics procaine and tetracaine. Fluorescene dynamics measurements indicate that the hydrocarbon core of the bulk lipid in electroplax is not significantly affected by binding cholinergic ligands to membranebound AcChR. However, the hydrophobic region of the membrane is perturbed by both local anesthetics and one cholinergic ligand, d-tubocurarine. Pyrene was also incorporated into lipid vesicles prepared from T. californica electroplax lipids. The fluorescence lifetimes and quenching values of these lifetimes yielded results similar to those obtained with both sensitized and “desensitized” membrane preparations. The d-tubocurarine effect on the Tl⁺ quenching of the pyrene probe is ascribed to direct interaction of d-tubocurarine with the lipids. These findings favor a mechanism in which perturbation of the hydrophobic (lipid) environment of the AcChR in membranes by local anesthetics and even d-tubocurarine may influence the receptor conversion: sensitized state ? desensitized state.     

Lipid bilayers and liposomes in reconstitution experiments with cholinergic proteolipid from torpedo electroplax

The cholinergic proteolipid from $\text{Torpedo}$ electroplax was used in reconstitution experiments in artificial membranes being incorporated directly into the membrane-forming solution or into liposomes (proteoliposomes) which interacted with lecithin bilayers. In both cases the membrane became reactive to acetylcholine by a decrease in resistance and an increase in the frequency and amplitude of minute current fluctuations of 3·10^?11 to 4·10^?10mho. The injection of d-tubocurarine prod$\text{u}$ ced an increase in membrane resistance and a decrease in the amplitude of the current fluctuations. These results suggest that the cholinergic proteolipid is reconstituted in an active form in the bilayer.     

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.     

Detection with Monoclonal Antibodies of a 15-kDa Proteolipid in Both Presynaptic Plasma Membranes and Synaptic Vesicles in Torpedo Electric Organ

A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15-kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15-kDa proteolipid band on Western blots of purified mediatophore were selected. A 15-kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15-kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti-15-kDa proteolipid monoclonal antibodies.     

cAMP, not Ca/calmodulin, regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Interactions of polyclonal anti-Electrophorus nicotinic receptor antisera with Torpedo nicotinic receptor

Polyclonal antisera raised against solubilized and purified nicotinic acetylcholine receptor (nAcChoR) from Electrophorus electroplax and a polyclonal anti-alpha-bungarotoxin antiserum have been studied by the use of four different radioimmunoassay protocols. The results indicate unique sensitivities of different assay techniques in analysis of antibody-antigen interactions, and serve as a model for immunological study of other integral membrane proteins.     

The incorporation of partially delipidized proteolipid proteins from Torpedo marmorata electroplax into liposomes

The preparation of proteoliposomes from pure phospholipids and partially delipidized proteolipid proteins from Torpedo marmorata electroplax is described. The resulting vesicles are morphologically different from their lipid counterparts, the main feature being a reduction in the number of lamellae. These structures are highly permeable toward Rb⁺, K⁺, or glucose. The association between the proteolipid proteins and the phospholipids is not modified by increasing the amount of acidic phospholipids or cholesterol in the liposomes. The partially delipidized proteolipid proteins are capable of reducing the phasetransition temperature of dipalmitoylphosphatidylcholine. It is suggested that during the liposome formation procedure there is an interaction between the proteolipid proteins and the lipids, probably via hydrophobic associations. This gives rise to highly permeable, more fluid structures compared to pure phospholipid vesicles.     

An electrophoretic analysis of proteolipids from different rat brain subcellular fractions

Proteolipid proteins were extracted from adult rat brain subcellular fractions and purified by chromatography on Sephadex LH-60. Polyacrylamide gel electrophoresis of the delipidized proteins, in the presence or absence of 8 M urea, was carried out with all fractions. The distribution of the various types of proteolipid proteins was studied and their molecular weight calculated by the Ferguson relationship. Several bands of proteolipid proteins were found in the five membrane fractions analyzed. Some of them, such as the 17.5 K and 37 K components were very prominent in mitochondria and synaptosomes. The 30 K component was found in myelin-derived membranes and in microsomes, while the 20 K and 25 K proteolipid proteins were present in all subcellular fractions. The 30 K component (proteolipid protein (PLP)), typical of the purified myelin membranes, showed a similar distribution to that of 2′,3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37) activity, while the other major proteolipid protein present in all subcellular fractions (25 K) did not show such parallelism, indicating that it might not be an exclusive component of myelin. The electrophoretic pattern of microsomal proteolipid proteins did not show the high molecular weight components (aggregates of PLP) which are found in myelin. Furthermore, the 30 K component showed a smaller $\text{Y}{}_0$ value than that of the 30 K found in myelin. Thus the presence of 30 K proteolipid protein in microsomes should not be considered as being due to myelin contamination.     

Pyrenesulfonyl azide as a fluorescent label for the study of protein-lipid boundaries of acetylcholine receptors in membranes

Acetylcholine receptor (AcChR) enriched membrane fragments from Torpedo californica electroplax were labeled by in situ photogenerated nitrenes from a hydrophobic fluorescent probe, pyrene-1-sulfonyl azide. Preferential photolabeling of membrane proteins, mainly AcChR, has been achieved and there is a pronounced exposure of the 48,000 and 55,000 molecular weight subunits of AcChR to the lipid environment of the membrane core. Covalent attachment of the photogenerated fluorescence probe does not perturb the α-neurotoxins' binding properties of membrane-bound AcChR or the desensitization kinetics induced by prolonged exposures to cholinergic agonists. Non-covalent photoproducts can be conveniently removed from labeled membrane preparations by exchange into lipid vesicles prepared from electroplax membrane lipids. Fluorescence features of model pyrene sulfonyl amide derivatives, such as fine vibrational structure of emission spectra or fluorescence lifetimes, are highly sensitive to the solvent milieu. The covalently bound probe shows similar fluorescence properties in situ. PySA photoproducts have great potential to spectroscopically monitor neurotransmitter induced events on selected AcChR subunits exposed to the hydrophobic environment of membranes.     

ISOLATION AND PURIFICATION OF CHOLINERGIC RECEPTOR PROTEOLIPIDS FROM RAT GASTROCNEMIUS TISSUE1

Four organic solvent extraction methods have been used to isolate receptor proteolipids from rat gastrocnemius tissue. The resulting proteolipids have high binding capacities and specificities for a number of cholinergic ligands including acetylcholine, α-bungarotoxin. tubocurarine and deca-methonium. The proteolipids have been purified by repeated Sephadex LH-20, Sepharose-CL and affinity chromatography in organic solvent systems. Protein, carbohydrate and lipid phosphorous analysis of the receptors throughout the purification procedure show that the relative proportions of these components varied with extraction method. The purified receptor proteolipid resulting from wet tissue extraction has been characterized as a somatic, nicotinic cholinergic receptor by its specificity and binding kinetics toward a variety of drugs and toxins. The presence of carbohydrate in the receptor proteolipid preparations throughout the purification procedure may indicate that the cholinergic receptor from rat gastrocnemius tissue is a glycoproteolipid.     

Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.     

Author index

The membrane-bound ATPase activity of Bacillus subtilis was inhibited by dicyclohexylcarbodiimide (DCCD). The DCCD-reactive proteolipid of B. subtilis was extracted, from labelled or untreated membranes containing F₁ or depleted of F₁, with neutral or acidic chloroform/methanol. Purification of the [₁₄C]DCCD-binding proteolipid was attempted by column chromatography on methylated Sephadex G-50 and on DEAE-cellulose. The maximal amount of DCCD which could be bound to the purified proteolipid was found to exceed the amount bound by the purified proteolipid extracted from membranes labelled with the lowest [₁₄C]DCCD concentration required for maximal inhibition of the membrane-bound ATPase activity. The radioactive protein peaks eluted by gel filtration and ion-exchange chromatography were analysed by urea-SDS polyacrylamide slab gel electrophoresis and autoradiography. Radioactivity was incorporated into two components of M_r 18 000 and 6000 when proteolipid was purified by methylated Sephadex. The 6000 polypeptide was always present, whatever the extraction and purification procedures. However, the 18 000 polypeptide was present in largest quantity only when proteolipid was extracted from membranes containing F₁ and purified by methylated Sephadex. When proteolipid was purified on DEAE-cellulose this [₁₄C]DCCD binding component of M_r 18 000 was absent.     

Two pools of cholesterol in acetylcholine receptor-rich membranes from Torpedo

The acetylcholine receptor (AChR)-containing electroplax membranes from Torpedo californica have a relatively high cholesterol content. Reconstitution studies suggest that this cholesterol may be important in preserving or modulating the function of the acetylcholine receptor-channel complex. We have manipulated cholesterol levels in intact Torpedo AChR-rich membrane fragments using small, unilamellar phosphatidylcholine liposomes. Conditions have been established that allow further subfractionation of sucrose gradient purified Torpedo electroplax membranes into AChR-rich and ATPase-rich populations and that, at the same time, achieve cholesterol depletion without phospholipid back exchange or fusion. The incubation of membranes with excess liposomes could only achieve about a 50% reduction in the molar ratio of cholesterol to phospholipid. In no case was the number of cholesterol molecules per AChR oligomer reduced below 36. The remaining cholesterol could not be depleted either by longer incubations or by multiple, sequential depletions. Cholesterol depletion was accompanied by a significant increase in bulk membrane fluidity as measured by electron spin resonance spectroscopy, but the equilibrium binding parameters of acetylcholine to its receptor were unaltered. This suggests strongly that there exist two pools of cholesterol in the AChR-rich Torpedo electroplax membrane: an easily depleted fraction that influences bulk fluidity, and a tightly-bound fraction perhaps surrounding the AChR oligomer.     

Thymopoietin, a Thymic Polypeptide, Specifically Interacts at Neuronal Nicotinic α-Bungarotoxin Receptors

alpha-Bungarotoxin (alpha-BGT), a snake venom polypeptide, interacts potently and specifically with a nicotinic receptor population in neuronal tissue. However, the identity of this site is unclear, because, unlike at the neuromuscular junction and in electroplax, in nervous tissue the toxin does not block nicotinic cholinergic responses. Therefore, we sought endogenous compounds other than acetylcholine that could interact with the neuronal alpha-BGT site. In the present experiments, thymopoietin, a polypeptide isolated from the thymus, is shown to inhibit potently alpha-BGT binding to brain membranes in a dose-dependent manner (IC50 = 3.1 nM). This effect was not shared by a wide variety of other peptides, including thysplenin, a closely related polypeptide. Thymopoietin did not inhibit the binding of other radioligands known to interact with different populations of cholinergic receptors, such as [3H]nicotine and [3H]methylcarbachol, which bind to nicotinic receptors, or [3H]quinuclidinylbenzilate, which binds to muscarinic receptors. These results show that thymopoietin potently and specifically affects 125I-alpha-BGT binding to brain membranes and suggest that thymopoietin might be an endogenous ligand for alpha-BGT receptors in neuronal tissue.     

Characterization of acetylcholine receptor isolated from Torpedo californica electroplax through the use of an easily removable detergent, β-d-octylglucopyranoside

Non-ionic detergents used for the solubilization and purification of acetylcholine receptor from Torpedo californica electroplax may remain tightly bound to this protein. The presence of detergent greatly hinders spectrophotometric and hydrodynamic studies of the receptor protein. β-d-Octylglucopyranoside, however, is found to be effective in solubilizing the receptor from electroplax membranes with minimal interference in the characterization of the protein. The acetylcholine receptor purified from either octylglucopyranoside or Triton X-100-solubilized extracts exhibits identical amino acid compositions, α-Bungarotoxin and (+)-tubocurarine binding parameters, and subunit distributions in SDS-polyacrylamide gels. The use of octylglucopyranoside allows for the assignment of a molar absorptivity for the purified receptor at 280 nm of approx. 530 000 $\text{M}{}^{\mathrm{?1}}\text{·}\text{cm}{}^{\mathrm{?1}}$. Additionally, successful reconstitution of octylglucopyranoside-extracted acetylcholine receptor into functional membrane vesicles has recently been achieved (Gonzales-Ros, J.M., Paraschos, A. and Martinez-Carrion, M. (1980) Proc.Natl. Acad. Sci. U.S.A. 77, 1796–1799).Removal of octylglucopyranoside by dialysis does not alter the specific toxin and antagonist binding ability of the receptor or its solubility at low protein concentrations. Sedimentation profiles of the purified acetylcholine receptor in sucrose density gradients reveal several components. Sedimentation coefficients obtained for the slowest sedimenting species agree with previously reported molecular weight values. Additionally, the different sedimenting forms exhibit distinctive behavior in isoelectric focusing gels. Our results suggest that both the concentration and type of detergent greatly influence the physicochemical behavior of the receptor protein.     

Neurotoxin-modulated uptake of sodium by highly purified preparations of the electroplax tetrodotoxin-binding glycopeptide reconstituted into lipid vesicles

Summary Using the dialysable detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), the tetrodotoxin-binding protein from the electroplax of the electric eel has been purified to a high degree of both chemical homogeneity and toxin-binding activity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the best preparations showed only a single microheterogeneous band atM _r approximately 260,000, despite attempts to visualize smaller bands by sample overloading. Upon dialysis, this material became incorporated into the membranes of small unilamellar vesicles, and in this form the purified protein exhibited tetrodotoxin-binding properties similar to the component in the original electroplax membrane. Furthermore, in the presence of activator neurotoxins the vesicles were able to accumulate isotopic sodium in a manner similar to that previously described for less active or less pure preparations of vesicles containing either mammalian or eel electroplax toxinbinding proteins. Quantitative consideration of the isotopic transport activity of this pure material, along with the high degree of purity of the protein, strongly suggests that the 260-kDa glycopeptide from electroplax is necessary and sufficient to account for the sodium channel function seen in these studies, and eliminates the possible involvement of smaller peptides in the channel phenomena observed.