Torpedo Californica Research Articles

Probing the Active Site of Acetylcholinesterase by Molecular Dynamics of Its Phosphonate Ester Adducts

Molecular dynamics (MD) simulations using CHARMM were performed for the solution structures of the pentacoordinate and tetracoordinate PSCS and PRCS adducts of Torpedo californica (Tc) acetylcholinesterase (AChE) formed with 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) to assess the molecular origins of stereoselectivity of phosphonylation. MD simulations were also carried out for the PSCS transients in soman-inhibited trypsin to evaluate the differences in the mode of operation of the two enzymes. Parameters for the pentacoordinate phosphonate fragments were constructed from results of an ab initio calculation at the 6-31G* level for a model compound, and those for the tetracoordinate phosphonate fragments were from MNDO calculations. Starting equilibrium structures for the above and for analogous structures for chymotrypsin were generated and energy-optimized in program YETI. The stereoselectivity of AChE for the levorotatory diastereomers of soman amounts to >5.6 kcal/mol difference in transition state free energies and can be rationalized based on the results of the MD calculations: There is a predominant conformation of transient forms of the PSCS diastereomer of soman-inhibited AChE in which every ligand in phosphorus is stabilized by an optimal binding feature of the active site. In contrast, the phosphonyl fragment in the PRCS diastereomer may be accommodated with equal difficulty, at least, two different ways: In the most favorable conformation, the phosphoryl oxygen is engaged in weak interactions with constituents of the oxyanion hole if adjustments in the Cα backbone and substantial motions of Trp84, Trp233, Phe288, and Phe290 are allowed. The remarkable efficiency of F- departure from the pentacoordinate transition states of phosphonylated AChE cannot be explained by general base catalysis by HisH+440. Leaving group departure from these structures must be promoted by electrostatic forces, “push” from Glu199 and “pull” from the oxyanion hole, in addition to steric strain. One of the distinguishing features of the crystal structure of TcAChE is the short H-bonds in the catalytic triad. The His440 Nδ---OOCβ Asp327 bond distance is 2.5 Å (2.8 Å resolution) in AChE and 0.2 Å shorter than the corresponding H-bond in trypsin and chymotrypsin (1.5 Å resolution). This distance increased to 2.7 Å during the dynamics simulation. However, the average H-bond distances are further shortened by 0.05−0.3 Å in energy-minimized structures of the adducts of AChE covalently modified by soman at the pentacoordinate and tetracoordinate intermediate stage. MD simulations of the optimized structures of native AChE and its adducts gave insight into how the skeletal motions accommodate an overcrowded active site particularly in the pentacoordinate adducts. The steric relief is only partial and is balanced by a repositioning of Glu199 toward the catalytic triad and phosphonyl fragment. This subtle reorientation of active-site residues should be relevant to the prominent catalytic efficiency of AChE.

Compared structures of the free nicotinic acetylcholine receptor main immunogenic region (MIR) decapeptide and the antibody-bound [A76]MIR analogue: a molecular dynamics simulation from two-dimensional NMR data.

Monoclonal antibodies against the main immunogenic region (MIR) of the muscle acetylcholine receptor (AChR) are capable of inducing experimental myasthenia gravis (MG) in animals. The epitope of these antibodies has been localized between residues 67 and 76 of the AChR alpha-subunit. The conformation in solution of the Torpedo californica MIR peptide and of its [A76] MIR analogue have been analyzed using molecular modeling based on nmr interproton distances and J-derived phi dihedral angles. Molecular dynamics simulations including dimethyl-sulfoxide as explicit solvent have been carried out on the free MIR peptide. Calculation of the structure of the [A76]MIR analogue bound to an anti-MIR monoclonal antibody have been performed in the presence of water molecules. A tightly folded structure appears for both peptides with alpha beta-folded N-terminal N68-P-A-D71 sequence of type I in the free state and type III in the mAb6-bound state. The C-terminal sequence is folded in two different ways according to the result in the free and bound state of the peptides: two overlapping beta/beta or beta/alpha turns result in a short helical sequence in the free MIR peptide, whereas the bound analogue is folded by uncommon hydrogen bond closing an 11-membered cycle. This structural evolution is essentially the result of the reorientation of the hydrophobic side chains that are probably directly involved in peptide--antibody recognition.

Interaction of partially unfolded forms of Torpedo acetylcholinesterase with liposomes.

A water-soluble dimeric form of acetylcholinesterase from electric organ tissue of Torpedo californica was obtained by solubilization with phosphatidylinositol-specific phospholipase C of the glycophosphatidylinositol-anchored species, followed by purification by affinity chromatography. The water-soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristoylphosphatidylcholine. We previously showed that either chemical modification or exposure to low concentrations of guanidine hydrochloride converted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In the present study, it was shown that such molten globule species, whether produced by mild denaturation or by chemical modification, interacted efficiently with small unilamellar vesicles. Binding was not accompanied by significant vesicle fusion, but transient leakage occurred at the time of binding. The bound acetylcholinesterase reduced the transition temperature of the vesicles slightly, and NMR data suggested that it interacted primarily with the head-group region of the bilayer. The effects of tryptic digestion of the bound acetycholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass approximately 5 kDa, remained associated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268-Lys 315. This polypeptide contains the longest hydrophobic sequence in the protein, Leu 274-Met 308, as identified on the basis of hydropathy plots. Inspection of the three-dimensional structure of acetylcholinesterase reveals that this hydrophobic sequence is largely devoid of tertiary structure and is localized primarily on the surface of the protein. It is suggested that this hydrophobic sequence is aligned parallel to the surface of the vesicle membrane, with nonpolar residues undergoing shallow penetration into the bilayer.

The handedness of the subunit arrangement of the nicotinic acetylcholine receptor from Torpedo californica.

Cross-linking an alpha-neurotoxin with a known three-dimensional structure and with photoactivatable groups in known positions to native membrane-bound acetylcholine receptor reveals its quaternary structure, including the handedness of its circular subunit arrangement. Photolabelling with alpha-neurotoxin carrying the photoactivatable group at position Lys46 is inhibited by the competitive antagonist (+)-tubocurarine in a biphasic manner, indicating that it reacts with both alpha-subunits that were shown to have different affinities for this antagonist [Neubig, R. R. & Cohen, J. B. (1979) Biochemistry 18, 5464-5475]. Lys46 is located on loop III of the neurotoxin. The other information necessary for the elucidation of the handedness was provided by the recent finding that the central loop of the toxin (loop II) is oriented towards the central pore of the receptor, securing the overall orientation of the bound toxin [Machold, J., Utkin, Y. N., Kirsch, D., Kaufmann, R., Tsetlin, V. & Hucho, F. (1995b) Proc. Natl Acad. Sci. USA 92, 7282-7286]. Looking at the receptor from the synaptic side of the postsynaptic membrane, it was concluded that the clockwise subunit arrangement is alpha H-gamma-alpha L-delta-beta (alpha H and alpha L are the alpha-subunits binding (+)-tubocurarine with high and low affinity, respectively). Its mirror image alpha alpha L-gamma-alpha H-beta-delta could thus be excluded.

The expression of Na+/K+-ATPase ?-subunit cRNA injected into Xenopus oocytes is affected by coinjection with ?-subunit cRNA

The cRNA for Torpedo californica Na+/K(+)-ATPase beta-subunit (cRNA beta) was injected into Xenopus oocytes alone or with the cRNA for the Na+/K(+)-ATPase alpha-subunit (cRNA alpha). When cRNA beta was injected alone, the amount of the beta-subunit that accumulated in oocytes increased with increasing amounts of injected cRNA beta. When cRNA beta and cRNA alpha were injected simultaneously, less beta-subunit accumulated than when cRNA beta was injected alone, whereas the Na+/K(+)-ATPase activity increased markedly. The decrease in the accumulation of the beta-subunit was dose-dependent upon the cRNA alpha. The mutant beta-subunit unable to assemble with the alpha-subunit accumulated in oocytes independently of cRNA alpha, suggesting that post-translational control mechanisms may serve to reduce the accumulation of the beta-subunit.

Cysteine string protein, a DnaJ family member, is present on diverse secretory vesicles

Synaptic vesicles are the secretory organelles responsible for the regulated secretion of neurotransmission. Proteins associated with or integral components of the lipid bilayer likely represent important components in regulated secretion. CSP (cysteine string protein) is a 34kDa protein which copurifies with cholinergic synaptic vesicles from the marine ray Torpedo californica. In Drosophila melanogaster deletion of the CSP gene causes impaired presynaptic neuromuscular transmission. A rat brain complementary DNA (cDNA) encoding CSP was isolated and sequence analysis predicts a protein with 86% identity to Torpedo CSP. Rat CSP contains a “J domain” as well as a cysteine rich “string” region. The J domain “fingerprints” the CSP family as members of the universally conserved DnaJ/hsp40 (heat shock protein) chaperone family. Polyclonal antisera raised against a seventeen amino acid peptide representing the carboxy terminus of rat CSP detected a 35 kDa immunoreactive protein in a rat brain synaptic vesicle enriched preparation. A 35 kDa immunoreactive protein that comigrates electrophoretically with rat brain CSP was also detected in zymogen granule membranes. These results establish the presence of a CSP in rat brain and in the zymogen granule of the rat pancreas and suggest that CSPs have a role in exocrine and neural secretion.

Primary sequence, tissue specificity and mRNA expression of the Na+,K+ -ATPase ?1 subunit in the European eel (Anguilla anguilla)

The entire amino acid coding sequence of the Na(+),K(+)-ATPase β1 isoform was cloned from the gill of the European eel (Anguilla anguilla) by a PCR based method. The amino acid sequence translated from the nucleotide sequence shared 61.4 and 56.2% homology respectively with previously published Na(+),K(+)-ATPase β1 isoform sequences from the clawed toad (Xenopus laevis) and the ray (Torpedo californica) an elasmobranch fish. The size of the Na(+),K(+)-ATPase β1 mRNA transcript in eel tissues was demonstrated to be 2.35 Kb. Detectable levels of Na(+),K(+)-ATPase β1 mRNA were found at some level in all tissues except liver and cardiac muscle. The level of branchial Na(+),K(+)-ATPase β1 mRNA was observed to increase after the adaptation of fresh water eels to normal or double concentration sea water.

Primary sequence, tissue specificity and expression of the Na +,K +-ATPase α 1 subunit in the European eel ( Anguilla anguilla)

The entire cDNA nucleotide sequence of the Na +,K +-ATPase α 1 isoform was cloned from the gills of the European eel ( Anguilla anguilla) by a PCR based method. The amino acid sequence translated from the sequence shared 89.4 and 85.6% homology respectively with previously published Na +,K +-ATPase α subunit sequences from elasmobranch ( Torpedo californica) and teleost ( Catostomus commersoni) fish. The size of Na +,K +-ATPase α 1 mRNA transcripts in eel tissues was demonstrated to be 3.5 kb, except in the ovary where a 3.7 kb transcript existed. Na +,K +-ATPase α 1 mRNA was present at some level in all tissues investigated with the exception of cardiac and skeletal muscle where no Na +,K +-ATPase α l mRNA was detectable. The level of branchial Na +,K +-ATPase α 1 mRNA increased after the adaptation of freshwater eels to normal or double concentration seawater.

Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor.

A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis.

Synthesis of nitrodiazirinyl derivatives of neurotoxin II from Naja naja oxiana and their interaction with the Torpedo californica nicotinic acetylcholine receptor.

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) from Naja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of (2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy)acet ic acid followed by chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [125I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane-bound nicotinic acetylcholine receptor (AChR) from Torpedo californica. The pattern of labeling the receptor's alpha, beta, gamma, or delta subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series of p-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.

Effects of halothane on the nicotinic acetylcholine receptor from Torpedo californica

To determine whether the binding of anesthetics to key membrane receptors is a plausible mode of action, we modeled the effect of the general anesthetic halothane in the nicotinic acetylcholine receptor membrane system isolated from Torpedo californica. Our results demonstrated that halothane inhibits the binding of [ 3H]phencyclidine ([ 3H]PCP) to the acetylcholine receptor. The inhibition was reversible, concentration dependent, and had an equilibrium dissociation constant ( K d ) of 2.2% atm halothane at 25 °. Double-reciprocal plots of the halothane effects at various phencyclidine (PCP) concentrations imply that, under equilibrium conditions, halothane inhibits [ 3H]PCP binding competitively. In contrast, results from kinetic studies showed that the rate of PCP dissociation is highly sensitive to halothane with EC 50 = 0.8% atm halothane in nitrogen. Several possible interpretations are discussed; however, the basic observation was that the kinetics of [ 3H]PCP binding to the nicotinic acetylcholine receptor was affected by halothane at low concentrations in this model system.

Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients.

Although microaneurysms are a clinicopathological hallmark of diabetic retinopathy, there have been few ultrastructural studies of these important lesions. As a result, knowledge of the mechanisms involved in the pathogenesis of microaneurysms remains fragmentary. This study provides histological and ultrastructural evidence of various stages in microaneurysm formation within the retinal vasculature. The eyes of three type II diabetic patients, obtained within 24 hours of death, were studied by the trypsin digest technique. Eyes from two further type II diabetics were fixed in 2.5% glutaraldehyde within 12 hours of death and processed for electron microscopy. In the trypsin digest preparations, small saccular and fusiform microaneurysms were observed in the peripheral retinal. In the central retina, the microaneurysms ranged in morphology from thin walled, cellular forms to dense, acellular, hyalinised forms. Ultrastructurally, four distinct groups of microaneurysm were observed. Type I showed an extensive accumulation of polymorphonuclear cells into the lumen. The endothelium remained intact, although pericytes were invariably absent. Type II microaneurysms were typified by large numbers of red blood cells (RBCs) in the lumen. Endothelial cells and pericytes were completely absent. The type III microaneurysm was also non-perfused and contained aggregates of irregularly shaped RBC profiles and RBC breakdown products. Recanalisation by new vessels into the occluded lumen was observed in one microaneurysm. Type IV microaneurysms were almost or completely sclerosed, with extensive fibrosis and lipid infiltration into the lumen and basement membrane wall. This investigation describes several distinctive stages in the formation of microaneurysms during diabetic retinopathy. With reference to the pathogenesis of retinal microaneurysms, the interaction of various cell types is discussed and the significance of vascular cell death and localised hypertensive events highlighted.

A New Class of Photoactivatable and Cleavable Derivatives of Neurotoxin II from Naja naja oxiana. Synthesis, Characterisation, and Application for Affinity Labelling of the Nicotinic Acetylcholine Receptor from Torpedo californica

A new series of photoactivatable and cleavable derivatives of neurotoxin II from the cobra Naja naja oxiana is investigated which can be used for mapping the surface topology of the nicotinic acetylcholine receptor from Torpedo electric tissue. The preparation and characterisation of five toxin derivatives, each with a radioactive 125I-azidosalicylamidoethyl-1,3′-dithiopropyl group in a defined position within the primary structure, are described. The photoinduced cross-linking reaction of the toxin derivatives with membrane-bound receptor is investigated. The photoactivatable group located at position K25 reacts almost exclusively with the δ subunit of the receptor, whereas the K15 derivative reacts with the α and β subunits. The other derivatives did not react with the receptor to any significant extent. It is shown that, with respect to the receptor subunits, the cross-linking pattern depends on the length and chemical nature of the cross-linking group.

Structure-Functional Effects of a Series of Alcohols on Acetylcholinesterase-Associated Membrane-Vesicles - Elucidation of Factors Contributing to the Alcohol Action

Structure-functional effects of a series of alcohols on acetylcholinesterase (AChE) derived from Torpedo californica were investigated. No significant effect on the soluble form of the enzyme was observed, but a marked biphasic effect on the activity of the membrane-associated form of the enzyme was found: it increased to maxima of 115 ± 2% at 40 mM ethanol, 123 ± 2% at 10 mM propanol, and 114 ± 2% at 10 mM t-butanol; declined with further increases in alcohol concentration; and then leveled off. Significantly, no increase was observed with n-butanol. The effect on membrane-associated AChE activity was in the order of propanol >t-butanol > ethanol >n-butanol, suggesting that the site of action of alcohol was small and could not accommodate straight-chain alcohols longer than propanol. Comparison of the results for soluble AChE and membrane-associated AChE activity indicated the involvement of the membrane in alcohol action. Fluorescence steady-state polarization (P) measurements of Torpedo membrane vesicles showed that membrane fluidity increased with increasing [alcohol], as indicated by a decrease in polarization (ΔP = −0.010 ± 0.003, −0.062 ± 0.003, −0.017 ± 0.002, and −0.011 ± 0.002 in the presence of 60 mM ethanol, propanol, n-butanol, and t-butanol, respectively). Alcohol-induced increase in membrane fluidity was in the order propanol >n-butanol >t-butanol, ethanol, suggesting that both lipid solubility and molecular size of alcohols could be important if alcohols must fit into membrane lipid domains in order to affect membrane fluidity. The biphasic effect of alcohol action on membrane-associated AChE activity is discussed in terms of membrane fluidity, lipid solubility, molecular size of alcohol, enzyme-lipid interaction, site of alcohol action, and a direct interaction of alcohol with the enzyme.

A new class of photoactivatable and cleavable derivatives of neurotoxin II from Naja naja oxiana. Synthesis, characterisation, and application for affinity labelling of the nicotinic acetylcholine receptor from Torpedo californica.

A new series of photoactivatable and cleavable derivatives of neurotoxin II from the cobra Naja naja oxiana is investigated which can be used for mapping the surface topology of the nicotinic acetylcholine receptor from Torpedo electric tissue. The preparation and characterisation of five toxin derivatives, each with a radioactive 125I-azidosalicylamidoethyl-1,3'-dithiopropyl group in a defined position within the primary structure, are described. The photoinduced cross-linking reaction of the toxin derivatives with membrane-bound receptor is investigated. The photoactivatable group located at position K25 reacts almost exclusively with the delta subunit of the receptor, whereas the K15 derivative reacts with the alpha and beta subunits. The other derivatives did not react with the receptor to any significant extent. It is shown that, with respect to the receptor subunits, the cross-linking pattern depends on the length and chemical nature of the cross-linking group.

Functional consequences of substitution of the disulfide-bonded segment, Cys127-Cys150, located in the extracellular domain of the Na,K-ATPase beta subunit: Arg148 is essential for the functional expression of Na,K-ATPase.

The Cys127-Cys150 disulfide-bonded loop (L1) of the Torpedo californica Na,K-ATPase beta 1 subunit was substituted with the corresponding loop of the rat beta 1, mouse beta 2, or pig H,K-ATPase beta subunit. All the substituted mutant beta subunits assembled with the Na,K-ATPase alpha subunit in a trypsin-resistant manner. The mutants with L1 from the Na,K-ATPase beta subunit isoforms (rat beta 1 and mouse beta 2) each formed a functional complex with the Na,K-ATPase alpha subunit. On the other hand, the complex of the alpha subunit with the mutant beta subunit that was substituted with the pig H,K-ATPase beta subunit L1 was inactive as to ATP hydrolysis. Ser131 and Phe148 located within L1 of the pig H,K-ATPase beta subunit-substituted mutant were back-mutated to Pro131 and Arg148, respectively. The Phe148 to Arg mutation restored the ability of the mutant beta subunit substituted with the H,K-ATPase beta subunit L1 to form a functional complex with the alpha subunit. These results suggested that the Cys127-Cys150 loop of the Na,K-ATPase beta 1 subunit, especially Arg148, plays a critical role in the functional expression of Na,K-ATPase.

Differential agonist-induced displacement of quinacrine and ethidium from their respective histrionicotoxin-sensitive binding sites on the Torpedo acetylcholine receptor.

Fluorescence spectroscopy was used to begin to localize the agonist inhibitory binding site on the nicotinic acetylcholine receptor (AcChR) from Torpedo californica. High concentrations of three cholinergic agonists, suberyldicholine (SubCh), acetylcholine (AcCh), and carbamylcholine (CCh), differentially inhibited the binding of two noncompetitive inhibitors (NCIs), quinacrine and ethidium, which bind at distinctly different loci on the desensitized AcChR at zero membrane potential. The agonist-induced inhibition of quinacrine binding occurred at significantly lower (17-fold) concentrations than the inhibition of ethidium binding. Schild plots of SubCh inhibition of ethidium and quinacrine binding showed the competitive nature of the agonist inhibition of the binding of these two NCIs. The quenching constants for short-range quenching of receptor-bound quinacrine and ethidium fluorescence by spin-labeled acetylcholine were about the same as their inhibition constants for agonist-induced displacement of AcChR-bound quinacrine and ethidium. The results demonstrate that agonists can directly bind to both the quinacrine and the ethidium binding sites, albeit at different agonist concentrations. Because the agonist-induced displacement of receptor-bound quinacrine occurs at significantly lower concentrations than the displacement of ethidium, the quinacrine binding site is more likely than the ethidium binding site to form part of the agonist inhibitory binding site.

Purification and characterization of HSP70 proteins from Torpedo electric organ

Members of the HSP70 family were purified from electric organ tissue of Torpedo californica by chromatography on an ATP-agarose column. A 70 kDa protein was the major component purified. The identity of this protein as HSP70 was established by Western blotting with monoclonal antibodies raised against purified and recombinant human HSP70. Sequencing of proteolytic fragments revealed that they possessed high sequence homology with a wide spectrum of members of the HSP70 family from bacteria through to humans. Two minor components, which co-purified with the principal 70 kDa band on the ATP-agarose column, may correspond to Torpedo BiP and to the stress-induced version of HSP70.

Matrix-assisted laser desorption ionization (MALDI) and post source decay (PSD) product ion mass analysis localize a photolabel crosslinked to the delta-subunit of nAChR protein by neurotoxin II

In an attempt to probe the molecular topology of the ion gate in the nicotinic AChR protein, the high affinity (Kd > 10−11 molar) ligand neurotoxin II (NT II), a 61 residue peptide from the venom of Naja naja oxiana, was photocrosslinked to the nAChR of Torpedo californica. The photoactivatable group was a (125I)-p-azidosalicylamidoethyl-1,3-dithiopropyl (ASED) moiety located at the K25 residue of NT II. Specific labeling occurred at the delta-subunit which, after SDS-PAGE, was subjected to tryptic digestion and reverse phase high performance liquid chromatography (rpHPLC) separation. Although the radioactivity profile of the collected HPLC fractions exhibited two sharp maxima suggesting rather specific crosslinking site(s), attempts to localize these sites by Edman degradation failed. Thus, matrix-assisted laser desorption ionization (MALDI) mass spectrometry was employed in the hope of circumventing the difficulties encountered with the conventional protocols. This technique, in conjunction with the opt...

Analysis of exposed regions on the main extracellular domain of mouse acetylcholine receptor alpha subunit in live muscle cells by binding profiles of antipeptide antibodies.

To study the structural organization of the main extracellular domain of the nicotinic acetylcholine receptor (AChR) alpha subunit in live muscle cells, we examined the native membrane-bound receptors in cultured mouse skeletal muscle cells for their ability to bind a panel of antibodies against uniform-sized overlapping synthetic peptides which collectively represent this entire domain. The binding profile indicated that the regions alpha 23-49, alpha 78-126, alpha 146-174, and alpha 182-210 are accessible to binding with antibody. Residues alpha 23-49, alpha 78-126, and alpha 194-210 contain binding regions for alpha-neurotoxin and some myasthenia gravis autoantibodies. A comparison of this binding profile with the profile obtained for membrane-bound Torpedo californica AChR in isolated membrane fractions showed some similarities as well as significant differences between the subunit organization in the isolated membrane fraction and that in the membrane of live muscle cells. Regions alpha 89-104 and alpha 158-174, which are exposed in the isolated membrane fraction, are also exposed in the live cell. On the other hand, regions alpha 23-49, and alpha 182-210, which are exposed in the live cell, are not accessible in the isolated membrane and, furthermore, the region alpha 1-16, which has marginal accessibility in the cell, becomes highly accessible in the membrane isolates. The exposed regions defined by this study may be the primary targets for the initial autoimmune attack on the receptors in experimental autoimmune myasthenia gravis.