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The antigen designated as Chol-1 beta, detected by an antiserum specific for cholinergic neurons, has been purified to homogeneity from ganglioside mixtures extracted from Torpedo electric organ and pig brain. The final products from the two sources behaved identically in a wide range of tests and gave coincident immunopositive and Ehrlich-positive spots after thin layer chromatography in seven different solvent systems; they were thus considered to be identical and to constitute a single, pure chemical species. Gas-chromatographic analysis revealed the presence of long-chain bases, glucose, galactose, N-acetylgalactosamine, and sialic acid in integral molar ratios of 1:1:2:1:3; the compound's reactivity to cholera toxin after Vibrio cholerae sialidase treatment on thin layer chromatography and the recovery of GM1 as sole product of exhaustive sialidase treatment identified it as a member of the gangliotetrahexosyl series. From the products of partial enzymatic desialylation and treatment with beta-galactosidase and a comparison of the compound's immunoreactivity to anti-Chol-1 antisera with that of other trisialogangliosides of defined molecular structure, we were able to assign a disialosyl residue alpha-Neu5Ac-(2----8)-alpha-Neu5Ac-(2----3)- to the inner galactose, and we suggest GalNAc as a possible site of linkage of the third sialic acid.

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The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible.

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The nicotinic acetylcholinergic receptor has been isolated and purified from extracts of the electric organ of the fish Torpedo fuscomaculata. The isolation procedure involves (a) a series of purification steps including preparation of membrane fragments, extraction of receptors with non-ionic detergents and chromatofocusing; (b) a novel fluorimetric titration assay. The purified receptor is isolated following a 9-fold purification with an overall yield of 12% and a specific activity of 4027 nM.g-1. Gel electrophoresis in the presence of sodium dodecylsulphate produced only one major band with molecular weight of 44,600 associated with the alpha-subunit. A comparison is made with other established procedures. Affinity chromatography on cobratoxin CNBr-Sepharose CL4B produced a 6.8-fold purification, 5% yield and 2900 nM.g-1 specific activity, while in ion-exchange chromatography on DEAE Sepharose 6B gave a 4.7-fold purification, 3% yield and specific activity of 1988 nM.g-1.

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Upon SDS PAGE of isolated mediatophore, an acetylcholine-translocating protein, a doublet at 15 kDa was identified. Amino acid sequencing after CNBr cleavage gave a 17 residue-long peptide completely homologous with a sequence of the proton-translocating proteolipid from bovine chromaffin granules. A 51-mer oligodeoxynucleotide corresponding to this sequence was used to screen a library of electric lobe cDNAs constructed in lambda Zap II. A positive recombinant clone was isolated and found to encode the complete sequence of a 15.5 kDa protein highly homologous to the bovine chromaffin or yeast vacuolar ATPase proteolipid. In vitro translation of sense RNA transcripts of the clone indeed yielded a single 15 kDa proteolipid. Northern blot analysis showed that the 1.3 kb mRNA encoding this protein is significantly expressed in nervous tissues but not in electric organ or liver of Torpedo marmorata.

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The electric organs of two species of skate have been examined morphologically, physiologically and biochemically. They can be easily dissociated into innervated or denervated component electrocytes by a Torpedo Ringer's solution containing 1% collagenase. Collagenase treatment did not, however, separate the Schwann cell cover capping the synaptosomes. Isolated electrocytes generate normal MEPP frequencies and show evoked responses for two days in Torpedo Ringer's. The nerve terminals retain excitability and transmitter release properties up to the time of separation. Since isolated terminals and denervated electrocytes show normal ultrastructural characteristics for up to 12 h, the skate electric organ provides several preparations which are not attainable with Torpedo tissue. Acetylcholine (ACh) content of supernatant fractions containing the synaptosomes was comparable to that found in Torpedo (sps.). Collagenase specifically eliminates the basal lamina associated with the synaptic junctional region. Neuronal cell death and synaptic terminal degeneration were also noted in the adult organs of both species. The skate electric organ is ideally suited for the study of cholinergic development and transmission.

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Analysis of the binding of monoclonal antibodies (mAbs) by Torpedo nicotinic acetylcholine receptor (AChR) has demonstrated that a region of the alpha-subunit between alpha-156 and alpha-179 is exposed on the cytoplasmic surface of the nicotinic post-synaptic membrane. A panel of mAbs was produced that recognized sodium dodecyl sulfate-denatured subunits of the Torpedo AChR. Antibodies recognizing alpha-subunit were distinguished in terms of their ability to bind alpha-subunit fragments generated by Staphylococcus aureus V8 protease: an 18-kDa fragment beginning at Val-46, a 20-kDa fragment beginning at Ser-173/Ser-162, and a 10 kDa fragment beginning at Asn-339. Three mAbs, selected for binding to each of the V8-protease alpha-subunit fragments, respectively, were characterized in detail. The location of epitopes recognized by both anti-V8-18 and anti-V8-20 mAbs was determined to be within alpha-156 to alpha-179 by isolation of small immunoreactive peptides from proteolytic digests of the alpha-subunit, while the mAb reactive to V8-10 was bound to an epitope within alpha-339 to alpha-386. Quantitative evaluation of binding of the anti-V8-18 and anti-V8-20 mAbs to overlapping synthetic peptides corresponding to alpha-147 to alpha-179 localized the epitopes to distinct portions of this region. Further screening of the panel of mAbs using these synthetic peptides revealed three additional mAbs that bind in this region. The mAbs that bound the three distinct V8-protease alpha-subunit fragments were shown to bind to native AChR by indirect immunofluorescence on frozen sections of Torpedo electric organ. Binding to the native AChR was to the cytoplasmic surface of the AChR since the mAbs could bind to AChR in native vesicles, in which the AChR is oriented right-side-out, only after permeabilization of the vesicles by alkaline treatment or after scrambling of the orientation of the AChR by solubilization and reconstitution into liposomes. The location of the mAb-binding sites at the cytoplasmic surface of the AChR was visualized directly by freeze-etch immunoelectron microscopy. The identification of alpha-156 and alpha-179 as containing a cytoplasmic exposed sequence implies the existence of two non-hydrophobic transmembrane sequences between the site of N-glycosylation (Asn-141) and Cys-192, a site alkylated by the cholinergic affinity labels.

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The Torpedo electrocyte is a flattened syncytium derived from skeletal muscle, characterized by two functionally distinct plasma membrane domains. The electrocyte is filled up with a transversal network of intermediate filaments (IF) of desmin which contact in an end-on fashion both sides of the cell. In this work, we show that polyclonal antibodies specific for lamin B recognizes a component of the plasma membrane of Torpedo electrocyte. This protein which thus shares epitopes with lamin B has a relative molecular mass of 54 kD, an acidic IP of 5.4. It is localized exclusively on the cytoplasmic side of the innervated membrane of the electrocyte at sites of IF-membrane contacts. Since our previous work showed that the noninnervated membrane contains ankyrin (Kordeli, E., J. Cartaud, H. O. Nghiêm, L. A. Pradel, C. Dubreuil, D. Paulin, and J.-P. Changeux. 1986. J. Cell Biol. 102:748-761), the present results suggest that desmin filaments may be anchored via the 54-kD protein to the innervated membrane and via ankyrin to the noninnervated membrane. These findings would represent an extension of the model proposed by Georgatos and Blobel (Georgatos, S. D., and G. Blobel. 1987a. J. Cell Biol. 105:105-115) in which type III intermediate size filaments are vectorially inserted to plasma and nuclear membranes by ankyrin and lamin B, respectively.

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To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++-activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.

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In order to further molecular investigations on the binding capacity of acetylcholine receptors, a method was developed for the affinity chromatography of the nicotinic acetylcholine receptor. Reversibly binding cholinergic ligand groups were used as affinity ligands, instead of the well known snake venom alpha-toxins. These ligands are small in size, chemically well defined and fixed to long spacer chains (at least 40 nm). One ligand, with a pharmacologically stabilizing effect on the receptor, was a derivative of gallamine. Another, with a depolarizing effect, resembled carbamoylcholine and a third was a derivative of decamethonium. The receptor proteins were isolated from Torpedo marmorata electric organs. Preparation included solubilization with a non-ionic detergent, alkaline treatment to extract peripheral membrane proteins and affinity purification. The receptor proteins eluted from the three affinity resins were identical in their assembly of subunits (alpha, beta, gamma and delta) but of different purity. Receptor proteins were obtained on a large scale within a short time and under mild conditions for elution with the affinity ligands of the decamethonium or the gallamine type. This was a considerable advantage compared to the use of alpha-bungarotoxin.

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The voltage-sensitive sodium channel from eel electroplax is formed of a polypeptide of 208,321 Da, to which is attached ca. 85 kDa of carbohydrate. Sialic acid is a prominent constituent, contributing ca. 113 negative charges to the protein surface. We here demonstrate that antibodies raised against the bacterial antigen alpha-(2----8)-polysialic acid, specific for polymers of ten or more consecutive sialic acid residues, react specifically and with high affinity to the electroplax sodium channel. In extracts of electroplax membranes, the sodium channel is the only protein that demonstrates this immunoreactivity, suggesting the presence of a polysialosyl-sialyltransferase specifically committed to this unique post-translational modification of the sodium channel. Polysialic acid is rare in vertebrates, having previously been found only associated with neural-cell adhesion molecules, present in the developing neuromuscular system. The other prominent source is the capsular polysaccharide of highly pathogenic meningitis bacteria. Antibodies to the bacterial antigen thus provide highly specific affinity markers for the sodium channel. The high avidity of these antibodies and the ratio of sialic acid residues to consensus glycosylation sites suggest that the terminal chains are well over ten sialosyl residues in length, potentially extending 10-30 nm into the extracellular environment.

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Polyclonal rabbit antibodies were raised against 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS), an inhibitor of a variety of anion transport proteins. These antibodies specifically recognize SITS-reacted erythrocyte band 3 in immunoprecipitations and Western blots. In Western blots of SITS-reacted membrane proteins derived from vesicles of the electric organ of Torpedo californica (known to express a SITS-sensitive Cl- channel) the antibodies recognized two major species of approximately 93 kDa and approximately 105 kDa. The approximately 93 kDa protein was identified as the alpha-subunit of the Na,K-ATPase. The approximately 105 kDa protein (designated sp105) is a glycoprotein which binds to wheat-germ agglutinin and concanavalin A and is present as a disulphide-linked homodimer under non-reducing conditions. A partial amino acid sequence and a polyclonal antibody were used to clone the corresponding cDNA. sp105 is encoded in electroplax by two abundant mRNAs of approximately 6 and approximately 6.8 kb. A hybridizing mRNA of approximately 5 kb was over 200-fold and over 500-fold less abundant in brain and heart respectively. Sequence analysis of the cDNA predicted a novel protein of 697 amino acids containing eight potential N-linked glycosylation sites. Analysis of hydrophobicity indicated the presence of at least one, and possibly three, putative membrane-spanning domains. When expressed from the Sp6 message in Xenopus laevis oocytes, the protein was inserted into membranes, glycosylated and processed to form a dimer. However, no increase in 36Cl uptake or in membrane conductance could be detected. We found no effect of hybrid depleting the specific message on expression of the Torpedo electroplax Cl- channel in oocytes. Thus we conclude that this novel electroplax membrane protein is probably not a functional part of the chloride channel.

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About two-thirds of the antibodies to the nicotinic acetylcholine receptor (AChR) in myasthenic patients, and in rats immunized with intact AChR, bind to the main immunogenic region (MIR) on the alpha-subunit. We tested all available anti-MIR monoclonal antibodies (mAbs) by competition experiments for binding on the intact AChR from Torpedo electric organ and human muscle. Practically complete competition between all possible paired combinations of anti-MIR mAbs was found. As a consequence, the MIR must be a very concrete and small region. Furthermore, the location of the MIR relative to some other less immunogenic regions was also determined.

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The number of free cysteines in each polypeptide of acetylcholine receptor from the electric organ of Torpedo californica has been assessed by alkylating the native protein with N-ethylmaleimide and iodoacetamide during homogenization of the tissue and alkylating the polypeptides with N-ethylmaleimide as they were unfolded in solutions of dodecyl sulfate. The cysteines unavailable for alkylation could be accounted for as specific cystines, connecting positions in the amino acid sequences of the individual polypeptides. Unreduced, alkylated polypeptides of acetylcholine receptor were digested with thermolysin or trypsin. Cystine-containing peptides in the chromatograms of the digests were identified electrochemically by the use of a dual gold/mercury electrode. Three thermolytic peptides and three tryptic peptides have been isolated from these digests and shown to contain intact cystines that were originally present in the native protein. The majority of these peptides contained an intact, intramolecular cystine connecting two cysteines in locations homologous to cysteines 128 and 142 from the alpha polypeptide. Each of these cystines from each of the polypeptides of acetylcholine receptor was isolated in at least one peptide, respectively. Each of these cystine-containing peptides also contained glucosamine. It can be concluded that each asparagine in the sequence Asn-Cys-Thr/Ser, which occurs in the respective, homologous location in every polypeptide, is glycosylated even though a cystine sits between the asparagine and the threonine or serine. In addition, the existence of the cystine connecting the adjacent cysteines, alpha 192 and alpha 193, in the alpha subunit of acetylcholine receptor [Kao, P. N., & Karlin, A. (1986) J. Biol. Chem. 261, 8085-8088] has been confirmed.

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By use of an antiserum raised against presynaptic plasma membrane purified from the purely cholinergic electromotor system of Torpedo marmorata we have been able to identify a group of antigenically-related minor gangliosides (collectively designated Chol-1) that appear to be exclusively localized on cholinergic neurons. The cholinergic-specificity of these antigens has been shown by the following findings: a) The anti-Chol-1 antiserum induces a selective complement-mediated lysis of the cholinergic subpopulation of mammalian brain synaptosomes; b) Section of the fimbria, which causes a massive degeneration of cholinergic terminals in the hippocampus, leads to a concomitant depletion of the level of the Chol-1 gangliosides in the hippocampus; c) The anti-Chol-1 serum can be used to immunostain cholinergic elements in the central and peripheral nervous systems of the rat. The discovery of a cell surface cholinergic-specific antigen has provided a new and effective tool with which to study the cholinergic neuron. For instance, we have immuno-isolated cholinergic synaptosomes from rat cortex and used this preparation to study transmitter coexistence. Our results indicate that approximately 75% of the cortical cholinergic neurons also express the neuropeptide VIP. Furthermore, we are investigating the expression of Chol-1 in patients affected by diseases such as ALS which primarily involve central cholinergic neurons.

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As an initial step in characterizing the function of basal lamina components during muscle cell differentiation and innervation in vivo, we have determined immunohistochemically the pattern of expression of three components--laminin, proteins related to agrin (an acetylcholine receptor (AChR)-aggregating protein), and a heparan sulfate proteoglycan--during the development of chick embryo hindlimb muscles. Monoclonal antibodies against agrin were used to purify the protein from the Torpedo ray and to characterize agrin-like proteins from embryonic and adult chicken. In early hindlimb buds (stage 19), antibodies against laminin and agrin stained the ectodermal basement membrane and bound to limb mesenchyme with a generalized, punctate distribution. However, as dorsal and ventral premuscle masses condensed (stage 22-23), mesenchymal immunoreactivity for laminin and agrin-like proteins, but not the proteoglycan, became concentrated in these myogenic regions. Significantly, the preferential accumulation of these molecules in myogenic regions of the limb preceded by 1-2 days the appearance of muscle-specific proteins, myoblast fusion, and muscle innervation. All three basal lamina components were preferentially associated with all AChR clusters from the time we first observed them on newly formed myotubes at stage 26. Localization of these antigens in three-dimensional collagen gel cultures of limb mesenchyme, explanted prior to innervation of the limb, paralleled the staining patterns seen during limb development in the embryo. These results indicate that basal lamina molecules intrinsic to limb mesenchyme are early markers for myogenic and synaptic differentiation, and suggest that these components play important roles during the initial phases of myogenesis and synaptogenesis.

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We have studied the ganglioside content and pattern of synaptic vesicles isolated from the electric organs of two species of Torpedinidae, Torpedo californica and Torpedo marmorata. The ganglioside concentrations were high relative to protein content (77 and 58 micrograms of N-acetylneuraminic acid/mg of protein, respectively), owing to the low protein-to-lipid ratio; however, they were also appreciable in relation to phospholipid (15.6 and 10.0 micrograms of N-acetylneuraminic acid/mg of phospholipid). The fact that a membrane fraction that separated from synaptic vesicles of T. californica on a controlled-pore glass-bead column and constituted the main potential source of contamination in this preparation had a lower ganglioside content and a different TLC pattern than synaptic vesicles indicated the relatively high purity of the latter. Most of the gangliosides from synaptic vesicles of both species migrated on TLC in the vicinity of standards with three or more sialic acids. Synaptosomes from T. marmorata had a higher lipid N-acetylneuraminic acid/phospholipid ratio and a different TLC pattern than synaptic vesicles. Considering these results and other data appearing recently in the literature, we suggest that reexamination of synaptic vesicles from mammalian brain for the possible presence of gangliosides is warranted.

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Two methods for extracting calelectrin, a Ca2+-regulated membrane-binding protein from the electric organ of Torpedo marmorata, have been compared and the more promising one was modified to increase the yield to 7-8 mg.kg-1 wet weight of tissue, that is 4-5 times greater than the original method. The calelectrin so obtain could be resoloved into a minor component (designated L-calelectrin) eluted from an anion-exchange column at relatively low ionic strength (100 mM NaCl) and a major component (H-calelectrin) eluted at higher ionic strength (300 mM NaCl). The two forms were also separated by chromatography on a hydrophobic resin. Electrophoresis on cellulose acetate indicated that L-calelectrin had a lower mean isoelectric point that the H-form and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that under reducing conditions (presence of 5% beta-mercaptoethanol) both forms migrated as single species, the L-form having a lower apparent relative molecular mass (Mr 32,000) that the H-form (34,000). Under non-reducing conditions, there was no change in the migration of L-calelectrin but the H-form was resolved into two components of Mr 34,000 and 32,000. The addition of 2 mM Ca2+ had no effect on the migration of either form. Both forms were equally recognized by an anti-calelectrin antiserum and were microheterogeneous with respect to their isoelectric points (pH 4.3-5.5) in two-dimensional gel electrophoresis. Physical measurements were carried out on the major H-form. The Stokes radius was estimated to be 3nm, corresponding to an apparent Mr of 44,000. It was unaffected by changes in ionic strength, pH or Ca2+ concentration. Analytical ultracentrifugation gave a sedimentation constant of 2.9 S and an apparent Mr of 36,000. Measurements of circular dichroism indicated that 78% of the molecule was in the alpha-helix configuration and 22% in random coil. Ca2+ had no significant effect on the conformation.

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Antibodies were raised against a synthetic peptide corresponding to residues 927-938 of the eel electroplax sodium channel primary sequence. This segment, lying between putative internal repeat domains II and III, is postulated to be exposed on the cytoplasmic surface of the membrane in several recent models of channel tertiary structure and on the external surface in another. The antiserum and affinity-purified IgG derived from it specifically recognize the peptide and the eel sodium channel in a solid-phase radioimmunoassay and bind to a single diffuse band of 260-280 kDa on Western blots of eel electroplax membrane proteins. All reactions are blocked by co-incubation of the antibodies with the synthetic peptide (1 microM). The antibody immunoprecipitates the solubilized channel in a form that retains its characteristic high-affinity binding of saxitoxin. In eel electroplax, the antibodies label only the innervated membrane known to contain sodium channels; at the ultrastructural level, this labeling is exclusively associated with the cytoplasmic surface of the membrane. Sodium channels containing the epitope are not seen in the postsynaptic membrane or in the membrane of the presynaptic nerve terminal. Segment 927-938 of the eel sodium channel is accessible on the surface of the protein in its solubilized form and is exposed in the cytoplasmic face of the innervated membrane of the electroplax in situ. This location is consistent with 3 models of channel structure but not with a fourth.

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Desmin, the muscle-specific intermediate filament protein was purified from the main electric organ of Electrophorus electricus. It is shown that pure desmin can be separated into 5 isoforms presenting different isoelectric points. These isoforms have similar molecular weight, react with an antibody directed against desmin and generate identical peptides after digestion with protease V8 from Staphylococcus aureus.

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