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At least 19 recordsLinked to original sources
PubMed1983

Purification of acetylcholine receptors from the muscle of Electrophorus electricus.

Muscle from the electric eel Electrophorus electricus contains acetylcholine receptors at 50 times the concentration of normal mammalian muscle and fully one-tenth the concentration of receptors in its electric organ tissue. Receptor is organized much more diffusely over the surface of Electrophorus muscle cells than is the case in normally innervated mammalian skeletal muscle. Receptor was purified from Electrophorus muscle by affinity chromatography on cobra toxin-agarose and found to contain subunits which correspond immunochemically to the alpha, beta, gamma, and delta subunits of receptor from electric organ tissue of Torpedo californica. Receptor purified from Electrophorus muscle appears virtually identical with receptor purified from Electrophorus electric organ tissue.

Animals↗
PubMed1979

Immunochemical similarities between subunits of acetylcholine receptors from Torpedo, Electrophorus, and mammalian muscle.

Polypeptide chains composing acetylcholine receptors from the electric organs of Torpedo californica and Electrophorus electricus were purified and labeled with 125I. Immunochemical studies with these labeled chains showed that receptor from Electrophorus is composed of three chains corresponding to the alpha, beta, and gamma chains of receptor from Torpedo but lacks a chain corresponding to the delta chain of Torpedo. Experiments suggest that receptor from mammalian muscle contains four groups of antigenic determinants corresponding to all four of the Torpedo chains. Binding of 125I-labeled chains was measured by quantitative immune precipitation and electrophoresis. Antisera to the following immunogens were used: denatured alpha, beta, gamma, and delta chains of Torpedo receptor, native receptor from Torpedo and Electrophorus electric organs and from rat and fetal calf muscle, and human muscle receptor (from autoantisera of patients with myasthenia gravis). The four chains of Torpedo receptor were immunologically distinct from one another and from higher molecular weight chains found in electric organ membranes. Antibodies to these chains reacted very efficiently with native Torpedo receptor, but the reverse was not true. Antibodies to native receptor from Torpedo and Electrophorus reacted slightly with each of the chains of the corresponding receptor. However, cross-reaction between chains and antibodies to any native receptor was most obviuos with the alpha chain of Torpedo or the corresponding alpha' chain of Electrophorus. Antiserum to alpha chains exhibited higher titer aginst receptor from denervated rat muscle. Antibodies from myasthenia gravis patients did not cross-react detectably with 125I-labeled chains from electric organ receptors. Most interspecies cross-reaction occurred at conformationally dependent determinants whose subunit localization could not be determined by reaction with the denatured chains.

Animals↗
PubMed1987

Monoclonal antibodies against acetylcholinesterase from electric organs of Electrophorus and Torpedo.

We studied the reactivity of monoclonal antibodies (mAbs) raised against acetylcholinesterase (AChE) purified from Electrophorus and Torpedo electric organs. We obtained IgG antibodies (Elec-21, Elec-106, Tor-3E5, Tor-ME8, Tor-1A5), all of them directed against the catalytic subunit of the corresponding species, with no significant cross-reactivity. These antibodies do not inhibit the enzyme and recognize all molecular forms, globular (G) and asymmetric (A). Tor-ME8 reacts specifically with the denatured A and G subunits of Torpedo AChE, in immunoblots. Several hybridomas raised against Electrophorus AChE produced IgM antibodies (Elec-39, Elec-118, Elec-121). These antibodies react with the A forms of Electrophorus electric organs and also with a subset of dimers (G2) from Torpedo electric organ. In addition, they react with a number of non-AChE components, in immunoblots. In contrast, they do not recognize AChE from other Electrophorus tissues or A forms from Torpedo electric organs.

Acetylcholinesterase↗
PubMed1978

Binding of antibodies to acetylcholine receptors in Electrophorus and Torpedo electroplax membranes.

Antisera against purified acetylcholine receptors from the electric tissues of Torpedo californica and of Electrophorus electricus were raised in rabbits. The antisera contain antibodies which bind to both autologous and heterologous receptors in solution as shown by an immunoprecipitation assay. Antibodies in both types of antisera bind specifically to the postjunctional membrane on the innervated surface of the intact electroplax from Electrophorus electric tissue as demonstrated by an indirect immunohistochemical procedure using horseradish peroxidase conjugated to anti-rabbit IgG. Only anti-Electrophorus receptor antisera, however, cause inhibition of the receptor-mediated depolarization of the intact Electrophorus electroplax. The lack of inhibition by anti-Torpedo receptor antibodies, which do bind, suggests that the receptor does not undergo extensive movement during activity. The binding of anti-Torpedo antibodies to receptor-rich vesicles prepared by subcellular fractionation of Torpedo electric tissue was demonstrated by both direct and indirect immunohistochemical methods using ferritin conjugates. These vesicles can be conveniently collected and prepared for electron microscopy on Millipore filters, a procedure requiring only 25 micrograms of membrane protein per filter. In addition, it was possible to visualize the binding of anti-Torpedo receptor antibodies directly, without ferritin. These anti-Torpedo receptor antibodies, however, do not inhibit the binding of acetylcholine or of alpha-neurotoxin to receptor in Torpedo microsacs but do inhibit binding of alpha-neurotoxin to Torpedo receptor in Triton X-100 solution. It is likely that the principal antigenic determinants on receptor are at sites other than the acetylcholine-binding sites and that inhibition of receptor function, when it occurs, may be due to a stabilization by antibody binding of an inactive conformational state.

Acetylcholine↗
PubMed1984

Synthesis in vitro of precursors of the catalytic subunits of acetylcholinesterase from Torpedo marmorata and Electrophorus electricus.

We translated poly(A-rich messenger RNA prepared from the electric organs of Electrophorus electricus and Torpedo marmorata in a reticulocyte lysate system. In the case of Electrophorus, which appears to contain only one type of acetylcholinesterase catalytic subunit, an anti-(Electrophorus acetylcholinesterase) antiserum precipitated a single 65-kDa polypeptide from the products translation obtained in vitro. In the case of Torpedo, where a number of distinct catalytic subunits corresponding to different fractions of the enzyme have been described, an anti-(Torpedo acetylcholinesterase) antiserum precipitated two main polypeptides, 61 kDa and 65 kDa, both of which could be displaced by unlabelled purified Torpedo acetylcholinesterase. Synthesis in vitro thus appears to produce a single type of precursor of the acetylcholinesterase catalytic subunit for Electrophorus, and at least two distinct precursors for Torpedo, suggesting that several mRNAs code for the catalytic subunits in the latter species.

Acetylcholinesterase↗
PubMed1976

Adenosine triphosphate in cholinergic vesicles isolated from the electric organ of Electrophorus electricus.

Synaptic vesicles have been isolated from the electirc organ of the bony fish Electrophorus electricus using sucrose step gradients and zonal centrifugation. Although the acetylcholine (ACh) content of the Electrophorus electric organ is only 2% of that of Torpedo, ACh and ATP can readily be measured in the peak fractions using the leech microassay and the firefly luciferin luciferase assay respectively. The protein content of the vesicle fraction in experiments with Electrophorus was much higher than with Torpedo, but a possible contamination of this fraction with mitochondrial or cytoplasmic particles could be excluded. The ACh to ATP ratio of 10.8 is close to that found for cholinergic vesicles isolated from Torpedo and also to that of other amine storing granules.

Acetylcholine↗
PubMed1982

Subunit structure of the acetylcholine receptor from Electrophorus electricus.

The amino-terminal amino acid sequences of the four major peptides (Mr 41,000, 50,000, 55,000, and 62,000) present in purified preparations of Electrophorus electricus nicotinic acetylcholine receptor (AcChoR) have been determined for 24 cycles by automated sequence analysis procedures yielding four unique polypeptide sequences. The sequences showed a high degree of similarity, having identical residues in a number of positions ranging between 37% and 50% for specific pairs of subunits. Comparison of the sequences obtained with those of the subunits of similar molecular weight from Torpedo californica AcChoR revealed an even higher degree of homology (from 46% to 71%) for these two highly diverged species. Simultaneous sequence analysis of the amino termini present in native, purified Electrophorus AcChoR showed that these four related sequences were the only ones present and that they occur in a ratio of 2:1:1:1, with the smallest subunit ("alpha 1") being present in two copies. Genealogical analysis suggests that the subunits of both Torpedo and Electrophorus AcChoRs derive from a common ancestral gene, the divergence having occurred early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as the highly conserved structure of the AcChoR complex along animal evolution, suggest that each of the subunits evolved to perform discrete crucial roles in the physiological function of the AcChoR.

Amino Acid Sequence↗
PubMed1986

Single-channel current recordings of acetylcholine receptors in electroplax isolated from the Electrophorus electricus Main and Sachs' electric organs.

Extensive chemical kinetic measurements of acetylcholine receptor-controlled ion translocation in membrane vesicles isolated from the electroplax of Electrophorus electricus have led to the proposal of a minimum model which accounts for the activation, desensitization, and voltage-dependent inhibition of the receptor by acetylcholine, suberyldicholine, and carbamoylcholine. Comparison of chemical kinetic measurements of the dynamic properties of the acetylcholine receptor in vesicles with the properties of the receptor in cells obtained from the same organ and animal have been hampered by an inability to make the appropriate measurements with Electrophorus electricus electroplax cells. Here we report a method for exposing and cleaning the surface of electroplax cells obtained from both the Main electric organ and the organ of Sachs and the results of single-channel current recordings which have now become possible. The single-channel current recordings were made in the presence of either carbamoylcholine or suberyldicholine, as a function of temperature and transmembrane voltage. Both the channel open times and the single-channel conductance were measured. The data were found to be consistent with the model based on chemical kinetic measurements using receptor-rich membrane vesicles prepared from the Main electric organ of E. electricus.

Animals↗
PubMed1976

Immunochemical studies on the large polypeptide of electrophorus electroplax (Na+ + K+)-ATPase.

Antibodies against Lubrol-solubilized Electrophorus electroplax (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and its 96 000-dalton polypeptide (P96) were raised in rabbits. The P96 antibody does not cross react with the (Na+ + K+)-ATPase from mammalian species and tissues, but it cross reacts with the (Na+ + K+)-ATPase from both Electrophorus electroplax and brain. The combination of enzyme with anti-P96 is found to inhibit phosphoryl enzyme formation to the same extent that it inhibits enzyme activity. The rate of K+-sensitive dephosphorylation of phosphoryl enzyme appears to be unchanged. These are also found to be true with the antibody against the whole enzyme. Upon tryptic digestion of the enzyme-anti-P96 complex only the large polypeptide of the enzyme is protected. In the case of enzyme-anti-Lubrol-solubilized enzyme complex, both the large and small polypeptides are protected, whereas preimmune sera are without any protecting effect. The data indicate that the phosphoryl acceptor polypeptide and the Lubrol-solubilized electroplax (Na+ + K+)-ATPase from which the polypeptide is derived are phylogenetically distinct from those of the mammalian (Na+ + K+)-ATPases. The selective tryptic resistance of the enzyme-anti-P96 complex indicates that the two polypeptides are spatially well separated, possibly on opposite sides of the membrane.

Adenosine Triphosphatases↗
PubMed1997

A major second messenger mediator of Electrophorus electricus electric tissue is CaM kinase II.

Electric tissue of the electric eel, Electrophorus electricus, has been used extensively as a model system for the study of excitable membrane biochemistry and electrophysiology. Membrane receptors, ion channels, and ATPases utilized by electrocytes are conserved in mammalian neurons and myocytes. In this study, we show that Ca2+ predominates as the major mediator of electric tissue phosphorylation relative to cyclic AMP and cyclic GMP-induced phosphorylation. Mastoparan, a calmodulin inhibitor peptide, and a peptide corresponding to the pseudosubstrate region of mammalian calmodulin-dependent protein kinase II (CaMKII (281-302)) attenuated Ca(2+)-dependent phosphorylation in a dose-dependent manner. These experiments demonstrated that calmodulin-dependent protein kinase II activity predominates in electric tissue. The Electrophorus kinase was purified by a novel affinity chromatography procedure utilizing Ca2+/calmodulin-dependent binding to the CaMKII (281-302) peptide coupled to Sepharose. The purified 51 kDa calmodulin-dependent protein kinase II demonstrated extensive autophosphorylation and exhibited a 3- to 4-fold increase in Ca(2+)-independent activity following autophosphorylation. Immunofluorescent localization experiments demonstrated calmodulin to be abundant in electrocytes, particularly subjacent to the plasma membrane. Calmodulin-dependent protein kinase II had a punctate distribution indicating that it may be compartmentalized by association with vesicles or the cytoskeleton. As the primary mediator of phosphorylation within electric tissue, CaM kinase II may be critical for the regulation of the specialized electrophysiological function of electrocytes.

Animals↗
PubMed1998

Electrophorus electricus as a model system for the study of membrane excitability.

The stunning sensations produced by electric fish, particularly the electric eel, Electrophorus electricus, have fascinated scientists for centuries. Within the last 50 years, however, electric cells of Electrophorus have provided a unique model system that is both specialized and appropriate for the study of excitable cell membrane electrophysiology and biochemistry. Electric tissue generates whole animal electrical discharges by means of membrane potentials that are remarkably similar to those of mammalian neurons, myocytes and secretory cells. Electrocytes express ion channels, ATPases and signal transduction proteins common to these other excitable cells. Action potentials of electrocytes represent the specialized end function of electric tissue whereas other excitable cells use membrane potential changes to trigger sophisticated cellular processes, such as myofilament cross-bridging for contraction, or exocytosis for secretion. Because electric tissue lacks these functions and the proteins associated with them, it provides a highly specialized membrane model system. This review examines the basic mechanisms involved in the generation of the electrical discharge of the electric eel and the membrane proteins involved. The valuable contributions that electric tissue continues to make toward the understanding of excitable cell physiology and biochemistry are summarized, particularly those studies using electrocytes as a model system for the study of the regulation of membrane excitability by second messengers and signal transduction pathways.

Animals↗
PubMed1983

Comparison of the subunit structure of acetylcholine receptors from muscle and electric organ of Electrophorus electricus.

The acetylcholine receptors of the electric organ and muscle tissues of Electrophorus electricus are composed of alpha, beta, gamma, and delta subunits. Receptor subunits from the two tissues were compared by peptide mapping with monoclonal antibodies, an affinity-labeling reagent, and a lectin to characterize particular peptide fragments. These experiments indicate that the corresponding receptor subunits from the two tissues are extensively homologous or identical throughout their amino acid sequences. Small differences in the electrophoresis of peptide fragments of alpha subunits between the two tissues occurred on fragments which bound labeled lectin. These results suggest that the acetylcholine receptors in electric organ and muscle tissues of Electrophorus differ in structure only by minor posttranslational modifications perhaps involving carbohydrate.

Animals↗
PubMed1976

Torpedo marmorata acetylcholinesterase; a comparison with the Electrophorus electricus enzyme. Molecular forms, subunits, electron microscopy, immunological relationship.

Electron microscopy, sequential degradation by hydrolytic enzymes and the physical-chemical properties of the molecular forms of Torpedo acetylcholinesterase indicate that these molecules are structurally related to each other in the same way as the molecular forms of Electrophorus acetylcholinesterase: all are derived from a complex structure in which three tetrameric groups of subunits are associated with a rod-like 'tail'. In aged preparations the catalytic subunits are split into fragments in a manner similar to those of Electrophorus acetylcholinesterase. Immunological cross-reaction between both enzymes demonstrates the occurrence of common antigenic sites. The enzymes from the two sources, however, are different in their molecular weights and susceptibility to hydrolytic enzymes. Also, Torpedo acetylcholinesterase does not precipitate with either isologous or heterologous antibodies.

Acetylcholinesterase↗
PubMed1978

Active-site catalytic efficiency of acetylcholinesterase molecular forms in Electrophorus, torpedo, rat and chicken.

The active sites of acetylcholinesterase multiple forms from four widely different zoological species (Electrophorus, Torpedo, rat and chicken) were titrated using a stable, irreversible phosphorylating inhibitor (O-ethyl-S2-diisopropylaminoethyl methyl-phosphonothionate). In all cases, we found that within a given species, the molecular forms we examined were equivalent in their catalytic activity per active site. As pure preparations of the molecular forms of Electrophorus acetylcholinesterase were available, we were able to establish that one inhibitor molecule binds per monomer unit for each of them. This had already been shown by several authors for the tetrameric globular form, but not for the tailed molecules. Analysis of the phosphorylation reaction showed that they are equally reactive. Under our experimental conditions, their turnover number per site was 4.4 x 10(7) mol of acetylthiocholine hydrolysed . h-1 at 28 degrees C, pH 7.0. The corresponding value was less than half for Torpedo (1.64 x 10(7) mol . h-1), and again lower for rat (1.32 x 10(7) mol . h-1) and chicken (1.05 x 10(7) mol . h-1). In the case of rat acetylcholinesterase, the activity per active site of solubilized (with or without Triton X-100) and membrane-bound enzyme were identical. We discuss the implications of these findings with respect to the quaternary structure of acetylcholinesterase, and to the physico-chemical state and physiological properties of its molecular forms.

Acetylcholinesterase↗
PubMed1987

An immunoglobulin M monoclonal antibody, recognizing a subset of acetylcholinesterase molecules from electric organs of Electrophorus and Torpedo, belongs to the HNK-1 anti-carbohydrate family.

An immunoglobulin M (IgM) monoclonal antibody (mAb Elec-39), obtained against asymmetric acetylcholinesterase (AChE) from Electrophorus electric organs, also reacts with a fraction of globular AChE (amphiphilic G2 form) from Torpedo electric organs. This antibody does not react with asymmetric AChE from Torpedo electric organs or with the enzyme from other tissues of Electrophorus or Torpedo. The corresponding epitope is removed by endoglycosidase F, showing that it is a carbohydrate. The subsets of Torpedo G2 that react or do not react with Elec-39 (Elec-39+ and Elec-39-) differ in their electrophoretic mobility under nondenaturing conditions; the Elec-39+ component also binds the lectins from Pisum sativum and Lens culinaris. Whereas the Elec-39- component is present at the earliest developmental stages examined, an Elec-39+ component becomes distinguishable only around the 70-mm stage. Its proportion increases progressively, but later than the rapid accumulation of the total G2 form. In immunoblots, mAb Elec-39 recognizes a number of proteins other than AChE from various tissues of several species. The specificity of Elec-39 resembles that of a family of anti-carbohydrate antibodies that includes HNK-1, L2, NC-1, NSP-4, as well as IgMs that occur in human neuropathies. Although some human neuropathy IgMs that recognize the myelin-associated glycoprotein did not react with Elec-39+ AChE, mAbs HNK-1, NC-1, and NSP-4 showed the same selectivity as Elec-39 for Torpedo G2 AChE, but differed in the formation of immune complexes.

Acetylcholinesterase↗
PubMed1983

The A12 acetylcholinesterase and polypeptide composition of electric organ basal lamina of Electrophorus and some Torpedinae fishes.

Basal lamina (BL) of Torpedo, Discopyge and Electrophorus electric organs was purified in order to establish polypeptide composition and association with acetylcholinesterase (AChE). Results indicate that BL presents a distinct peptide pattern and that the A12 form of AChE is directly attached to it. Comparison of the species studied demonstrated similarities both in polypeptide composition and AChE content of the purified BL. Extractions of BL with solutions of high ionic strength, guanidine-HCl and acetic acid indicated the differential solubilization of various domains of BL polypeptides.

Acetylcholinesterase↗
PubMed2001

Expression of muscle-specific myosin heavy chain and myosin light chain 1 in the electric tissue of Electrophorus electricus (L.) in comparison with other vertebrate species.

Myosin light and heavy chains from skeletal and cardiac muscles and from the electric organ of Electrophorus electricus (L.) were characterised using biochemical and immunological methods, and compared with myosin extracted from avian, reptilian, and mammalian skeletal and cardiac muscles. The results indicate that the electric tissue has a myosin light chain 1 (LC1) and a muscle-specific myosin heavy chain. We also show that monoclonal antibody F109-12A8 (against LC1 and LC2) recognizes LC1 of myosin from human skeletal and cardiac muscles as well as those of rabbit, lizard, chick, and electric eel. However, only cardiac muscles from humans and rabbits have LC2, which is recognized by antibody F109-16F4. The data presented confirm the muscle origin of the electric tissue of E. electricus. This electric tissue has a profile of LC1 protein expression that resembles the myosin from cardiac muscle of the eel more than that from eel skeletal muscle. This work raises an interesting question about the ontogenesis and differentiation of the electric tissue of E. electricus.

Animals↗
PubMed1985

Scanning electron microscopy of the electric organs of Electrophorus electricus L. II. Organs of Sachs and Hunter.

The surfaces of the organs of Sachs and Hunter of Electrophorus electricus L. were examined by scanning electron microscopy. Special attention was directed to morphological details of the electrocyte to provide a better understanding of its anterior and posterior faces. Some aspects of the microanatomy of these organs, which differ markedly from those of the main electric organ, provide new information on the structure as revealed previously by light and transmission electron microscopy. The relief, mainly expressed by papillae, is related to the actual membrane area, which is important for calculations of specific resistance and conductance. Information is also presented on the general organization of the tissue, in particular the distribution of the connective elements and external configuration of synaptic terminals. Shrinkage in preparation of tissue was evaluated and correction made whenever necessary. Correction factors for actual membrane area were calculated for anterior and posterior faces of electrocytes from both organs.

Animals↗