Biochemical characterization of the tetrodotoxin binding protein from Electrophorus electricus

Biochemical properties of a detergent-solubilized tetrodotoxin binding component from Electrophorus electricus have been examined and compared with those found for the membrane-bound protein. The toxin binding component was solubilized with high efficiency by a variety of nonionic detergents and with lower efficiency by sodium cholate and deoxycholate. Detergent-solubilized preparations bound tetrodotoxin and saxitoxin tightly and specifically, and this binding was observed to be rapidly and irreversibly blocked by carboxylate-modifying reagents. Inactivation by carbodiimide and glycine ester or by a trimethyloxonium salt could be prevented by tetrodotoxin occupancy of the binding site. Tetrodotoxin binding activity in both solubilized preparations and in membranes was found to be highly resistant to proteases. In contrast, the activity was extremely sensitive to the action of phospholipase A2. The biochemical properties of the tetrodotoxin binding component solubilized in mixed lipid-detergent micelles are similar to those found in native membranes, with respect to the characteristics of equilibrium toxin binding and to the sensitivity of toxin binding activity to chemical modification and degradative enzymes. There were some differences with respect to the kinetics of tetrodotoxin binding. In addition, the tetrodotoxin binding component from eel is shown to behave as a glycoprotein, being selectively absorbed to resins coupled to concanavalin A, wheat germ agglutinin, Lens culinaris lectin, and ricin with the appropriate glycoside.     

Properties of the tetrodotoxin binding component in plasma membranes isolated from Electrophorus electricus.

The biochemical properties of the electrically excitable sodium channels in the electroplaque of Electrophorus electricus were investigated using tritiated tetrodotoxin (TTX) as a specific membrane probe. Membrane fragments from the electroplaque were isolated essentially by differential centrifugation and characterized with respect to the plasma membrane markers acetylcholine receptors, acetylcholinesterase, (Na+ + K+)ATPase, and [3H]TTX binding. Equilibrium binding studies showed that [3H]TTX bound to a single population of noninteracting receptor sites with an apparent dissociation constant of 6 +/- 1 X 10(-9) M. The toxin-membrane complex dissociated with a first-order rate constant of 0.012 sec-1. Studies on the pH dependence of complex formation demonstrated the requirement for an ionizable, functional group with a pK of 5.3 and this group has been shown to be a carboxyl. Treatment of the membranes with trimethyloxonium tetrafluoroborate, a carboxyl group modifying reagent, resulted in an irreversible loss in the binding of [3H]TTX, which could be prevented by low concentrations of TTX or saxitoxin. This decrease was due to a reduction in the total number of binding sites and not to a decrease in toxin binding affinities. The relative binding affinities of various monovalent alkali metal and polyatomic cations for the TTX-receptor site showed that this site displayed cation discrimination properties which were similar to those reported previously for the electrically excitable sodium channel in intact nerve fibers. A possible role for this site in the ion selectivity of the sodium channel is proposed.     

Half-of-the-sites reactivity of the membrane-bound Electrophorus electricus acetylcholine receptor

Equilibrium binding studies of the interaction of activators (decamethonium, carbamylcholine) and inhibitors (d-tubocurarine, α-bungarotoxin) of membrane electrical potential changes in electroplax membrane preparations from $\text{Electrophorus electricus}$ have been carried out at 4°C, in cel Ringer solution, pH 7.0. The properties of the interaction of these chemical mediators with the membrane-bound receptor appear to be similar to those observed with regulatory enzymes which exhibit an allosteric mechanism involving ligand-induced conformational changes. The data presented here show that activators and inhibitors compete for only one-half the available membrane sites. The experiments also provide additional support for the interpretation of kinetic studies which indicated that electroplax membranes contain two different binding sites, one for activators and one for inhibitors of electrical membrane potential changes.     

Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2

The effects of phospholipase A₂ treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [³H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A₂ from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by $\text{β-}\text{bungarotoxin}$. (2) Phospholipase A₂ from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A₂ treatment on [³H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.     

Phospholipid and fatty acid composition of tetrodotoxin receptor-rich membrane fragments from Electrophorus electricus

To study the interaction of voltage-sensitive Na+-channels with membrane lipids, the phospholipid and fatty acid composition of highly purified membrane fragments from the remarkably differentiated plasma membrane of Electrophorus electricus has been analyzed. After density gradient fractionation and carrier free electrophoresis, fractions with up to 30 pmol tetrodotoxin binding/mg protein can be obtained, which may correspond to a 50% pure preparation of the extrasynaptic part of the excitable face. Phospholipid classes and cholesterol are separated by one-dimensional thin-layer chromatography in acidic and alkaline solvent systems. The following mean molar contents are found: 40% phosphatidylcholine, 23% phosphatidylserine, 30% phosphatidylethanolamine and 7% sphingomyelin. In a series of 11 animals, significant deviations from these mean values have been observed. The fatty acid composition of the phospholipids has been determined by gas chromatography. Phosphatidylcholine contains more than 50% 16:0, and about 20% unsaturated fatty acids in the C-18 group. Compared to other plasma membrane fractions, this phospholipid is the least differentiated. By contrast, phosphatidylethanolamine and phosphatidylserine show many characteristics in different membrane fractions, especially in their unsaturated components representing more than 50%. 22:6, as the major constituent in these fractions, accounts for a quarter to a third of all fatty acids in these fractions. 18:0 is the main saturated component in these two phospholipids with abundances of typically a quarter or less of all fatty acids. Knowledge of the lipid composition of these excitable membranes may help to conserve binding and structural properties when analyzing lipid-sensitive Na+-channels in vitro. It is also useful as a guideline for systematic reconstitution studies.     

Morphological study of the heart of Electrophorus electricus

Hearts of 7 Electrophorus electricus have been investigated on macroscopical and microscopical levels. Generally, the structure of the heart exhibits relative similarities with the other studied teleosts. The ventricular myocardium is mainly spongy and suggests that these structural features can offer an additional contraction power.     

Interaction and inhibition of acetylcholinesterase from Electrophorus electricus by nitrosamines

Kinetic analysis has shown that dimethylnitrosamine, dipropylnitrosamine, dibutylnitrosamine, and diphenylnitrosamine initially act as reversible competitive inhibitors with respect to the substrate, acetylthiocholine chloride. The inhibitor constants Ki vary from 21-30 microM for the aliphatic nitrosamines to 8.2 microM for the aromatic diphenylnitrosamine. With time they act as irreversible covalent inhibitors with dimethylnitrosamine producing 82% inactivation after 40 min. Pseudo-first-order kinetics are observed with the rate constant being proportional to the concentration of the nitrosamine and the order of reaction being equal to one. Fluorometry, gel chromatography, and equilibrium dialysis have been used to study the binding of the nitrosamines with acetylcholinesterase. Scatchard analysis indicates that dimethyl-, dipropyl-, and dibutylnitrosamine have a weaker affinity for the enzyme (Kd 5.6-8.08 microM) compared to diphenylnitrosamine (Kd 2.32 microM). In all cases the number of binding sites was four.     

Isolation of Membrane Fractions with Sodium Channels Sensitive to Veratridine and tetrodotoxin from the Electric Organ of the Eel Electrophorus electricus

The veratridine/tetrodotoxin-sensitive sodium influx was measured in membrane fractions isolated from the electric organ of Electrophorus electricus. The fractions were characterized, and the main biochemical markers and their acetylcholine receptor content were determined. The innervated and noninnervated faces of the electroplax were separated. The different biochemical criteria used indicate that the pre- and postsynaptic membranes of the innervated face were isolated. Sodium influx increased by veratridine and blocked by tetrodotoxin was found in fractions from the presynaptic membrane. Because some of the vesicles in this fraction are in the inside-out conformation, tetrodotoxin had to be applied to both faces of the vesicles so that sodium influx was blocked completely. The fractions from the innervated face of the electroplax contained sodium channels with sensitivities to tetrodotoxin and veratridine similar to those of fractions from other nerve membrane preparations.     

Alpha-actinin in the electric organ of Electrophorus electricus, L

We have identified Alpha-actinin from the electric organ of the Electrophorus electricus, L. It was analysed by polyacrylamide gel electrophoresis, and identified by immunoblotting. This protein was also found in a membrane fraction of the electric organ enriched with components of the cytoskeleton. Our results suggest that this protein might play a role either in the organization of the microfilaments or its interactions with the membrane to maintain a polarized electrocyte.     

Desmin heterogeneity in the main electric organ of Electrophorus electricus

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.     

Localization of actin in the electrocyte of Electrophorus electricus L.

Summary The cytoplasm of the electrocyte of Electrophorus electricus possesses a meshwork of 7-nm thick filaments distributed throughout the cell. Observation of stereopairs of transmission electron micrographs shows association of the filaments with the plasma membrane and the membranes of cytoplasmic organelles. Intense fluorescence, indicative of the presence of actin, was observed in the cytoplasm of electrocytes incubated in the presence of NBD-phallacidin or anti-actin antibodies.     

The morphological study of the respiratory organ of Electrophorus electricus

The respiratory organ of Electrophorus electricus is located in the oral cavity and is formed by papillar structures. The papilla consists basically of a cartilaginous central nucleus and is bounded by a connective layer. The results reveal structural adaptations for the respiration among fishes which have efficient respiratory circulation related with the venous blood.