Tetrodotoxin Binding Protein Research Articles

Purification and some properties of a tetrodotoxin binding protein from the blood plasma of kusafugu, Takifugu niphobles

A tetrodotoxin binding protein has been purified from the plasma of the puffer fish kusafugu, Takifugu niphobles, through DEAE-cellulose treatment, ammonium sulfate fractionation, Sephadex gelfiltrations and Sephacryl S-200 and Cellulofine A-500 column chromatography. Final purification by HPLC on a TSK G-3000 SL column yielded a protein which showed only a single protein peak. The molecular weight of the protein was estimated to be 116,000 and 91,000 by SDS-PAGE and mass spectrometry, respectively. A blast search on the amino-terminal amino acid sequence of the purified protein revealed that the protein had no homology to any other protein on data base.

The lobster nerve sodium channel: solubilization and purification of the tetrodotoxin receptor protein

Solubilization and purification of the tetrodotoxin (TTX) binding protein of the lobster walking-leg nerve Na + channel were carried out utilizing [ 3H]tetrodotoxin ([ 3H]tetrodotoxin) as a marker. The nerve membrane was solubilized with Lubrol-PX and the Na + channel protein was purified with diethylaminoethyl Bio-Gel A, Bio-Gel hydroxylapatite powder and two Sepharose 6B columns. Care was taken to keep the temperature of the Na + channel preparation as close to 1°C as possible and to use solutions (pH 7.5) that contain Na channel protectors, i.e., egg phosphatidylcholine/Lubrol-PX mixture, TTX, EDTA, EGTA, phenylmethylsulfonyl fluoride, pepstatin A, iodoacetamide, antipain, phosphoramidon, soybean trypsin inhibnitor, leupeptin and bacitracin. From an initial specific binding of 20.1 pmol of [ 3H]TTX/mg protein for the solubilized membrane, the binding increased to 1241 pmol/mg protein for the most active fraction of the lat Sepharose 6B column. The [ 3H]TTX specific binding of the Sepharose 6B fractions correlated with a large peptide, of M r 260 000 (240–280K), although other peptides were also present in lesser amounts.

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

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

Single-channel properties of the reconstituted voltage-regulated Na channel isolated from the electroplax of Electrophorus electricus.

The tetrodotoxin-binding protein purified from electroplax of Electrophorus electricus has been reincorporated into multilamellar vesicles that were used for patch recording. When excised patches of these reconstituted membranes were voltage clamped in the absence of neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties qualitatively and quantitatively similar to those reported for Na channels from nerve and muscle cells, including uniform single-channel conductances of the appropriate magnitude (approximately equal to 11 pS in 95 mM Na+), mean open times of approximately equal to 1.9 msec, and 7-fold selectively for Na+ over K+. Currents averaged from many depolarizations showed initial voltage-dependent activation and subsequent inactivation. In the presence of batrachotoxin, channels were observed with markedly different properties, including conductances of 20-25 pS (95 mM Na+), mean open times of approximately equal to 28 msec, and no indication of inactivation. Collectively, these findings indicate that the tetrodotoxin-binding protein of electroplax is a voltage-regulated sodium channel.

Immunocytochemical localization of sodium channel distributions in the excitable membranes of Electrophorus electricus.

The tetrodotoxin binding protein, a major component of the Na+ channel, has been purified from the electric organ of the South American eel Electrophorus electricus. Antibodies to this protein were raised in rabbits and their specificity was demonstrated by a highly sensitive radioimmunoassay and by immunoprecipitation procedures. These antibodies were used to examine the distribution of the binding protein in the eel electroplax membranes and along myelinated nerve axons. The distribution of the antigen was determined by using the peroxidase-antiperoxidase technique at both the light and electron microscopic levels. In the electrocytes of the electric organ, only the innervated face showed staining in experimental material. The stained regions of electroplax plasmalemma included the caveolae of the innervated surface while caveolae of the non-innervated surface did not stain. Thus, the innervated surface including caveolae exclusively contains the Na+ channels. Along myelinated axons, staining was limited to the nodal zone of the node of Ranvier. The paranodal and internodal zones did not stain for the binding protein. Limited diffusion of primary IgG and subsequent reactants into the paranodal and internodal sites was eliminated as a possible source of focal staining at nodes because mechanically demyelinated preparations also exhibited focal nodal staining. Thus, this tetrodotoxin binding protein component of the Na+ channel is located solely within the nodal zone of the node of Ranvier.

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.

Electron microscopic visualization of the tetrodotoxin-binding protein from Electrophorus electricus.

Preparations of highly purified tetrodotoxin-binding protein (sodium channel) from the electric organ of the eel Electrophorus electricus were examined in negatively stained preparations. Structures observed in preparations exhibiting the highest tetrodotoxin binding tended to aggregate into ordered clusters with a unique ribbon-like conformation. The individual particles of these aggregates are elongated or rod-shaped, approximately 40 A wide and 170 A long. Stereoscopic imaging of the three-dimensional aspects of the structures revealed that the rod-like image is not an edge view of a flattened disc but represents a cylindrical structure. Individual rods in nonclustered forms were also observed but with greater frequency in preparations with lower specific activity. The dimensions of the particles suggest that they represent a protein core formed by perhaps one copy of the large glycopeptide previously identified as being part of the sodium channel. The structure of the sodium channel component visualized by negative staining is discussed in the context of the excitable properties it contributes to biological membranes.

Identification of a large molecular weight peptide associated with a tetrodotoxin binding protein from the electroplax of [formula omitted

We have previously described the purification of a tetrodotoxin binding protein from the electroplax of Electrophorus electricus . The preparation consisted of three peptides of M r ∼ 46,000, 59,000 and ∼ 300,000 daltons. Further investigation has now shown that the large peptide of M r ∼ 260,000 daltons is part of the tetrodotoxin binding component of the voltage-sensitive sodium channel.