Principal glycopeptide of the tetrodotoxin/saxitoxin binding protein from Electrophorus electricus: isolation and partial chemical and physical characterization

Abstract

Preparations of the tetrodotoxin (TTX) and saxitoxin binding protein isolated from the electroplax of Electrophorus electricus are of high specific activity (greater than or equal to 2000 pmol of TTX binding sites/mg of protein) and appear to be homogeneous in that they contain only the large polypeptide previously identified to make up part of the voltage-sensitive sodium channel [Agnew, W. S., Moore, A. C., Levinson, S. R., & Raftery, M. S. (1980) Biochem. Biophys. Res. Commun. 92, 860-866]. This permits the inference that the TTX binding site, thought to be associated with the mouth of the ion channel, is located on this peptide. This peptide presumably corresponds to the large peptide, designated the alpha-peptide subunit, of the synaptosomal sodium channel [Hartshorne, R. P., & Catterall, W. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4620-4624]. No convincing evidence for lower molecular weight peptides has yet been found for the electroplax protein. A rapid and convenient method is described for preparation of milligram quantities of the pure, sodium dodecyl sulfate (NaDodSO4) denatured form of the peptide, and its amino acid and carbohydrate compositions are reported. The peptide behaved anomalously on NaDodSO4-polyacrylamide gels. It was demonstrated that the molecular weight cannot be accurately quantified by this method but that the true value likely exceeds the value of 260 000 reported previously. The denatured peptide displayed an electrophoretic microheterogeneity which may be ascribed to variations in bulky carbohydrate substituents and an extremely high free mobility which is inferred to result from binding of unusually large amounts of NaDodSO4.

Traumatic Axonal Injury Induces Proteolytic Cleavage of the Voltage-Gated Sodium Channels Modulated by Tetrodotoxin and Protease Inhibitors

“…We performed immunocytochemistry to detect the identity of the processes traversing the cell-free 2 mm gap in the wells, evaluating immunostaining for microtubuleassociated protein 2 (MAP2), a specific marker for the dendrites and neuronal somata, and NaCh protein, which stains the axons in addition to the dendrites and neuronal somata. Cultures were fixed in 4% paraformaldehyde and 0.1 M PBS for 20 min, permeabilized with 0.1% Triton X-100 (PBST) for 20 min at room temperature (RT), and double labeled with monoclonal mouse anti-MAP2 antibody (Ab) (AP20; 1:500; Sigma) (Binder et al, 1986) and polyclonal rabbit anti-pan NaCh Ab (06 -811; 1:80; Upstate Biotechnology, Lake Placid, NY) specific for the intracellular III-IV loop of the NaCh ␣-subunit [ amino acid (aa) 1491-1508 of full length, aa 1-2005; P04775), a highly conserved segment of the intracellular III-IV loop (Miller et al, 1983;Dugandzija-Novakovic et al, 1995;Vabnick et al, 1996;Meier et al, 1997;Rasband et al, 1999) at 4°C overnight. After rinsing with PBS, the cultures were incubated with Alexa 594-conjugated anti-mouse IgG (Molecular Probes, Eugene, OR) and Alexa 488-conjugated anti-rabbit IgG (Molecular Probes) for 60 min at RT.…”

“…We used specific antibodies to the intracellular I-II loop on the NaCh ␣-subunit (aa 476 -485 of full length aa 1-2005; P04775), polyclonal rabbit anti-brain type II NaCh Ab (AB5206; 1:50; Chemicon, Temecula, CA) (Noda et al, 1986;Gordon et al, 1987;Westenbroek et al, 1989), and intracellular III-IV loop of the NaCh ␣-subunit (aa 1491-1508 of full length aa 1-2005; P04775), and polyclonal rabbit anti-pan NaCh Ab (06 -811; 1:80; Upstate Biotechnology) (Miller et al, 1983;DugandzijaNovakovic et al, 1995;Vabnick et al, 1996;Meier et al, 1997;Rasband et al, 1999) to evaluate immunoreactive changes with and without pharmacologic modulation. Treatment groups included control saline solution (CSS), TTX (Sigma), and a protease inhibitor (PI) mixture tablet Complete (Roche Diagnostics, Indianapolis, IN), which inhibits Ͼ90% of each protease activity of serine and cysteine (including calpain I and II) proteases, metalloproteases, Pronase, thermolysin, chymotrypsin, trypsin, and papain.…”

Distribution and lateral mobility of voltage-dependent sodium channels in neurons [published erratum appears in J Cell Biol 1989 May;108(5):preceding 2001]

“…This distribution of channels will determine some of the differential electrical properties of one part versus another part of a given excitable cell. A number of laboratories have recently isolated and characterized the sodium channel protein as an extremely carbohydrate-rich polypeptide of Mr 260 kD (9,22,26,36). Although localization of important receptors and some morphological and electrophysiological evidence on sodium channel distribution has been described (4-6, 17, 20, 43, 57), little is known concerning the mechanisms that regulate and maintain their surface localization.…”

Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide.

“…A DEAE fraction of crude eel electroplax membranes (prepared as described in ref. 22) was solubilized in 2% NaDodSO4. The proteins were separated by NaDodSO4/PAGE on a 5-15% acrylamide gradient, transferred to nitrocellulose paper, and probed with antibody as reported (23, 24).…”