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PubMed1991

Pharmacological and biochemical properties of saxiphilin, a soluble saxitoxin-binding protein from the bullfrog (Rana catesbeiana).

Supernatant fractions of various tissues and plasma from the North American bullfrog, Rana catesbeiana, specifically bind saxitoxin with high affinity. Binding of [3H]saxitoxin to bullfrog plasma follows single-site behavior with an equilibrium dissociation constant of Kd = 0.16 +/- 0.03 nM at 0 degrees C and a maximum binding capacity of 380 +/- 60 pmole/ml plasma. High-affinity binding of [3H]saxitoxin is chemically specific since it is unaffected by tetrodotoxin and a variety of cationic peptides, amino acids and drugs. The structure-activity dependence of binding to this site was investigated with eight different natural and synthetic derivatives of saxitoxin. Substitution of the carbamoyl side chain or the C-12 beta-hydroxyl group of saxitoxin with a hydrogen atom had little effect on binding affinity, but addition of a hydroxyl group at the N-1 position decreased the binding affinity from 430- to 710-fold in three different molecular pairs. High performance size exclusion chromatography of supernatant from bullfrog skeletal muscle showed that the [3H]saxitoxin-binding component migrates with an apparent molecular weight of Mr = 74,000 +/- 8000 or a Stokes radius of 35 +/- 2A. The [3H]saxitoxin-binding protein in skeletal muscle extract or plasma is retained on a cation-exchange column at pH 6.0, suggesting that the protein contains a region of exposed basic residues. Column isoelectric focusing of a sample from plasma indicated that the protein has a basic isoelectric point near pH = 10.7.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphibian Proteins↗
PubMedDate not supplied

Polyclonal anti-idiotypes induce specific anti-saxitoxin antibody responses.

Polyclonal BALB/C mouse and New Zealand White rabbit anti-idiotypic antibodies were raised by immunization with a protein G-purified burro anti-saxitoxin IgG antibody preparation. Following absorption of non-anti-idiotype reactivity, murine and rabbit IgG were purified by protein A chromatography and used to immunize BALB/C mice for the induction of anti-saxitoxin antibody responses. Unconjugated BALB/C anti-idiotypes did not induce significant anti-saxitoxin reactivity in BALB/C mice, even after repeated immunizations. However, BALB/C mice immunized with purified BALB/C anti-idiotypes conjugated to keyhole limpet hemocyanin, or with purified, unconjugated rabbit anti-idiotypes, as aluminum hydroxide precipitates, induced significant and specific anti-saxitoxin immune responses. Saxitoxin, a sodium channel blocker, can protect cells treated with veratridine and ouabain, whose respective actions are to open sodium channels and to block the activity of Na/K-ATPase. The anti-idiotype-induced anti-saxitoxin antibodies inhibited saxitoxin from protecting against cell death induced by veratridine and ouabain treatment. These and other published experimental results strengthen the concept of anti-idiotype-based vaccines in eliciting protective immunity against a variety of low molecular weight, nonproteinaceous biological and chemical toxins, whose extreme toxicity does not allow their use as safe immunogens.

Animals↗
PubMed1987

Binding of [3H]saxitoxin to the voltage-dependent Na channels and inhibition of 22Na influx in bovine adrenal medullary cells.

The binding characteristics of [3H]saxitoxin and its binding site were examined in bovine adrenal medullary cells. These cells showed a specific binding of [3H]saxitoxin which was saturable and reversible. Scatchard analysis showed a single class of high-affinity binding sites with an equilibrium dissociation constant of 5.8 nM and a maximum binding capacity of 427.2 fmoles/10(7) cells (124.2 fmoles/mg of cell protein). A Hill plot revealed that there were no co-operative interactions among the binding sites. Unlabeled saxitoxin inhibited the specific binding of [3H]saxitoxin as well as veratridine-induced 22Na influx with a similar potency as did tetrodotoxin. However, veratridine, aconitine and scorpion venom, at concentrations that increased 22Na influx, did not inhibit [3H]saxitoxin binding. These results indicate that saxitoxin binds to a specific site on voltage-dependent Na channels and inhibits the influx of 22Na. [3H]Saxitoxin would be useful for the detailed analysis of voltage-dependent Na channels in adrenal medullary cells.

Adrenal Medulla↗
PubMedDate not supplied

Paralytic shellfish poison reference materials: an intercomparison of methods for the determination of saxitoxin.

Within the framework of the European Commission's Measurements and Testing Programme (BCR) a project has been undertaken to develop shellfish reference materials for Paralytic Shellfish Poisons (PSP). In a preliminary phase of the project, an intercomparison study of methods was undertaken. In this exercise 18 laboratories were asked to analyse solutions of saxitoxin and PSP-containing shellfish extracts with a method of their choice. The study revealed that: all the methods considered (four HPLC methods, one ELISA method) were in principle adequate for the quantification of saxitoxin in solution in the absence of interfering substances (Coefficient of variation (CV) 33% at a concentration of 0.5 microgram/ml); three of the HPLC methods used were able to quantify saxitoxin in PSP-positive mussel extract, the fourth method gave significant overestimation; the CV of all HPLC results was 53% at a mean saxitoxin mass fraction of 2.06 mg/kg mussel meat, the recoveries varied from 59-173%; and the ELISA method grossly overestimated the saxitoxin content in mussel extract, probably due to cross reactions of the antibodies with other PSP. The feasibility of preparing a homogeneous batch of ampouled mussel extracts (CV 3.5% at a saxitoxin concentration of approximately 1.5 mg/kg shellfish), sufficiently stable for at least 4 months storage both at 4 degrees C and approximately 20 degrees C, was demonstrated. The performance of the different methods for the analysis of PSP other than saxitoxin has not yet been evaluated, due to the current lack of PSP standards. Some of the problems observed in the intercomparison study were partly due to the nature of the chromatographic columns used, the composition of the HPLC mobile phase (pH, ion strength), non-optimal conditions for derivatization and matrix interference. Following the outcome of this study, a three year multistage project involving 15-20 European laboratories has been initiated, aimed at improving the accuracy and comparability of PSP measurements as well as preparing reference materials for PSP.

Animals↗
PubMed1973

The binding of labelled saxitoxin to the sodium channels in nerve membranes.

1. Tritium labelled saxitoxin has been prepared and purified, and its binding both to intact rabbit vagus nerves and to a solubilized preparation of garfish olfactory nerve membranes has been examined.2. In intact and solubilized nerves there is a saturable binding component of magnitude equal to that previously obtained for labelled tetrodotoxin.3. This component of bound saxitoxin is displaced competitively by tetrodotoxin, and it is concluded that the two toxins bind to the same site.4. The saturable saxitoxin (STX) interaction with the nerve membrane is reversible and can be described by the equation STX + R right harpoon over left harpoon STX.R where R is the binding site or receptor. With the solubilized preparation of garfish nerve membranes the saxitoxin-receptor reaction rates are almost four times faster than those of tetrodotoxin. The half-life of the saxitoxin-receptor complexes is 13 sec compared with 44 sec for the tetrodotoxin-receptor complex.5. A number of agents were tested for their ability to displace the labelled saxitoxin. Calcium and thallous ions each produced significant reversible reduction in binding, with apparent equilibrium dissociation constants of about 20-30 mM. Toxin binding is also inhibited reversibly in acidic solutions by protons competing with toxin for a binding site with a pK(a) of 5.6-5.9. All three ions are known to block sodium currents in myelinated nerve at similar concentrations. Our experiments indicate that they do so at the site of toxin binding.6. Lidocaine and veratrine do not affect the binding of saxitoxin.

Animals↗
PubMed1994

Oxidation of saxitoxin: detection and biological activity of its reaction products.

We characterized the oxidation products of saxitoxin by high-performance liquid chromatography and thin-layer chromatography. We observed several changes in the saxitoxin and neosaxitoxin chromatographic profile, as well as changes in the biological activities of the saxitoxin oxidized product. Toxins were heated in the presence of oxidizing agent, and at least five oxidation products were detected. Spectrophotometric measurements with Folin-Ciocalteau reagent confirmed the formation of these oxidation products. The excitation maximum of each of the oxidized components was determined by spectrophotofluorometry scans, and differed from the parent compound. The oxidized saxitoxin inhibited 3H-saxitoxin binding to neuroblastoma-glioma hybrid cells (NG108-15) to a lesser extent than saxitoxin, and had no toxic effect in mice. Formation of oxidation products suggests the possible, non-enzymatic transformation of saxitoxin and neosaxitoxin.

Animals↗
PubMed1987

Inaction of saxitoxin-oximes on the sodium channel of frog skeletal muscle fibers.

Three oximes of saxitoxin, saxitoxin oxime, saxitoxin methyloxime, and saxitoxin carboxymethyloxime, were synthesized in which the oxime functions replaced the ketone function on C-12 of saxitoxin. On the voltage-clamped single frog muscle fibers these oximes were very weak or inactive in blocking the sodium channel. The results indicate that the hydrated ketone function in saxitoxin is essential for blockade of the sodium channel, probably through a hydrogen bonding mechanism with some receptor groups.

Animals↗
PubMed2003

A comparison of the mu-conotoxins by [3H]saxitoxin binding assays in neuronal and skeletal muscle sodium channel.

Sodium channels from rat brain, rat skeletal muscle, chick brain, and eel electroplax were compared by using the mu-conotoxins GIIIA, PIIIA, and StIII and [3H]saxitoxin. Rat skeletal muscle and eel electroplax sodium channels are equally sensitive to GIIIA, PIIIA, and StIII, displacing >90% of the [3H]saxitoxin binding sites in rat skeletal muscle and eel electroplax membranes and exhibiting inhibitory concentrations at half-maximal percentage specific binding (IC50) of 0.97 nM for GIIIA, 1.3 nM for PIIIA in rat skeletal muscle, and concentrations of 3.5 nM for GIIIA and 2.8 nM for PIIIA in eel electroplax. PIIIA and GIIIA at all concentrations inhibit only up to 10% of [3H]saxitoxin binding sites in chick brain membranes. Mu-conotoxin StIII at all concentrations inhibits only up to 10% of [3H]saxitoxin binding sites in rat brain membranes and displays two-site binding inhibition of the [3H]saxitoxin binding sites in rat skeletal muscle. PIIIA displaces >60% of the [3H]saxitoxin binding sites (IC50 of 44 nM) while GIIIA blocks out only 30% of the binding sites in rat brain sodium channels (IC50 of 69 nM). Thus, sodium channel subtypes can be classified into two categories: mu-conotoxin sensitive (i.e., subtypes predominantly found in rat skeletal muscle and eel electroplax) and insensitive (i.e., subtypes predominantly found in rat and chick brain).

Amino Acid Sequence↗
PubMed2001

Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalis.

Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 +/- 0.97 nmol x mg(-1) protein and a K(d) of 14.6 +/- 0.33 nm for [(3)H]saxitoxin in equilibrium binding assays. [(3)H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 microm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish.

Amino Acid Sequence↗
PubMed1986

Specific inhibition of [3H] saxitoxin binding to skeletal muscle sodium channels by geographutoxin II, a polypeptide channel blocker.

Geographutoxin II (GTX II), a peptide toxin isolated from Conus geographus, inhibited [3H]saxitoxin binding to receptor sites associated with voltage-sensitive Na channels in rat skeletal muscle homogenates and rabbit T-tubular membranes with K0.5 values of 60 nM for homogenates and 35 nM for T-tubular membranes in close agreement with concentrations that block muscle contraction. Scatchard analysis of [3H]saxitoxin binding to T-tubular membranes gave values of KD = 9.3 nM and Bmax = 300 fmol/mg of protein and revealed a primarily competitive mode of inhibition of saxitoxin binding by GTX II. The calculated KD values for GTX II were 24 nM for T-tubules and 35 nM for homogenates, respectively. In rat brain synaptosomes, GTX II caused a similar inhibitory effect on [3H]saxitoxin binding at substantially higher concentrations (K0.5 = 2 microM). In contrast, binding of [3H]batrachotoxin A 20-alpha-benzoate and 125I-labeled scorpion toxin to receptor sites associated with Na channels in synaptosomes was not affected by GTX II at concentrations up to 10 microM. Furthermore, [3H]saxitoxin binding to membranes of rat superior cervical ganglion was only blocked 10% by GTX II at 10 microM. These results indicate that GTX II interacts competitively with saxitoxin in binding at neurotoxin receptor site 1 on the sodium channel in a highly tissue-specific manner. GTX II is the first polypeptide ligand for this receptor site and the first to discriminate between this site on nerve and adult muscle sodium channels.

Amino Acid Sequence↗
PubMed1980

[Determination of saxitoxin in canned shellfish (author's transl)].

Poisonings by saxitoxin-containing shellfish occur regularly in shore areas. The reason is increased growth of the dinoflagellates Gonyaulax tamarensis and Gonyaulax catenella. Due to the widespread consumption of canned shellfish these kinds of poisoning also occurs in continental areas. Therefore it is necessary to determine saxitoxin in canned shellfish products. Because of their sensitivity fluorospectrophotometric determinations of saxitoxin are preferred. However, the methods described in the literature can only be applied to fresh shellfish. Consequently a method for the determination of saxitoxin in canned shellfish was developed. This method offers the advantage that parallel with the fluorophotometric determination a biotest with mice can be carried out with the same extract for forensic corroboration of the results. The extent of saxitoxin occurence in Spanish canned shellfish in Austria in the years 1976-1979 is described. Apparently the producers of canned shellfish were able to solve this problem since mid - 1978.

Animals↗
PubMed1981

Glycoprotein characteristics of the sodium channel saxitoxin-binding component from mammalian sarcolemma.

The saxitoxin-binding component of the excitable membrane sodium channel exhibits glycoprotein characteristics as evidenced by its specific interaction with various agarose-immobilized lectins. The detergent-solubilized saxitoxin-binding component interacts quantitatively with immobilized wheat germ agglutinin and concanavalin A and fractionally with immobilized Lens culinaris hemagglutinin and Ricinus communis agglutinin. These lectins preferentially bind N-acetylglucosamine and sialic acid (wheat germ agglutinin), mannose (concanavalin A and Lens cunilaris) and galactose (Ricinus communis). Removal of terminal sialic acid residues by neuraminidase markedly decreases binding to immobilized wheat germ agglutinin but uncovers sites capable of interacting with lectins specific for galactose and N-acetylgalactosamine. beta-N-acetylglucosaminidase, an exoglycosidase, has no effect on the binding of the channel protein to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component appears to be a glycoprotein containing terminal sialic acid residues and internal mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine residues. The toxin binding site is spatially separated from the binding sites for the lectins studied. The effect of these sugar moieties must be considered when evaluating the biophysical parameters of the sodium channel.

Animals↗
PubMed1984

Sodium channels in vertebrate hearts. Three types of saxitoxin binding sites in heart.

The affinity of saxitoxin binding to cardiac sarcolemmal and cytosolic fractions was examined across species. In amphibia (frog) the plasma membrane site demonstrated a high affinity (Kd approx. 5 X 10(-9) M) but the majority of the total sites in the homogenate appeared to be high affinity soluble sites (Kd approx. 2 X 10(-9) M). Chicken and turtle cardiac plasma membrane fractions bound [3H]saxitoxin with 500-fold less affinity (Kd values of approx. 2 X 10(-6) M). No binding was seen in the cytosol. The affinity of cardiac sarcolemmal binding in amphibians correlates quantitatively with the K0.5 for the inhibition of sodium currents. Physiological correlation of the low affinity saxitoxin sites in chicken and turtle with toxin concentrations necessary to inhibit the sodium current remains unclear. The hypothesis that frog cytosolic saxitoxin binding sites originated from sarcolemma during homogenization is examined. The presence of three types of saxitoxin binding sites in cardiac preparations supports the existence of sodium channel subtypes.

Animals↗
PubMed1982

Saxitoxin binding sites in frog-myocardial cytosol.

Cytosolic fractions of frog heart homogenates contain large amounts of a soluble, large molecular weight protein that binds the specific neurotoxin saxitoxin with the same high affinity as does the plasma membrane. Another neurotoxin, tetrodotoxin, which ordinarily is competitive with saxitoxin, does not displace saxitoxin from the cytosolic sites or from plasma membrane-enriched vesicular fractions even when its concentration exceeds that of saxitoxin by a factor of 1000. Thus, cytosolic sites are similar to membrane sites in this respect. The vesicular fraction accounts quantitatively for the amount of saxitoxin bound by whole ventricles, so that no appreciable losses seem to occur. Therefore, the cytosolic site probably is a membrane site precursor, although other possibilities cannot be ruled out. In any case, the occurrence of a soluble molecule closely related to the sodium channel provides opportunities for further study of the structure of the sodium channel.

Animals↗
PubMed1980

Saxitoxin binding to sodium channels in head extracts from wild-type and tetrodotoxin-sensitive strains of Drosophila melanogaster.

Extracts prepared from heads of Drosophila melanogaster show high-affinity binding (KD = 1.9 nM) of [3H]saxitonin, a compound known to bind to and block voltage-sensitive sodium channels in other organisms. The interaction between saxitoxin and the Drosophila saxitoxin receptor is non-cooperative and reversible with a half-life of 18.3 s for binding at 4 degrees C. The saturable binding is specifically inhibited by tetrodotoxin with a K1 = 0.30 nM. The number of saturable binding sites in the extract is 97 fmol/mg protein. Since approx. 50% of the binding activity is recovered in the extract, the number of binding sites in the head is estimated to be 6.4 fmol/mg head. Nerve conduction in Drosophila larvae is completely blocked after 20 min in a bathing solution containing 200 nM tetrodotoxin. A comparison between the binding and the electrophysiological studies in Drosophila and other organisms suggests that the Drosophila saxitoxin receptor is part of the voltage-sensitive sodium channel involved in the propagation of action potentials. A mutant (ttxs), which is abnormally sensitive to dietary tetrodotoxin, is shown to be indistinguishable from wild type with respect to [3H]saxitonin-binding properties and physiological sensitivity to tetrodotoxin. These studies provide techniques which can be used to identify mutants with defects in the saxitoxin-binding component of the sodium channel.

Animals↗
PubMed1982

HPLC separation and comparative toxicity of saxitoxin and its reaction products.

A chromatographic method was developed that was used to purify saxitoxin and separate it from its chemically modified products and the reagents used in the reactions. The separation time is about 10 minutes. Using differential-refractive-index detection, quantitation of the products (+/- 10%) can be done on 30-100 microgram of toxin. A simple bioassay with crab leg nerves in vitro was used in conjunction with the chromatography to determine, within a factor of two, the inhibition binding constants of saxitoxin and its products. The binding constant for saxitoxin at ambient temperature, 18-21 degree C, is Ki approximately 80 nM. The acid-hydrolysis product has Ki approximately 8 microM under the same conditions. The chemistry of saxitoxin was investigated using the chemical and bioassays.

Animals↗
PubMed1990

High-affinity binding sites for [3H]saxitoxin are associated with voltage-dependent sodium channels in portal vein smooth muscle.

Saturable, high-affinity binding sites for [3H]saxitoxin were identified in equine portal vein smooth muscle membranes. These sites had a dissociation constant of 0.29 nM and a maximal binding capacity of 115 fmol.mg-1 of protein. A similar dissociation constant was obtained with cells prepared from rat portal vein. Specific binding of [3H]saxitoxin was completely displaced by unlabelled saxitoxin and tetrodotoxin, with inhibition constants of 0.42 and 2.10 nM, respectively. Tetrodotoxin blocked the fast Na+ current in single cells of rat portal vein in a concentration-dependent manner, with an IC50 of 3.15 nM. These results suggest that the high-affinity binding sites for tetrodotoxin and saxitoxin may be associated with voltage-dependent Na+ channels in vascular myocytes.

Amphibian Proteins↗
PubMed1998

Comparative analyses by HPLC and the sodium channel and saxiphilin 3H-saxitoxin receptor assays for paralytic shellfish toxins in crustaceans and molluscs from tropical North West Australia.

The increased frequency and distribution of red tides requires the development of high-throughput detection methods for paralytic shellfish toxins (PST). Community ethics also requires that there be a reduced reliance upon the standard mouse bioassay. A biomolecular assay such as the sodium channel 3H-saxitoxin binding assay can satisfy both of these requirements but may be compromised by cross-reactivity with the structurally unrelated tetrodotoxins (TTX). This study utilised the sodium channel assay but also an alternative 3H-saxitoxin binding assay based upon a saxiphilin isoform from the centipede Ethmostigmus rubripes to screen for PSTs. Saxiphilin is a novel transferrin which binds saxitoxin (STX) but differs from the sodium channel in not having any measurable affinity for TTX. A detailed analysis of toxin composition was achieved by high performance liquid chromatography (HPLC). Various crustaceans and molluscs accumulate PSTs and TTX, thus proving useful biomarkers for these toxins in their immediate environment and an ideal challenge to the detection and analysis of PSTs in this presumptive screening program. Also, there has been little investigation of PSTs in invertebrates from the Indian Ocean so this region was selected to extend our knowledge of the distribution of these toxins. 190 crabs and shellfish encompassing 31 species were collected from reefs along the North-West Australian coast and tested for PSTs and TTX by sodium channel and saxiphilin bioassays as well as HPLC. PSTs were detected in 18 species of crabs and shellfish of the 31 species tested. Eight of these species have not been previously described as toxic, these being the crabs Euzanthus exsculptus, Lophozozymus octodentatus, Metopograpsus frontalis, Pilumnus pulcher, Platypodia pseudogranulosa and Portunus pelagicus, and the molluscs Tectus fenestratus and Trochus hanleyanus. By HPLC, only one or both of STX and decarbamoyl-STX was detected in any extract. Some extracts markedly inhibited 3H-saxitoxin binding by the sodium channel but not by saxiphilin. The close agreement between toxin quantification by the PST specific methods of HPLC and the saxiphilin bioassay is indicative that the additional toxicity detected by the sodium channel assay is TTX.

Amphibian Proteins↗