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PubMed1988

[3H]saxitoxin as a marker for canine deep muscular plexus neurons.

The objectives of this study are 1) to examine the potential of [3H]saxitoxin binding as a marker for the neuronal membranes in canine small intestinal muscle membrane preparations, 2) to develop a synaptosomal preparation from deep muscular plexus, and 3) to partially characterize [3H]saxitoxin binding to this fraction. A purified synaptosomal fraction, relatively low in the smooth muscle plasma membrane marker enzyme 5'-nucleotidase but enriched in [3H]saxitoxin binding (2,592 fmol/mg), was obtained on sucrose density gradient. Vasoactive intestinal peptide immunoreactivity was also highest (51.82 pmol/mg protein) in this fraction. The binding was rapid at 20 degrees C with quick and complete dissociation after the addition of excess unlabeled tetrodotoxin (TTX). Scatchard analysis of the saturation binding data revealed a single population of binding sites (Bmax = 5,705 fmol/mg protein). The affinity constants calculated from the kinetic and saturation data were in close agreement (Kd = 0.26 and 0.69 nM, respectively). TTX competed for the binding (Ki = 2.1 nM), whereas veratridine and guanidinium hydrochloride did not. Monovalent and divalent cations had differential effects on the binding.

Animals↗
PubMed2002

Mutual binding inhibition of tetrodotoxin and saxitoxin to their binding protein from the plasma of the puffer fish, Fugu pardalis.

The mutual binding inhibition of tetrodotoxin and saxitoxin to their binding protein from the plasma of Fugu pardalis was investigated by HPLC. The values for the half inhibitory concentration of tetrodotoxin (1.6 microM) binding to this protein (1.2 microM) for saxitoxin, and of saxitoxin (0.47 microM) binding to that (0.30 microM) for tetrodotoxin were 0.35 +/- 0.057 microM and 81 +/- 16 microM (n = 2), respectively.

Animals↗
PubMed1991

Purification and partial sequencing of saxiphilin, a saxitoxin-binding protein from the bullfrog, reveals homology to transferrin.

Plasma from the bullfrog, Rana catesbeiana, contains a soluble component of unknown function that specifically binds the neurotoxin, [3H]saxitoxin, with a Kd of approximately 0.2 nM. Saxiphilin, the protein responsible for this activity, was purified approximately 440-fold from bullfrog plasma by column chromatography on heparin-Sepharose followed by chromatofocusing. The purified saxiphilin preparation exhibits a binding capacity of 9.6 nmol/mg protein and a Kd of 0.32 nM for [3H]saxitoxin. Analysis of the preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a predominant band migrating with an apparent Mr of approximately 89,000 which is similar to the expected size of saxiphilin previously estimated by nondenaturing size exclusion chromatography. Amino-terminal sequencing of the approximately 89-kDa protein and sequencing of four different tryptic peptide fragments revealed that each of the partial saxiphilin sequences can be aligned by homology with members of the transferrin protein family with sequence identity as high as 69%. The available sequence corresponding to conserved residues that comprise part of the two Fe3+ binding sites in lacto-transferrin show several substitutions in saxiphilin, suggesting that saxiphilin is not an Fe(3+)-binding protein. Saxiphilin appears to be a monomeric approximately 89-kDa protein that is evolutionarily related to transferrin but which binds saxitoxin instead of Fe3+.

Amino Acid Sequence↗
PubMed1980

Size characteristics of the solubilized saxitoxin receptor of the voltage-sensitive sodium channel from rat brain.

The saxitoxin receptor of the voltage-sensitive sodium channel from rat brain was solubilized with Triton X-100 and stabilized with phosphatidylcholine. The size characteristics of the detergent . phospholipid . receptor complex were studied by gel filtration and sucrose gradient sedimentation in H2O and D2O. The complex has Stokes radius = 80 A, S20,W = 12 S, v = 0.82 ml/ g, and Mr = 601,000 +/- 48,000. Assuming v = 0.73 ml/g for the saxitoxin receptor protein, the mass of the complex consists of 47.4% detergent and phosphatidylcholine and 52.6% saxitoxin receptor protein with Mr = 316,000 +/- 63,000.

Amphibian Proteins↗
PubMedDate not supplied

Determination of decarbamoyl saxitoxin and its analogues in shellfish by prechromatographic oxidation and liquid chromatography with fluorescence detection.

Oxidation and chromatographic conditions for detecting the decarbamoyl analogues of several paralytic shellfish poison (PSP) toxins were studied. Prechromatographic oxidation with periodate or hydrogen peroxide under slightly alkaline conditions was used as previously reported for the parent PSP toxins. Both periodate and hydrogen peroxide oxidations produced 2 fluorescent products separable by liquid chromatography for each decarbamoyl (dc) toxin (dc-saxitoxin, dc-neosaxatoxin and dc-gonyautoxins 2 and 3). Decarbamoyl saxitoxin produced the same 2 products as did dc-neosaxitoxin but in different ratios. One of these products was the same as the one obtained with neosaxitoxin after periodate oxidation. Decarbamoyl gonyautoxins 2 and 3 (together) produced 2 products, one of which was the same as the major product obtained with gonyautoxins 1 and 4 (together) after periodate oxidation. Decarbamoyl gonyautoxins 1 and 4 were not available for study. The method was used to detect dc-saxitoxin and dc-gonyautoxins 2 and 3 in shellfish extracts.

Animals↗
PubMed1969

Differences between the effects of saxitoxin (paralytic shellfish poison) and tetrodotoxin on the frog neuromuscular junction.

1. End-plate potentials (e.p.p.) have been recorded from the neuromuscular junctions of frog sartorius and extensor longus dig. IV muscles, using intracellular micropipettes. Either curare or MgCl(2) were present in the Ringer solution, to keep the e.p.p. amplitude below the threshold for a muscle action potential and contraction.2. It has been shown that saxitoxin (paralytic shellfish poison) usually caused a progressive reduction in the amplitude of the e.p.p. Occasionally, when it was applied in the presence of MgCl(2), the e.p.p. disappeared abruptly.3. Tetrodotoxin usually caused the e.p.p. to disappear abruptly. Occasionally, when applied in the presence of curare, the e.p.p. declined progressively for a short time before disappearing abruptly.4. It is concluded that at the frog neuromuscular junction the preferential site of action of saxitoxin is at the nerve terminals, but tetrodotoxin preferentially blocks nerve conduction at a site proximal to the junction.5. It is suggested that this preparation would be a convenient and reliable test object for distinguishing saxitoxin from tetrodotoxin.

Animals↗
PubMed1997

New saxitoxin analogues from the freshwater filamentous cyanobacterium Lyngbya wollei.

Along with decarbamoylsaxitoxin and decarbamoylgonyautoxin-2 and -3, six new saxitoxin analogues were isolated from the freshwater mat-forming filamentous cyanobacterium Lyngbya wollei collected from Guntersville Reservoir on the Tennessee River in Alabama. Their structures were determined by electrospray ionization mass spectrometry and several NMR techniques. Five of the toxins contain an acetyl moiety attached to the side chain, which is the first report of these saxitoxin analogues. In three of the toxins a hydrated ketone at C-12 was reduced to alpha-alcohol. The presence of acetate in the side chain resulted in a sevenfold to 17-fold times decrease in mouse toxicity compared to their carbamoyl counterparts, while the reduction at C-12 resulted in a complete loss of mouse toxicity.

Animals↗
PubMed1984

Reconstitution of highly purified saxitoxin-sensitive Na+-channels into planar lipid bilayers.

Highly purified Na+-channels isolated from rat brain have been reconstituted into virtually solvent-free planar lipid bilayer membranes. Two different types of electrically excitable channels were detected in the absence of any neurotoxins. The activity of both channels was blocked by saxitoxin. The first channel type is highly selective for Na+ over K+ (approximately 10:1), it shows a bursting behavior, a conductance of 25 pS in Na+-Ringer and undergoes continuous opening and closing events for periods of minutes within a defined range of negative membranes voltages. The second channel type has a conductance of 150 pS and a lower selectivity for Na+ and K+ (2.2:1); only a few opening and closing events are observed with this channel after one voltage jump. The latter type of channel is also found with highly purified Na+-channel from Electrophorus electricus electroplax. A qualitative analysis of the physicochemical and pharmacological properties of the high conductance channel has been carried out. Channel properties are affected not only by saxitoxin but also by a scorpion (Centruroides suffusus suffusus) toxin and a sea anemone (Anemonia sulcata) toxin both known to be selective for the Na+-channel. The spontaneous transformation of the large conductance channel type into the small one has been considered; the two channel types may represent the expression of activity of different conformational states of the same protein.

Animals↗
PubMed1992

Analysis of saxitoxin in urine by continuous-flow fast-atom bombardment mass spectrometry.

An improved method of saxitoxin analysis in urine using continuous-flow fast-atom bombardment mass spectrometry was developed. Parameters studied were matrix composition, matrix flow, temperature of probe tip, probe-tip design and sample extraction. Optimal detection was obtained using the following matrix composition: 5% glycerol, 0.5% acetic acid, 0.025% sodium dodecylsulfate, 0.1% polyethylene glycol (PEG) 400 and 0.5% PEG 300; probe-tip temperature: (approximately 55 degrees C); flow rate: 5 or 8 microL per min.; probe tip: Olson-Hogge design. The STX standard was detected at 200 pg with signal-to-noise ratio of 11. The percent recovery of saxitoxin from human urine after clean-up on a weak cation exchange column was 75%.

Humans↗
PubMed2005

First report of saxitoxin in Finnish lakes and possible associated effects on human health.

This study is the first report of saxitoxin in cyanobacterial blooms in Finland. Bloom samples (n = 50) were collected from Finnish freshwater sites during summer months of 2002 and 2003. These samples were screened for the presence of paralytic shellfish toxins (PSTs) using the Jellett rapid PSP screening test. Samples testing positive for PSTs (n = 7) were further analyzed with saxiphilin- and voltage-gated sodium channel [(3)H]-STX-binding radioreceptor assays and liquid chromatography using fluorescence and mass spectrometric analysis. The results indicated that saxitoxin (STX) was the only PST analogue in the samples and that it was present in high concentrations, as much as 1 mg L(-1). Microscopic analysis revealed that 95%-100% of the phytoplankton in the positive samples consisted of Anabaena lemmermannii. The trophic status of lakes in which STX-containing blooms were found varied from oligotrophic to hypertrophic. All the lakes had high nitrogen-to-phosphorus ratios. In some instances, samples had been collected from sites where swimmers had reported adverse health effects, and in three such cases, reported adverse health effects were associated with sites from which samples testing positive for STX had been received. Symptoms of fever, eye irritation, abdominal pains, and skin rash were reported in children aged 2-10 years after exposure to the water. These were not the adverse human symptoms typical of STX poisoning; rather, they represented acute effects often reported following recreational exposure to cyanobacterial blooms.

Abdominal Pain↗
PubMed1993

Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitive sodium channels: semiautomated assay for saxitoxins, brevetoxins, and ciguatoxins.

In the present study we have developed an assay for the detection of sodium channel-specific marine toxins based upon mitochondrial dehydrogenase activity in the presence of veratridine and ouabain. This cell bioassay allows detection of either sodium channel enhancers, such as the brevetoxins and the ciguatoxins, or sodium channel blocking agents, such as the saxitoxins. The assay responds in a dose dependent manner and differentiates the toxic activity as either sodium channel blocking or enhancing. In addition, the assay is highly sensitive, with present detection limits of 2 ng/ml for either saxitoxins or brevetoxins (PbTx-1 and PbTx-3). Assay response to a ciguatoxic extract and to brevetoxins is rapid, allowing dose dependent detection within 4 to 6 h. The method is simple, utilizes readily available reagents, uses substantially less sample than required for mouse bioassay, and is well within the scope of even modest tissue culture facilities. This cell-based protocol has the potential to serve as an alternate and complementary method to the standard mouse bioassay.

Animals↗
PubMed1994

Antibody-antigen binding constants determined in solution-phase with the threshold membrane-capture system: binding constants for anti-fluorescein, anti-saxitoxin, and anti-ricin antibodies.

Affinities of various monoclonal and polyclonal antibodies for fluorescein-containing antigens, saxitoxin and ricin, were determined by using a light addressable potentiometric sensor-based system (Threshold). The dissociation constants, determined from Scatchard plots, ranged from 2 x 10(-7) to approximately 3 x 10(-12) M. Dissociation constants for fluorescein and saxitoxin were compared with values determined by independent means. This technique was found to be quick, simple, reproducible, and accurate.

Antibodies↗
PubMed1983

The active guanidinium group of saxitoxin and neosaxitoxin identified by the effects of pH on their activities on squid axon.

The relative potencies of saxitoxin at pH 7.25 and 8.25 have been determined on the squid giant axon under voltage-clamp conditions or by Vmax of the propagated action potential. Of the two guanidinium groups in saxitoxin, the 7, 8, 9 group has been identified as the biologically active group. The evidence lies in the demonstration of a quantitative agreement between the relative abundance of the protonated, positively charged form of that group at pH's 7.25 and 8.25 (ratio 1.80) with the relative potencies (ratio 1.79) of the toxin. The 1, 2, 3 group is excluded by the lack of agreement between the relative abundance of the protonated form (ratio 1.00) and the relative potencies at these pH's. The 1, 2, 3 group is further excluded by the observation that neosaxitoxin is equally potent at pH 6.50 and 7.25, in spite of a difference of 6-fold in the abundance of a deprotonated hydroxyl group on N-1 which should have influenced the potency.

Animals↗
PubMed1990

Isolation of two saxitoxin-sensitive sodium channel subtypes from rat brain with distinct biochemical and functional properties.

Two different 3H-saxitoxin-binding proteins, with distinct biochemical and functional properties, were isolated from rat brain using a combination of anion exchange and lectin affinity chromatography as well as high resolution size exclusion and anion exchange HPLC. The alpha subunits of the binding proteins had different apparent molecular weights on SDS-PAGE (Type A: 235,000; Type B: 260,000). When reconstituted into planar lipid bilayers, the two saxitoxin-binding proteins formed sodium channels with different apparent single-channel conductances in the presence of batrachotoxin (Type A: 22 pS; Type B: 12 pS) and veratridine (Type A: 9 pS; Type B: 5 pS). The subtypes were further distinguished by scorpion (Leiurus quinquestriatus) venom which had different effects on single-channel conductance and gating of veratridine-activated Type A and Type B channels. Scorpion venom caused a 19% increase in single-channel conductance of Type A channels and a 35-mV hyperpolarizing shift in activation. Scorpion venom doubled the single-channel conductance of Type B channels and shifted activation by at least 85 mV.

Amphibian Proteins↗
PubMed1976

Structural characteristics of the saxitoxin receptor on nerve.

The effects of uranyl ion (UO22+; at low concentrations binds specifically to phosphate groups) and the cationic dye methylene blue (MB+; binds strongly to carboxyl groups) on saxitoxin (STX) potency in crayfish axon has been studied by means of intracellular microelectrodes. At pH 6.00 +/- 0.05 and 13.5 mM Ca2+, addition of 10.0 muM UO22+ + 5.0 nM STX had only slightly, if any, less effect on the spike's maximum rate of rise [0.79 +/- 0.04 (viz., mean +/- SEM) of control value] than did addition of 5.0 nM STX alone (0.72 +/- 0.05). Under the same conditions of pH and Ca2+ concentration, 1.0 mM MB+ had approximately the same effect: 1.0 mM MB+ + 5.0 nM STX, 0.76 +/- 0.03; 5.0 nM STX alone, 0.70 +/- 0.04. However, at pH 7.00 +/- 0.05 and lower Ca2+ concentrations, 1.0 mM MB+ significantly reduced STX potency. Using 6.0 mM Ca2+: 1.0 mM MB+ + 5.0 nM STX, 0.92 +/- 0.01; 5.0 nM STX alone, 0.68 +/- 0.08. Using 3.0 mM Ca2+, the corresponding values were 0.94 +/- 0.03 and 0.67 +/- 0.04. It is concluded that: (1) In accord with previous suggestions, the ionized acidic group known to exist in the Na channel (and to which a guanidinium group of STX appears to bind) is very likely a carboxyl group and not a phosphate group. (2) The accessible part of the Na channel mouth serving as the saxitoxin receptor probably does not include phospholipid in its structure proper.

Action Potentials↗
PubMed2006

Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: deconvolution strategy.

Abstract Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X1-X2-hinge-X3-X4-NH2 (capped) and X1-hinge-X2-X3 (uncapped), where X1 through X4 are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a central hinge residue in small peptides increases the opportunity for side chain interactions, providing a distinct advantage for hinge peptide libraries over conventional peptide libraries. Hinge peptide libraries are a rich source of novel ligands for modulation of biomechanisms. The library subsets uncovered in this study may possess peptides that will lead to effective therapies to neurotoxin poisoning.

Biological Assay↗
PubMed1984

A competitive displacement assay to detect saxitoxin and tetrodotoxin.

An assay is described which detects saxitoxin (STX) and tetrodotoxin (TTX) by their competitive displacement of [3H]saxitoxin from its receptor in rat brain membranes. The assay has a sensitivity of 0.15 ng STX/ml and 0.8 ng TTX/ml for buffer samples. The assay was also applied to detection of these toxins in unextracted human plasma and found to have a sensitivity of 0.5 ng STX/ml and 0.6 ng TTX/ml. The competitive displacement assay appears to be the most sensitive procedure yet for detection of STX and TTX.

Amphibian Proteins↗
PubMed1983

Saxitoxin and ouabain binding activity of isolated skeletal muscle membrane as indicators of surface origin and purity.

A simple biochemical method for identifying and distinguishing transverse tubule and sarcolemma membranes in preparations of skeletal muscle microsomes is proposed and evaluated. This method is based on the previous observation that the ratio of ouabain to saxitoxin binding sites is five-fold higher in the sarcolemma than the transverse tubule. We measured [3H]saxitoxin and [3H]ouabain binding to microsomes of frog, rat and rabbit muscle in the presence of detergents to expose latent sites. A high density fraction (30--40% sucrose) of the membranes was identified as transverse tubule on the basis of a low ouabain/saxitoxin ratio and its association with sarcoplasmic reticulum. A low density fraction (20--30% sucrose) was identified as transverse tubule containing variable amounts of sarcolemma as judged by a higher ratio of ouabain/saxitoxin sites. Our results suggest that this ratio can be used to determine the surface origin of muscle membrane preparations. Several different methods for purifying transverse tubules were compared by this technique.

Amphibian Proteins↗