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A peptide was isolated from the venom of the scorpion Tityus trivittatus. It is an isoform of the toxin TsTX-IV earlier described [Toxicon 37 (1999) 651] and identical to butantoxin [Arch. Biochem. Biophys. 379 (2000) 18], both isolated from the Brazilian scorpion Tityus serrulatus. This newly characterized peptide contains 40 amino acid residues with a molecular mass of [M+H(+)] 4507.0, cross-linked by four disulfide bridges, made between the cysteine pairs: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36 and Cys20-Cys38. It blocks in a completely reversible manner the Shaker B K(+)-channels, with a K(d) around 660nM. It belongs to the sub-family 12 and it is now being classified as alpha-KTx 12.2.

Two novel toxic peptides (Tc30 and Tc32) were isolated and characterized from the venom of the Brazilian scorpion Tityus cambridgei. The first have 37 and the second 35 amino acid residues, with molecular masses of 3,871.8 and 3,521.5, respectively. Both contain three disulfide bridges but share only 27% identity. They are relatively potent inhibitors of K(+)-currents in human T lymphocytes with K(d) values of 10 nM for Tc32 and 16 nM for Tc30, but they are less potent or quite poor blockers of Shaker B K(+)-channels, with respective K(d) values of 74 nM and 4.7 microM. Tc30 has a lysine in position 27 and a tyrosine at position 36 identical to those of charybdotoxin. These two positions conform the dyad considered essential for activity. On the contrary, Tc32 has a serine in the position equivalent to lysine 27 of charybdotoxin and does not contain any aromatic amino acid. Due to its unique primary sequence and to its distinctive preference for K(+)-channels of T lymphocytes, it was classified as the first example of a new subfamily of K(+)-channel-specific peptides (alpha-KT x 18.1). Tc30 is a member of the Tityus toxin II-9 subfamily and was given the number alpha-KT x 4.4.

By means of high performance liquid chromatography (HPLC) the soluble venom of the Amazonian scorpion Tityus cambridgei was fractionated into over 50 different components. Four toxic and/or lethal peptides to mice were obtained in pure form and sequenced. Mass spectrometry analysis showed molecular weights of 7310, 7151, 7259 and 7405, respectively, for toxins Tc48a, Tc49a, Tc54 and Tc49b. The N-terminal amino acid sequence was obtained for the three first toxins mentioned, whereas the full primary structure was determined for Tc49b. It contains 64 amino acid residues, closely packed by four disulfide bridges. Sequence comparison analysis showed similarities around 50% with other toxins from scorpions of the genus Tityus of Brazil. It is lethal to mice at doses of 20 microg per 20 g mouse. The toxin was shown to affect the Na(+)-currents permeability of rat cerebellum granular cells in culture. Almost a complete elimination of current was observed with 100 nM toxin concentration. This effect was partially reversible. Furthermore, this toxin does not modify the function of the Shaker B K(+)-channels expressed on Sf9 cells, nor does it modify the Na(+)-channel function in a similar manner as those reported for the alpha-scorpion toxins purified from other scorpions.

A novel crustacean toxin (Cn11) was isolated and characterized from the venom of the Mexican scorpion Centruroides noxius Hoffmann. It contains 63 amino acid residues and is stabilized by four disulphide bridges. It is lethal to crustaceans (Cambarellus montezumae), less toxic to insects (crickets) and non-toxic to mammals (mice) at the doses assayed. In neurons isolated from the X organ-sinus gland system of the crayfish Procambarus clarkii, it blocks the Na(+) currents with an estimated K(m) of 320 nmol l(-1), without affecting the Ca(2+) and K(+) currents. The voltage-gated tetrodotoxin-sensitive Na(+) current was recorded from X organ neurons in culture 24 h after plating using the whole-cell clamp configuration. The Na(+) current was isolated by blocking Ca(2+) currents with Cd(2+) and Cs(+) and K(+) currents with tetraethylammonium and 4-aminopyridine. Under control conditions, the Na(+) currents were activated at -40 mV with a maximum amplitude at 0 mV. In the presence of 1 micromol l(-1) Cn11, the Na(+) current amplitude was reduced by 75 % without apparent modifications to the gating mechanism. These findings suggest that Cn11 selectively blocks a Na(+) channel. It is the first representative of a new group of scorpion toxins specific for this molecular target.

We show here that ergtoxin (ErgTx) is a bona fide, specific blocker of the human ether-a-go-go-related gene (HERG) channels. It does not affect the function of either M-eag or M-elk channels. A chimeric construction containing a segment of the P-region of M-eag channel inserted into the HERG channel drastically diminished or completely abolished the inhibitory effect of ErgTx, whereas chimeras of the P-region of HERG channel into M-eag channels recovered the inhibitory effect. From the P-region point mutants of HERG channel assays, only the mutant N598Q shows about 25% decrement of the ErgTx inhibitory effect. ErgTx recognizes the P-region of HERG channels, blocking the channel function with a K(d) in the order of 12 nM.

A novel peptide from Centruroides noxius Hoffmann scorpion venom was isolated and sequenced. The 37 amino acid peptide belongs to the charybdotoxin sub-family (alphaKTx1) and was numbered member 11. alphaKTx1.11 has 75% sequence identity with iberiotoxin and 54% with charybdotoxin. alphaKTx1.11 revealed specificity for mammalian MaxiK channels (hSlo), thus, was named slotoxin. Slotoxin blocks the MaxiK pore-forming alpha subunit reversibly (K(d)=1.5 nM). Slotoxin association with alpha+beta (beta1 or beta4) channels was approximately 10 times slower than iberiotoxin and charybdotoxin, leading to a lack of effect on alpha+beta4 when tested at 100 nM for 5 min. Thus, slotoxin is a better tool to distinguish MaxiK alpha+beta complexes.

A new peptide, Tc1, containing only 23 amino acids closely packed by three disulfide bridges was isolated from the Amazonian scorpion Tityus cambridgei. It blocks reversibly the Shaker B K(+)-channels with a K(d) of 65 nM and displaces binding of noxiustoxin to mouse brain synaptosome membranes. It is the shortest known peptide from scorpion venom that recognizes K(+)-channels and constitutes a new structural subfamily of toxin, classified as alphaKTx 13.1.

A new antimicrobial peptide, hadrurin, was isolated from the venom of the Mexican scorpion Hadrurus aztecus, by gel filtration on a Sephadex G-50 column, followed by high performance liquid chromatography. It is a basic peptide composed of 41 amino-acid residues with a molecular mass of 4436 Da, and contains no cysteines. A model of the three-dimensional folding of hadrurin is compatible with that of an amphipatic molecule with two alpha-helical segments. Hadrurin demonstrates antimicrobial activity at low micromolar concentration, inhibiting the growth of bacteria such as: Salmonella thyphi, Klebsiella pneumoniae, Enterococcus cloacae, Pseudomonas aeruginosa, Escherichia coli and Serratia marscences. It also shows cytolytic activity when tested in human erythrocytes. Hadrurin and two analogs (C-terminal amidated, and all D-enantiomer) were chemically synthesized. They were used to study the possible molecular mechanism of action by testing their ability to dissipate the diffusion potential of liposomes of different compositions. The results obtained indicate that there are no specific receptor molecules for the action of hadrurin, and the most probable mechanism is through a membrane destabilization activity. It is surmised that hadrurin is used by the scorpion as both an attack and defense element against its prey and putative invasive microorganisms. It is a unique peptide among all known antimicrobial peptides described, only partially similar to the N-terminal segment of gaegurin 4 and brevinin 2e, isolated from frog skin. It would certainly be a model molecule for studying new antibiotic activities and peptide-lipid interactions.

A novel peptide, scorpine, was isolated from the venom of the scorpion Pandinus imperator, with anti-bacterial activity and a potent inhibitory effect on the ookinete (ED(50) 0.7 microM) and gamete (ED(50) 10 microM) stages of Plasmodium berghei development. It has 75 amino acids, three disulfide bridges with a molecular mass of 8350 Da. Scorpine has a unique amino acid sequence, similar only to some cecropins in its N-terminal segment and to some defensins in its C-terminal region. Its gene was cloned from a cDNA library.

The primary structure of a phospholipase A2, with unique structural and functional characteristics, was determined. The large subunit has 108 amino acid residues, linked by a disulfide bridge to the small subunit, which contains 17 residues. Its gene was cloned from a cDNA library. The nucleotide sequence showed that the same RNA messenger encodes both subunits, separated only by a pentapeptide, that is processed during maturation.

The antibody BCF2 generated against the mammal-specific toxin Cn2 of the scorpion Centuroides noxius Hoffmann neutralizes the effect of both the toxin and the venom. We cloned and sequenced the genes coding for the Fv fragment of BCF2. A three-dimensional (3D) model of the Fv fragment was generated using a knowledge-based approach. Furthermore, a 3D model of the complex Cn2-BCF2 was built using the nuclear magnetic resonance (NMR) structure of Cn2 and experimental results on a putative epitope region around the N and C termini. The initial complex conformations were submitted to a new refinement procedure of rigid-body energy minimization combined with flexible-side-chain molecular dynamics. The final complex, selected after an extensive evaluation, uses the loop 7-11 as the central part of the epitope. The generated complex allows the following conclusions: 1) the neutralizing capacity of BCF2 toward the venom of C. noxius might rather be caused by the high venom concentration and toxicity of Cn2 than by a broad specificity, 2) the region involved in the binding of Cn2 to the Na(+) channel, should overlap with the employed epitope region, and 3) contact residues SerL91, AsnL92, LeuH50, AspH56, TyrH95, and TyrH98 of BCF2 are candidates for mutations to broaden its specificity. Proteins 1999;37:130-143., (Copyright 1999 Wiley-Liss, Inc.)

Na+-channel specific scorpion toxins are peptides of 60-76 amino acid residues in length, tightly bound by four disulfide bridges. The complete amino acid sequence of 85 distinct peptides are presently known. For some toxins, the three-dimensional structure has been solved by X-ray diffraction and NMR spectroscopy. A constant structural motif has been found in all of them, consisting of one or two short segments of alpha-helix plus a triple-stranded beta-sheet, connected by variable regions forming loops (turns). Physiological experiments have shown that these toxins are modifiers of the gating mechanism of the Na+-channel function, affecting either the inactivation (alpha-toxins) or the activation (beta-toxins) kinetics of the channels. Many functional variations of these peptides have been demonstrated, which include not only the classical alpha- and beta-types, but also the species specificity of their action. There are peptides that bind or affect the function of Na+-channels from different species (mammals, insects or crustaceans) or are toxic to more than one group of animals. Based on functional and structural features of the known toxins, a classification containing 10 different groups of toxins is proposed in this review. Attempts have been made to correlate the presence of certain amino acid residues or 'active sites' of these peptides with Na+-channel functions. Segments containing positively charged residues in special locations, such as the five-residue turn, the turn between the second and the third beta-strands, the C-terminal residues and a segment of the N-terminal region from residues 2-11, seems to be implicated in the activity of these toxins. However, the uncertainty, and the limited success obtained in the search for the site through which these peptides bind to the channels, are mainly due to the lack of an easy method for expression of cloned genes to produce a well-folded, active peptide. Many scorpion toxin coding genes have been obtained from cDNA libraries and from polymerase chain reactions using fragments of scorpion DNAs, as templates. The presence of an intron at the DNA level, situated in the middle of the signal peptide, has been demonstrated.

The authors have developed a method for rapid detection of polymerase chain reaction (PCR) amplicons based on surface immobilized PNA-DNA hybrid probes ('molecular beacons') that undergo a fluorescent-linked conformational change in the presence of a complementary DNA target. Amplicons can be detected by simply adding a PCR reaction to a microtitre-well containing the previously immobilized probe, and reading the generated fluorescence. No further transfers or washing steps are involved. The authors demonstrate the specificity of the method for the detection of ribosomal DNA from Entamoeba histolytica.

Several site directed mutations were introduced into a synthetic Noxiustoxin (NTX) gene. Alanine scanning of the nonapeptide at the N-terminal segment of NTX (threonine 1 (T1) to serine 9 (S9)) was constructed and the recombinant products were obtained in pure form. Additionally, lysine 28 (K28) was changed to arginine (R) or glutamic acid (E), cysteine 29 was changed to alanine, and residues 37-39 (Tyr-Asn-Asn) of the carboxyl end were deleted. The recombinant mutants were tested for their ability to displace 125I-NTX from rat brain synaptosome membranes, as well as for their efficiency in blocking the activity of Kv1.1 K+ channels expressed in Xenopus laevis oocytes. The main results indicate that residues K6, T8 at the amino end, and K28 and the tripeptide YNN at the carboxyl end are involved in specific interactions of NTX with rat brain and/or Kv1.1 K+ channels.

Potassium-channel-blocking scorpion toxins (alpha-K-toxins) have been shown to be valuable tools for the study of potassium channels. Here we report two toxins, cobatoxin 1 and 2, of 32 amino acids, containing three disulphide bridges, that were isolated from the venom of the Mexican scorpion Centruroides noxius. Their primary sequences show less than 40% identity to other alpha-K-toxins. It is therefore proposed that they belong to subfamily 9. The cDNA of cobatoxin 1 encodes a putative signal peptide, a putative short propeptide, the mature peptide and two amino acids that are processed to leave cobatoxin 1 amidated at the C-terminus. In rat brain synaptosomal membranes cobatoxin 1 and cobatoxin 2 bind to a common binding site of alpha-K-toxins with Ki values of 109 pM and 87 pM, respectively. Moreover, they block the Shaker and Kv1.1 K+ channels with moderate affinities, with Kd values of around 0.7 microM and 4.1 microM (Shaker) and 0.5 microM and 1.0 microM (Kv1.1), respectively. A three-dimensional model of cobatoxin 1 was generated and used to interpret the obtained functional data on a structural basis.

Two novel peptides, named Pi4 and Pi7, were purified from the venom of the scorpion Pandinus imperator, and their primary structures were determined. These peptides have 38 amino acids residues, compacted by four disulfide bridges, instead of the normal three found in most K+-channel specific toxins. Both peptides contain 25 identical amino acid residues in equivalent positions (about 66% identity), including all eight half-cystines. Despite the fact that their C-terminal sequence comprising amino acid residues 27 to 37 are highly conserved (10 out of 11 amino acids are identical), Pi4 blocks completely and reversibly Shaker B K+ -channels (a Kv1.1 sub-family type of channel) at 100nM concentration, whereas Pi7 is absolutely inactive at this concentration. Similar effects were observed in binding and displacement experiments to rat brain synaptosomal membranes using 125I-Noxiustoxin, a well known K+-channel specific toxin. In this preparation Pi4 displaces the binding of radiolabeled Noxiustoxin with Ic50 in the order of 10 nM, whereas Pi7 is ineffective at same concentration. Comparative analysis of Pi4 and Pi7 sequences with those obtained by site directed mutagenesis of Charybdotoxin, another very well studied K -channel blocking toxin, shows that the substitution of lysine (in Pi4) for arginine (in Pi7) at position 26, might be one of the important 'point mutations' responsible for such impressive variation in blocking properties of both toxins, here described.

Scorpion venoms contain a variety of low mol. wt peptides toxic to different organisms. These peptides have been intensively studied because they represent excellent models for investigating structure-function relationships and they are also fine probes for studying ionic channel functions. This review deals with the biological and chemical aspects of toxic peptides that affect Na+ or K+ channels and the cloning of the cDNAs and genes encoding the main alpha and beta neurotoxins present in the venom of the three most dangerous species of Brazilian scorpion, Tityus bahiensis, Tityus stigmurus and Tityus serrulatus, and the Venezuelan scorpion Tityus discrepans. At least 16 different peptides specific for Na+ channels and five affecting K+ channels were isolated and characterized from the venom of these scorpions. The isolation of cDNAs and genes encoding four distinct toxins has permitted the elucidation of their nucleotide sequences as well as their genomic organization. Venoms and isolated toxins from scorpions of the genus Tityus were shown to enhance the secretory activity of the pancreas. Antisera obtained against venom of T. serrulatus show cross-reactivity with other species of the Brazilian scorpions.

We present an in-depth analysis of the structural and functional properties of Imperatoxin I (IpTxi), an approximately 15-kDa protein from the venom of the scorpion Pandinus imperator that inhibits Ca2+ release channel/ryanodine receptor (RyR) activity (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12185-12189). A cDNA library was prepared from the venomous glands of this scorpion and used to clone the gene encoding IpTxi. From a single continuous messenger RNA, the information coding for the toxin is translated into two mature polypeptide subunits after elimination of a basic pentapeptide. The IpTxi dimer consists of a large subunit (104-amino acid residues) with phospholipase A2 (PLA2) activity covalently linked by a disulfide bond to a smaller (27 amino acid residues), structurally unrelated subunit. Thus, IpTxi is a heterodimeric protein with lipolytic action, a property that is only shared with beta-bungarotoxins, a group of neurotoxins from snake venoms. The enzymatic subunit of IpTxi is highly homologous to PLA2 from bee (Apis mellifera) and lizard (Heloderma horridum) venoms. The small subunit has no significant similarity to any other known peptide, including members of the Kunitz protease inhibitors superfamily that target the lipolytic effect of beta-bungarotoxins. A synthetic peptide with amino acid sequence identical to that of the small subunit failed to inhibit RyR. On the other hand, treatment of IpTxi with p-bromophenacylbromide, a specific inhibitor of PLA2 activity, greatly reduced the capacity of IpTxi to inhibit RyRs. These results suggested that a lipid product of PLA2 activity, more than a direct IpTxi-RyR interaction, was responsible for RyR inhibition.

We present the complete amino acid sequence of Imperatoxin A (IpTx(a)), a 33-amino-acid peptide from the venom of the scorpion P. imperator which activates Ca2+ release channels/ryanodine receptors (RyR) of sarcoplasmic reticulum (SR). The amino acid sequence of IpTx(a) shows no homology to any scorpion toxin so far described, but shares some homology to the amino acid sequence of Tx2-9 and agelenin, two spider toxins that target neuronal P-type Ca2+ channels. We also describe the total synthesis of IpTx(a) and demonstrate that it efficiently activates RyRs with potency and affinity identical to those of native IpTx(a). The use of synthetic IpTx(a) should help in the identification of the structural motifs of RyR critical for channel gating.

A novel crustacean-specific toxin, Cn5, containing 66 amino acid residues was isolated from the venom of the scorpion Centruroides noxius Hoffmann. It is stabilized by four disulfide bridges, formed between Cys12-Cys65, Cys16-Cys41, Cys25-Cys46 and Cys29-Cys48. Toxicity tests revealed that Cn5 is a toxin that affects arthropods but not mammals. However, at high concentrations, Cn5 does displace the mammal-specific toxin Cn2 from rat brain synaptosomes. The concentration of Cn5 that produces half-maximal inhibition (IC50) was estimated to be 100 microM. Sequence comparison of Cn5 with toxin Cn2, a mammal-specific toxin from the same scorpion, showed the presence of two sequence stretches, at positions 30 to 38 and 49 to 58, where the majority of the differences are concentrated. On the three-dimensional structure of Cn5 it is demonstrated that these two sequence stretches form a continuous surface region near the site thought to bind to the sodium channel. We assume that this region might be implicated in determining species specificity.

1. Shaker B K+ channels, expressed in the insect cell line Sf9, were studied in zero K+, Na+ or N-methyl-D-glucamine (NMG)-containing solutions. In the absence of K+ ions on both sides of the membrane, the K+ conductance collapsed with the delivery of short depolarizing pulses that activated the channels. The collapse of the conductance was fully prevented when the channels were kept closed at a holding potential of -80 mV. 2. The fall in K+ conductance had the notable characteristic of being strikingly stable. At -80 mV or more negative holding potentials, the conductance never recovered (cells observed for up to 1 h). 3. The extent of collapse of the K+ conductance depended on the number of depolarizing activating pulses applied in zero K+ solutions. For moderate to low frequencies of pulsing (1 to 0.002 Hz), the extent of the collapse did not depend on the frequency. 4. K+, Rb+, Cs+ and NH4+ added to the external Na+ solution impeded the fall in K+ conductance. 5. TEA added to the external, zero K+, Na(+)-containing solution also precluded the fall of the conductance. The protection by TEA paralleled its block of the outward K+ currents recorded with standard recording solutions. 6. The fall in K+ conductance was prevented by depolarized holding potentials. 7. The K+ conductance that was thought to be irreversibly lost at -80 mV or more negative holding potentials was fully recovered, however, after a prolonged (tens of seconds to minutes) change in the holding potential to depolarized values (above -50 mV). Full recovery could be obtained at any time after the former halt of the conductance.

Here we describe the basic features of the interaction of K+ channels with Pi1, a recently described 35 amino acid scorpion toxin, which has four disulfide bridges instead of the three commonly found in all the other known scorpion toxins. We found that: (a) Pi1 blocks ShakerB from the outside with a 1:1 stoichiometry, and a Kd of 32 nM in zero external [K+]; (b) extracellular K+, Rb+ and Cs+ but not NH4+ ions strongly impede (destabilize) the block by this toxin; interestingly (c) the destabilizing binding of K+, Rb+, and Cs+ is described by a Hill coefficient n > 1; (d) external K+ is more effective than internal K+ to reduce the block by Pi1.

Scorpion toxins acting on sodium channels differ in their specificity. Toxic peptides specific towards mammals and arthropods (insects and/or crustaceans) have been described. Because of the similar three-dimensional fold of these peptides, the molecular base of their specificity is thought to reside in certain differences at the level of amino acid residues especially within or near the binding site of the toxin to the particular ion channel. The cDNA, amino acid sequence and biological activity of an insect-specific toxin, Cn10, from the scorpion Centruroides noxius Hoffmann is reported. The electrostatic potential surface around a three-dimensional model of Cn10 was calculated. It revealed that residues Tyr4, Lys13, Ile18, Leu19, Gly20, Lys43, Leu44, Thr57, Tyr58, Pro59, Thr64 and Cys65, situated at the side of the toxin proposed in the literature to bind to the sodium channel, constitute a positive surface region. Therefore, they may form the site that binds to the channel. Cn10 was included in a comparative analysis of two groups of natural variants, highly similar peptides of the genus Centruroides with specificities towards mammals or arthropods. A number of surface-accessible residues, consistently different between the two groups and situated near the putative binding site, may be of importance for the specificity of the analyzed toxins.

Among the small nuclear RNAs (snRNAs) involved in the spliceosomal processing of pre-mRNA, U6 is the most conserved. As a first evidence for the presence of the splicing machinery in the amitochondrial protozoan Entamoeba histolytica (Eh), we have cloned the u6 snRNA gene. We find that in this organism u6 is a single copy gene that is transcribed as a poly(A)- RNA molecule of approximately 105 nucleotides. We have mapped the 5' end of the U6 snRNA transcript, and identified typical elements of a putative polymerase III promoter. This is the first snRNA gene reported in Eh. Sequence analysis indicates that this gene contains all the conserved nucleotides known to be important for U6 snRNA function. These results, in conjunction with the earlier finding of genes that contain pre-mRNA introns, suggest that Eh has a functional spliceosomal complex.

A set of six synthetic overlapping oligonucleotides coding for noxiustoxin were coupled into a continuous DNA fragment by means of recursive polymerase chain reaction. The polymerase chain reaction product was digested with SalI and HindIII, ligated into the E, coli vector pCSP 105 and expressed as a fusion protein. The fusion protein was purified and digested with trypsin and the hydrolysis products were separated by high-performance liquid chromatography. Approximately 1.3 mg of recombinant noxiustoxin per liter of culture was obtained. Amino acid analysis and N-terminal amino acid sequence of the recombinant noxiustoxin confirmed the nucleotide sequence of the cloned DNA. Binding experiments using rat brain synaptosomal membranes revealed that recombinant noxiustoxin displaced bound radioactive native NTX with a similar efficiency to cold native noxiustoxin.

BCF2 is a murine hybridoma cell line that produces a neutralizing antibody against toxin 2 (Cn2) from the scorpion Centruroides noxius Hoffmann of Mexico. In this communication we report the preparation and use of the BCF2 antibody and its antigen binding fragments (Fab) in experiments aiming at obtaining protection of experimental albino mice (strain CD1) challenged with purified toxin Cn2, as well as, with whole soluble venom from C. noxius. The monoclonal antibody BCF2 in amounts of 1 mg neutralizes 28 LD50 of soluble venom of C. noxius, whereas the Fab fragments of BCF2 (1 mg) are capable of neutralizing 43 LD50 dose of the same venom. To reach the same level of neutralization, with the commercially available horse antiserum [F(ab')2], we need to use about ninefold more material.

Three novel toxic peptides were purified to homogeneity from the venom of the Australian taipan snake, Oxyuranus scutellatus scutellatus. On the basis of complete amino acid sequence analyses, two of these toxins belong to the family of short-chain alpha-neurotoxins found in elapid and hydrophid snake venoms and are the first postsynaptic neurotoxins identified in taipan venom. Radioligand binding studies confirm that taipan toxins 1 and 2 inhibit the binding of [125I]-alpha-bungarotoxin to nicotinic acetylcholine receptors in skeletal muscle with IC50 values of 2.4-2.5 nM but are 5-fold less potent in this assay than alpha-bungarotoxin or the two short-chain alpha-neurotoxins erabutoxin a and erabutoxin b. Taipan toxins 1 and 2 do not antagonize [125I]-alpha-bungarotoxin binding to central neuronal nicotinic receptors at concentrations up to 3 microM. We find that erabutoxin a and erabutoxin b do inhibit the binding of [125I]-alpha-bungarotoxin to central neuronal nicotinic receptors but are over 350-fold less potent than long-chain alpha-neurotoxins at these receptors. The novel alpha-neurotoxins from taipan venom do not inhibit the binding of [3H]nicotine to high-affinity nicotine receptors in brain, a property they share with alpha-bungarotoxin and the erabutoxins. The results demonstrate that at least two neuromuscular junction-blocking peptides are present in taipan venom. Nonconservative substitutions at position 32 in both taipan toxin 1 and 2 may be responsible for the observed decreases in affinities of the toxins of 5-fold for muscle receptors (compared to alpha-bungarotoxin) and over 10-fold for alpha-bungarotoxin-sensitive nicotinic receptors in brain (compared to the structurally similar short-chain alpha-neurotoxins erabutoxin a and erabutoxin b).

We have developed a method to detect PCR amplicons that contain a restriction enzyme site. The method is called Polymerase Chain Reaction-Hybridization and Enzymatic Release Assay (PCR-HERA). The PCR product is hybridized to a synthetic oligonucleotide that is immobilized on a microtitre plate. Specific cleavage of the resulting hybrid by a restriction endonuclease liberates a fluorescein labeled arm of the immobilized DNA. We demonstrate the use of PCR-HERA to detect species-specific sequences of extra-chromosomal, ribosomal DNA, of Entamoeba histolytica. This highly specific assay does not require any washing steps and can be easily automated.

A novel peptide was purified and characterized from the venom of the scorpion Pandinus imperator. Analysis of its primary structure reveals that it belongs to a new structural class of K+-channel blocking peptide, composed of only 35 amino acids, but cross-linked by four disulphide bridges. It is 40, 43 and 46% identical to noxiustoxin, margatoxin and toxin 1 of Centruroides limpidus respectively. However, it is less similar (26 to 37% identity) to toxins from scorpions of the geni Leiurus, Androctonus and Buthus. The disulphide pairing was determined by sequencing heterodimers produced by mild enzymic hydrolysis. They are formed between Cys-4-Cys-25, Cys-10-Cys-30, Cys-14-Cys-32 and Cys-20-Cys-35. Three-dimensional modelling, using the parameters determined for charybdotoxin, showed that is it possible to accommodate the four disulphide bridges in the same general structure of the other K+-channel blocking peptides. The new peptide (Pil) blocks Shaker B K+ channels reversibly. It also displaces the binding of a known K+-channel blocker, [125I]noxiustoxin, from rat brain synaptosomal membranes with an IC50 of about 10 nM.

Seven toxic peptides from the venom of Tityus bahiensis and Tityus stigmurus was isolated and sequenced, five of them to completion. The most abundant peptide from each of these two species of scorpion was 95% identical with that of toxin gamma from the venom of Tityus serrulatus. They were consequently named gamma-b and gamma-st respectively. The genes encoding these new gamma-like peptides were cloned and sequenced by utilizing oligonucleotides synthesized according to known cDNA sequences of toxin gamma, and amplified by PCR on templates of DNA purified from both T. bahiensis and T. stigmurus. They contain an intron of approx. 470 bp. Possible mechanisms of processing and expressing these peptides are discussed, in view of the fact that glycine is the first residue of the N-terminal sequence of T. stigmurus, whereas lysine is the residue at position 1 of toxin gamma from T. serrulatus and T. bahiensis. In addition, chemical characterization of the less abundant toxic peptides showed the presence of at least four distinct families of peptides in all three species of the genus Tityus studied. There is a large degree of similarity among peptides from different venoms of the same family. By using specific horse and rabbit antisera, the venoms of T. bahiensis, T. serrulatus and T. stigmurus were compared. They showed an extended degree of cross-reactivity. Thus these three species of scorpion have similar toxic components, the genes of which are similarly organized, processed and expressed.

Binding of [125I]-Noxiustoxin to rat brain synaptosome membranes and displacement with synthetic peptides corresponding to the amino acid sequence of Noxiustoxin, show that the N-terminal segment of this toxin is implicated in the recognition of brain K(+)-channels. Cleavage of NTX with cyanogen bromide, endopeptidase V8 from Staphylococcus aureus and endopeptidase Lys-C support these findings. On the contrary, a synthetic C-terminal tetradecapeptide of charybodotoxin (ChTX), shows that in this K(+)-channel blocker, the C-terminal region, rather than the N-terminal is capable of displacing 125[I]-NTX binding to brain membranes.

Seven peptides corresponding to the amino acid sequence of toxin 2 from the scorpion Centruroides noxius were chemically synthesized, purified and assayed in mice for their putative neutralizing properties against scorpion toxins. All the peptides were immunogenic and some produced neutralizing antibodies, as verified by injecting the antisera with toxin into naive animals. However, direct challenge of pre-immunized mice (with the longest synthetic peptides of 27 and 57 amino acid residues) revealed an unexpected sensitization phenomena: the animals did not resist injection of one LD50 of purified toxin 2 (5% survival), but pre-immunization of mice with native toxin protected 100% of the animals. These findings suggest that vaccine preparations with synthetic peptides corresponding to the amino acid sequence of certain toxins should be analyzed cautiously.

Using a cDNA library prepared from venomous glands of the Mexican scorpion Centruroides noxius Hoffmann the genes that encode toxins 1 and 2 were identified, cloned and sequenced. In view of the proposed mechanism for processing the mature peptides coded by these two genes, the corresponding peptide-toxins were sequenced de novo. Mass spectrometric and 1H-NMR analyses of the C-terminal peptide produced by enzymatic digestion of both toxins indicated that the last residue is serine-amide. Sequence comparison revealed that these two genes have a similarity of 56% and 80% at the amino acid and nucleotide levels, respectively. Small corrections to the published primary structures were introduced: Cn toxin 1 has an extra serine residue at position 65 and the residue in position 60 is a proline, while the amino acids at positions 34 and 35 of Cn 2 are, respectively, tyrosine and glycine. Sequence comparison of toxins from the genus Centruroides suggests the presence of at least three classes of distinct peptides in these venoms.

We have constructed an RNA molecule containing a hammerhead ribozyme that is under allosteric control. In the inactive state, the RNA enzyme is unable to cleave a suitable substrate. The formation of the active state of the ribozyme is triggered by a specific interaction with a DNA oligonucleotide effector that is complementary to a single-stranded loop in the RNA enzyme molecule. Other DNA or RNA molecules containing unrelated nucleotide sequences do not function as allosteric effectors. This work demonstrates the feasibility of designing RNA enzymes that are specifically activated in response to an artificially designed molecular recognition event. Such enzymes may have practical applications.