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L D Possani

Publications and source records attributed to L D Possani.

At least 127 records · Page 7Linked to original sources
PubMed1987

Blocking of the squid axon K+ channel by noxiustoxin: a toxin from the venom of the scorpion Centruroides noxius.

We have studied the selective effects of noxiustoxin (NTX), a fraction of the venom of the scorpion Centruroides noxius, on the K currents of perfused squid giant axons using the voltage-clamp technique. At concentrations below 1.5 microM, NTX blocked K currents in a voltage-independent manner, with little effect on their turning-on and turning-off kinetics. Above 1.5 microM, the block by NTX became voltage-dependent and could be partially removed by repetitive pulsing and strong depolarizations. Long repolarizations and more negative holding potentials favoured the slow restoration of channel block. Reduction of K currents by internally perfusing the fibers with solutions of low K+ concentration (200 mM), affected very little the removal of NTX-block during repetitive pulsing, suggesting that block removal depended on membrane potential and not on outward movements of K+ ions through open channels. In high extracellular K+ (300 mM) the blocking action of NTX was reduced and the instantaneous I-V characteristics showed a marked outward rectification. At 20 microM NTX, inward tail currents measured on step repolarizations to -70 mV were fully blocked, suggesting a direct interaction of the toxin with the open channel. The effects of the total venom Centruroides noxius Hoffmann was also studied. External application of 0.25 mg/ml of the venom caused a marked reduction of both Na and K currents, an effect similar to that of other scorpion venoms.

Animals↗
PubMed1987

Characterization of the actions of toxins II-9.2.2 and II-10 from the venom of the scorpion Centruroides noxius on transmitter release from mouse brain synaptosomes.

Toxic peptides II-9.2.2 and II-10, purified from Centruroides noxius venom, bear highly homologous N-terminal amino acid sequences, and both toxins are lethal to mice. However, only toxin II-10 is active on the voltage-clamped squid axon, selectively decreasing the voltage-dependent Na+ current. Here, we have tested toxins II-9 and II-10 on synaptosomes from mouse brain: both toxins increased the release of gamma-[3H]aminobutyric acid ([3H]GABA). Their effect was completely blocked by tetrodotoxin or by the absence of external Na+. Also, both toxins increased Na+ permeability in isolated nerve terminals. Besides the observation that toxin II-9 is active on synaptosomes, the effect of toxin II-10 in this preparation is opposite to that observed in the squid axon. Thus, our results reflect functional differences between the populations of Na+ channels in mouse brain synaptosomes and in the squid axon. The release of GABA evoked by these toxins from synaptosomes required external Ca2+ and was blocked by Ca2+ channel blockers (verapamil and Co2+). This latter observation is in sharp contrast to the releasing action of veratrine, which evoked release even in the absence of external Ca2+. Furthermore, the action of both C. noxius toxins was potentiated by veratrine, a result suggesting they have different mechanisms of action. Among drugs that release neurotransmitters by increasing Na+ permeability, it is noteworthy that scorpion toxins are the only ones yet reported to have a strict requirement for external Ca2+.

Animals↗
PubMed1987

Neurotoxins that act selectively on voltage-dependent cardiac calcium channels.

We searched for and found a toxin that acts specifically on cardiac calcium channels. This toxin, which we have designated TaiCatoxin (TCX), was purified from the venom of the Australian Taipan snake (Oxyuranus s. scutellatus), one of the world's most poisonous snakes. TCX is a highly charged, basic polypeptide with a molecular weight of 8,000. It blocks high- but not low-threshold cardiac calcium channels in a voltage-dependent manner and has no effect on potassium or sodium channels. The block occurs at nanomolar concentrations, is reversible, and is due to binding at an extracellularly facing site on the channel itself.

Animals↗
PubMed1986

Helodermatine, a kallikrein-like, hypotensive enzyme from the venom of Heloderma horridum horridum (Mexican beaded lizard).

We have purified and characterized the major N-benzoyl-L-arginine ethyl ester hydrolase from the venom of Heloderma horridum horridum. The enzyme belongs to the serine proteinase family, and its activity vs. peptide amide substrates and human high-molecular-weight kininogen suggests a similarity to the family of kallikreins. This interpretation is corroborated by its reactivity with the natural inhibitors soybean trypsin inhibitor and Kunitz-type bovine pancreatic trypsin inhibitor (aprotinin). Injection of the enzyme (2-16 micrograms/kg) into anesthetized rabbits leads to a rapid dose-dependent transient decrease of the arterial blood pressure. Like glandular kallikrein it specifically converts single-chain tissue type plasminogen activator into its double chain form. In contrast to other kallikrein-like enzymes from snake venoms it shows no thrombin-like or plasminogen activator activity. The enzyme is a single-chain glycoprotein (Mr 63,000). The N-terminal sequence revealed significant homology to pig pancreatic kallikrein and to kallikrein like enzymes from Crotalus atrox and Crotalus adamanteus venom. This enzyme, which we name Helodermatine, is the first purified from Sauria with kallikrein-like properties.

Amino Acid Sequence↗
PubMed1986

Biochemical characterization of the phospholipase A2 purified from the venom of the Mexican beaded lizard (Heloderma horridum horridum Wiegmann).

A phospholipase A2 was isolated from the venom of the mexican beaded lizard (Heloderma horridum horridum) by phenyl-Sepharose chromatography followed by Sephadex G-75 gel filtration and two additional steps on ion exchange resins (DE-32 cellulose). The affinity chromatographic method (PC-Sepharose 4B) reported for the isolation of other phospholipases [Rock, Ch. O., & Snyder, F. (1975) J. Biol. Chem. 250, 2564-2566; King, T. P., Alagon, A. C., Kwan, J., Sobotka, A. K., & Lichteinstein, L. M. (1983) Mol. Immunol. 20, 297-308; King, T. P., Kochoumian, L., & Joslyn, A. (1984) Arch. Biochem. Biophys. 230, 1-12] was uneffective for the separation of this enzyme. The monomeric form of the Heloderma phospholipase has an apparent Mr of 18 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 19 060 as calculated from amino acid analysis. It also contains on the order of 7% carbohydrates per mole of enzyme. The N-terminal amino acid sequence was shown to be very different from that of phospholipases isolated from mammalian pancreas and crotalids and elapids snake venoms. The first 39 amino acid residues at the N-terminal region have 56% homology with bee venom phospholipase but differ from the bee phospholipase in that its isoelectric point is acidic (pI = 4.5), instead of basic, and it has approximately 50 amino acid residues more in the molecule. The specificity of the enzyme is mainly A2 type with possible residual B-type activity. The enzymatic activity is Ca2+-dependent. Half-cystine alignment of the Heloderma phospholipase sequence with those of other known phospholipases shows the lack of an octadecapeptide at the N-terminal region, the existence of an extra hexapeptide at positions 42-47, and an exact correspondence of Heloderma Gly-12, Gly-14, His-36, and Asp-37 with Gly-30, Gly-32, His-48, and Asp-49 from other phospholipases shown to be important for Ca2+ binding (( Dijkstra, B. W., Drenth, J., Kalk, K. H., & Vandermaalen, P. J. (1978) J. Mol. Biol. 124, 53-60 )).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Noxiustoxin, a short-chain toxin from the Mexican scorpion Centruroides noxius, induces transmitter release by blocking K+ permeability.

Noxiustoxin (NTX), a 39 amino acid peptide purified from the venom of the Mexican scorpion Centruroides noxius, has been shown to block voltage-dependent K+ currents in the squid giant axon (Possani et al., 1982; Carbone et al., 1982). Although several other drugs known as K+ channel blockers in the squid axon also act on isolated nerve terminals to produce an increase in transmitter release, these releasing effects have not been shown to be related to a decrease of K+ permeability in synaptosomes (Vizi et al., 1977; Tapia and Sitges, 1982). In this work we show that NTX increases 3H-GABA release from perfused mouse brain synaptosomes. This effect was not blocked by TTX. Ca2+ channel blockers (verapamil or Co2+) or the absence of external Ca2+ prevents the releasing effect of this toxin. NTX does not seem to induce transmitter release by directly increasing Ca2+ permeability: The K+ ionophore valinomycin completely inhibits the release induced by NTX, as well as that evoked by the K+ channel blocker 4-aminopyridine; in contrast, the release evoked by a Ca2+ ionophore is not blocked by valinomycin. These findings strongly suggest that the releasing effect of NTX is mediated by a decrease in K+ permeability. External Ca2+ is needed only in order to couple this stimulus with the release process. Consistent with this hypothesis, we present evidence that NTX blocks the efflux of 86Rb+ from synaptosomes. An extended comparison of the effect of 4-aminopyridine with that of NTX is also reported.

4-Aminopyridine↗
PubMed1985

Scorpion toxins from Centruroides noxius and Tityus serrulatus. Primary structures and sequence comparison by metric analysis.

The complete primary structures of toxin II-14 from the Mexican scorpion Centruroides noxius Hoffmann and toxin gamma from the Brazilian scorpion Tityus serrulatus Lutz and Mello have been determined. Cleavage of toxin gamma after Met-6 with CNBr produced the 55-residue peptide 7-61, which maintained the four disulphide bonds but was not toxic to mice at a dose 3 times the lethal dose of native toxin gamma. Pairwise comparison by metric analysis of segment 1-50 of toxin gamma and the corresponding segments from two other South American scorpion toxins, five North American scorpion toxins, nine North African scorpion toxins and one Central Asian scorpion toxin showed that the three Brazilian toxins are intermediate between the North American and North African toxins. This result is consistent with the hypothesis that the South American and African continents were joined by a land connection in the distant past.

Amino Acid Sequence↗
PubMed1984

Selective modification of the squid axon Na currents by Centruroides noxius toxin II-10.

We have studied the effects of the purified toxin II-10, from the venom of the scorpion Centruroides noxius Hoffmann, on the Na and K currents of voltage clamped squid giant axons. Extracellular applications of 10 microM of toxin II-10 produced a selective depression of peak Na currents, with no significant effects on the time course of K currents. On pharmacologically separated Na currents, low concentrations of toxin II-10 (0.28-1 microM) caused a reversible decrease in inward and outward peak INa, with little effect on either the maintained level of the currents or their turning-off. At high concentrations (greater than 3 microM), toxin II-10 drastically reduced the peak conductance and increased both the level of the maintained conductance, and the time course of its turning-off. It is suggested that, when applied extracellularly on squid axons, toxin II-10 primarily reduces the peak Na conductance by modifying the activation of fast-inactivating Na channels. At high concentrations (10 microM), the toxin also modifies the rate constants of the transition from the inactivated to the second open state of the channel (Chandler and Meves, 1970) thus producing an increased level of the maintained Na conductance. It is also very likely, however, that peak conductance and maintained conductance reflect two separate populations of Na channels on which toxin II-10 has a differentiated action. Under these conditions, toxin II-10 would be the first reported toxin which can pharmacologically separate the two types of channels.

Amino Acid Sequence↗
PubMed1982

Electron microscopic evidence for scorpion toxin binding to synapses of rat brain cortex.

The venom from the scorpion Centruroides noxius Hoffman was fractionated by Sephadex G-50 gel filtration. The toxic fraction (n.II) was coupled to Sepharose-4B and used for purification of specific horse immunoglobulins. The purified immunoglobulins were linked to ferritin with glutaraldehyde and repurified in the same affinity column. This complex bound to the postsynaptic membrane of rat brain tissue previously exposed to toxin.

Animals↗
PubMed1982

Venom from two sub-species of Heloderma horridum (Mexican beaded lizard): general characterization and purification of N-benzoyl-L-arginine ethyl ester hydrolase.

The venom from the lizards Heloderma horridum horridum and Heloderma horridum alvarezi was obtained at a protein concentration of 80 mg/ml with a pH value of 6.9-7.0. The volume of venom obtained is approximately 0.5 ml per extraction. The i.p. LD50 value in mice for both sub-species is 2 mg/kg body weight. The electrophoretic pattern of the venom applied to polyacrylamide gels shows at least 18 protein bands and this pattern is constant for the same animal during all 12 months of the year, although different animals from the same population may present a slightly different pattern. The venom has the following enzymatic activities: phospholipase A, hyaluronidase, and Bz-Arg-OEt and Bz-Tyr-OEt hydrolase. Some of the venom components can be selectively and reversibly precipitated at acidic pH (4.7). The venom is very immunogenic and the sheep anti-sera against both sub-species cross-react quite extensively. A Bz-Arg-OEt hydrolase was purified from the venom of H. h. horridum by column chromatography in Sephadex G-75 followed by two steps on DEAE-cellulose columns at two different pH values (7.55 and 8.6). The last step was chromatography in a phenyl-sepharose column. The molecular weight of this enzyme, as obtained by SDS-gel electrophoresis, is approx. 65,000.

Animals↗
PubMed1980

Venom from the snake Bothrops asper Garman. Purification and characterization of three phospholipases A2.

The water-soluble venom of Bothrops asper Garman (San Juan Evangelista, Veracruz, México) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-l-arginine ethyl hydrolase, N-benzoyl-l-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded seven protein fractions, which were apparently less toxic than the whole venom (LD(50)=4.3mug/g mouse wt.). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH7.55) gave several fractions, two being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by gel disc electrophoresis yielding two more pure proteins designated phospholipase 2 and phospholipase 3. Analysis of phospholipids hydrolysed by these enzymes have shown that all three phospholipases belong to type A(2). Amino acid analysis has shown that phospholipase A(2) (type 1) has 97 residues with a calculated mol.wt. of 10978+/-11. Phospholipase A(2) (type 2) has 96 residues with a mol.wt. of 10959+/-11. Phospholipase A(2) (type 3) has 266 residues with 16 half-cystine residues and a calculated mol.wt of 29042+/-31. Automated Edman degradation showed the N-terminal sequence to be: Asx-Leu-Trp-Glx-Phe-Gly-Glx-Met-Met-Ser-Asx-Val- Met-Arg-Lys-Asx-Val-Val-Phe-Lys-Tyr-Leu- for phospholipase A(2) (type 2).

Amino Acids↗
PubMed1979

Purification and characterization of a phospholipase A2 from the venom of the coral snake, Micrurus fulvius microgalbineus (Brown and Smith).

A phospholipase A2 was purified from the Mexican coral snake Micrurus fulvius microgalbieus (Brown and Smith). Gel filtration of the soluble crude venom on Sephadex g-50 resolved five fractions, of which fraction II had 98% of the total phospholipase activity. This fraction was rechromatographed on a CM-cellulose column that resolved eight fractions, four of which had an important phospholipase activity. The first fraction (II-1) was homogeneous by polyacrylamide-gel electrophoresis and displayed a phospholipase specific activity of 920 units/mg of protein. The apparent molecular weight as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 14000. The amino acid analysis revealed the presence of 119 amino acid residues, with 12 half-cystines. the N-terminal sequence was shown to be Ser-Leu-Leu-Asx-Phe-Lys-Asx-Met-Ile-Glu-Ser-Thr..., which is homologous with that of phospholipases from other snake venoms.

Amino Acid Sequence↗