RESUMO
Sea anemones are a rich source of biologically active substances. In crayfish muscle fibers, Bunodosoma cangicum whole venom selectively blocks the I K(Ca) currents. In the present study, we report for the first time powerful hemolytic and neuroactive effects present in two different fractions obtained by gel-filtration chromatography from whole venom of B. cangicum. A cytolytic fraction (Bcg-2) with components of molecular mass ranging from 8 to 18 kDa elicited hemolysis of mouse erythrocytes with an EC50 = 14 microg/ml and a maximum dose of 22 microg/ml. The effects of the neuroactive fraction, Bcg-3 (2 to 5 kDa), were studied on isolated crab nerves. This fraction prolonged the compound action potentials by increasing their duration and rise time in a dose-dependent manner. This effect was evident after the washout of the preparation, suggesting the existence of a reversible substance that was initially masking the effects of an irreversible one. In order to elucidate the target of Bcg-3 action, the fraction was applied to a tetraethylammonium-pretreated preparation. An additional increase in action potential duration was observed, suggesting a blockade of a different population of K+ channels or of tetraethylammonium-insensitive channels. Also, tetrodotoxin could not block the action potentials in a Bcg-3-pretreated preparation, suggesting a possible interaction of Bcg-3 with Na+ channels. The present data suggest that B. cangicum venom contains at least two bioactive fractions whose activity on cell membranes seems to differ from the I K(Ca) blockade described previously.
Assuntos
Braquiúros/efeitos dos fármacos , Venenos de Cnidários/farmacologia , Hemólise/efeitos dos fármacos , Neurotoxinas/farmacologia , Anêmonas-do-Mar , Potenciais de Ação/efeitos dos fármacos , Análise de Variância , Animais , Cromatografia em Gel , Venenos de Cnidários/isolamento & purificação , Camundongos , Canais de Potássio/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacosRESUMO
Sea anemones are a rich source of biologically active substances. In crayfish muscle fibers, Bunodosoma cangicum whole venom selectively blocks the I K(Ca) currents. In the present study, we report for the first time powerful hemolytic and neuroactive effects present in two different fractions obtained by gel-filtration chromatography from whole venom of B. cangicum. A cytolytic fraction (Bcg-2) with components of molecular mass ranging from 8 to 18 kDa elicited hemolysis of mouse erythrocytes with an EC50 = 14 æg/ml and a maximum dose of 22 æg/ml. The effects of the neuroactive fraction, Bcg-3 (2 to 5 kDa), were studied on isolated crab nerves. This fraction prolonged the compound action potentials by increasing their duration and rise time in a dose-dependent manner. This effect was evident after the washout of the preparation, suggesting the existence of a reversible substance that was initially masking the effects of an irreversible one. In order to elucidate the target of Bcg-3 action, the fraction was applied to a tetraethylammonium-pretreated preparation. An additional increase in action potential duration was observed, suggesting a blockade of a different population of K+ channels or of tetraethylammonium-insensitive channels. Also, tetrodotoxin could not block the action potentials in a Bcg-3-pretreated preparation, suggesting a possible interaction of Bcg-3 with Na+ channels. The present data suggest that B. cangicum venom contains at least two bioactive fractions whose activity on cell membranes seems to differ from the I K(Ca) blockade described previously
Assuntos
Animais , Camundongos , Braquiúros/efeitos dos fármacos , Venenos de Cnidários/farmacologia , Hemólise/efeitos dos fármacos , Neurotoxinas/farmacologia , Anêmonas-do-Mar , Análise de Variância , Cromatografia em Gel , Venenos de Cnidários/isolamento & purificaçãoRESUMO
Muscarinic receptors are widely spread throughout the body, and are involved in the regulation of fundamental physiological processes, like the modulation of the heart rate, control of motor systems and modulation of learning and memory. In the central nervous system the cholinergic transmission is mainly mediated by muscarinic receptors; there are five subtypes that are all expressed in the brain of mammals (m1-m5). There are regional differences in their concentrations in the brain and more than one subtype is expressed in the same cell. It has been difficult to study their localization and function in vivo due to the lack of ligands that exclusively act on one subtype of the receptor. We studied the action of the muscarinic toxins MT1, MT2 and MT3, from the venom of the snake Dendroaspis angusticeps, on muscarinic receptors, by using the classical muscarinic radioligand 3H-NMS as reporter of the inhibition of its own binding, to either native or cloned receptors. We have also studied the in vivo effects on memory retention of the injection of the toxins into discrete brain regions. The muscarinic toxins appear to be invaluable tools to study receptor pharmacology, physiology and structure/function relationships. They would enable the design of new, more selective, pharmacological agents.
Assuntos
Receptores Muscarínicos/administração & dosagem , Receptores Muscarínicos/fisiologia , Toxinas Biológicas/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Venenos Elapídicos/administração & dosagem , Venenos Elapídicos/farmacologia , Humanos , Injeções Intraventriculares , Receptores Muscarínicos/metabolismo , Toxinas Biológicas/administração & dosagem , Toxinas Biológicas/metabolismoRESUMO
Copper binding to apolipoprotein B-100 (apo B-100) and its reduction by endogenous components of low-density lipoprotein (LDL) represent critical steps in copper-mediated LDL oxidation, where cuprous ion (Cu(I)) generated from cupric ion (Cu(II)) reduction is the real trigger for lipid peroxidation. Although the copper-reducing capacity of the lipid components of LDL has been studied extensively, we developed a model to specifically analyze the potential copper reducing activity of its protein moiety (apo B-100). Apo B-100 was isolated after solubilization and extraction from size exclusion-HPLC purified LDL. We obtained, for the first time, direct evidence for apo B-100-mediated copper reduction in a process that involves protein-derived radical formation. Kinetics of copper reduction by isolated apo B-100 was different from that of LDL, mainly because apo B-100 showed a single phase-exponential kinetic, instead of the already described biphasic kinetics for LDL (namely alpha-tocopherol-dependent and independent phases). While at early time points, the LDL copper reducing activity was higher due to the presence of alpha-tocopherol, at longer time points kinetics of copper reduction was similar in both LDL and apo B-100 samples. Electron paramagnetic resonance studies of either LDL or apo B-100 incubated with Cu(II), in the presence of the spin trap 2-methyl-2-nitroso propane (MNP), indicated the formation of protein-tryptophanyl radicals. Our results supports that apo B-100 plays a critical role in copper-dependent LDL oxidation, due to its lipid-independent-copper reductive ability.
Assuntos
Apolipoproteínas B/metabolismo , Cobre/metabolismo , Apolipoproteína B-100 , Apolipoproteínas B/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/metabolismo , Humanos , Cinética , Lipoproteínas LDL/isolamento & purificação , Lipoproteínas LDL/metabolismo , Compostos Nitrosos/metabolismo , Oxirredução , Detecção de Spin , Triptofano/metabolismoRESUMO
The selectivity of the muscarinic toxin MT3 from green mamba snake venom was corroborated by inhibition of the binding of [3H]NMS, a classical muscarinic radioligand, to native and cloned muscarinic receptors, showing 214-fold higher affinity for m4 than for m1 subtype, without significant binding to the others. The highest concentrations of MT3 sites (putative m4 receptors) in the rat brain were found in striatum and olfactory tubercle, intermediate concentration in dentate gyrus and CA1, and lower but still conspicuous levels in CA3 and frontal cortex. MT3 caused retrograde amnesia of an inhibitory avoidance task, when injected into the dorsal hippocampus of rats after training, suggesting a positive role of these MT3 sensitive sites, which are probably m4 muscarinic receptors, in memory consolidation of this task.
Assuntos
Encéfalo/metabolismo , Venenos Elapídicos/toxicidade , Memória/efeitos dos fármacos , Peptídeos/toxicidade , Receptores Muscarínicos/efeitos dos fármacos , Animais , Autorradiografia , Ligação Competitiva , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , N-Metilescopolamina/metabolismo , Neurotoxinas/toxicidade , Ensaio Radioligante , Ratos , Ratos Wistar , Receptor Muscarínico M4 , Receptores Muscarínicos/metabolismo , TrítioRESUMO
The partial amino acid sequence of the tetrameric isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21%. Each subunit displays the thirteen-stranded beta-barrel topology characteristic of legume lectins. The amino acid residues involved in metal- and sugar-binding are similar to those of other GalNAc-specific lectins, indicating that residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Fabaceae/química , Lectinas/química , Plantas Medicinais , Sequência de Aminoácidos , Simulação por Computador , Cristalografia por Raios X , Lectinas/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Lectinas de Plantas , Estrutura Secundária de ProteínaRESUMO
Acetylcholinesterases (AChEs) very sensitive to fasciculin inhibition (KiS in picomolar range) have a distinctive group of aromatic amino acids in the peripheral region (Y70, Y121, W279 in Torpedo AChE). Enzymes that lack these amino acids like butyrylcholinesterases (BChEs) or one or two of them like cobra venom, insect and chicken AChEs are 1000 to 1,000,000 times less sensitive. Fasciculin is a non-competitive inhibitor of the hydrolysis of choline and neutral esters by very sensitive AChEs. For the other group of enzymes, differences arise according to the type of substrate. Fasciculin still behaves as a non-competitive inhibitor with choline esters. In contrast, hydrolysis of phenylacetate was unaffected or slightly increased with BChEs and a partial competitive inhibition was observed with cobra venom and chicken enzymes.
Assuntos
Acetilcolinesterase/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Inibidores da Colinesterase/farmacologia , Venenos Elapídicos/enzimologia , Venenos Elapídicos/farmacologia , Ésteres/metabolismo , Animais , Galinhas , Inibidores da Colinesterase/metabolismo , Venenos Elapídicos/metabolismo , Hidrólise , Especificidade por SubstratoRESUMO
Norleucine methylester was coupled to carboxylates of fasciculin 2, a snake toxin that inhibits acetylcholinesterase (AChE). This neutralized negative charges but had no effect on the activity, suggesting that carboxyls do not participate in binding to AChE. Earlier results are discussed. Modification of three aromatic amino acids in the peripheral site of AChE, the binding site for fasciculin, decreased the affinity 100 to one million times. Neutralizing the charge of cationic groups of fasciculin lowered the affinity only three to seven times. A change in either the toxin or enzyme part of a binding site should have about the same effect. Since this was not so, it suggests that cationic groups of fasciculin do not bind to aromatic rings in the peripheral site.
Assuntos
Inibidores da Colinesterase/metabolismo , Venenos Elapídicos/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Ácidos Carboxílicos/química , Inibidores da Colinesterase/química , Inibidores da Colinesterase/isolamento & purificação , Inibidores da Colinesterase/toxicidade , Cromatografia Líquida de Alta Pressão , Venenos Elapídicos/química , Venenos Elapídicos/isolamento & purificação , Venenos Elapídicos/toxicidade , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
The venom of some Dendroaspis snakes contains small proteins (7500 mol. wt) that inhibit the binding of radiolabelled muscarinic antagonist to brain synaptomal membranes. There were no peptides described among muscarinic ligands until Adem et al. (Biochim. biophys. Acta 968, 340-345, 1988) reported that muscarinic toxins (MTxs), MTx1 and 2 were able to inhibit 3H-QNB binding to rat brain membranes. Since MTxs inhibit around half of specific binding of 3H-quinuclidinyl benzilate (3H-QNB) and 3H-N-methyl-scopolamine (3H-NMS), which do not discriminate between subtypes of muscarinic receptors, it has been proposed that MTxs might selectively bind to some subtype. MTx1 and 2 from Dendroaspis angusticeps almost completely inhibit the binding of 3H-pirenzepine (3H-PZ), a preferential M1 muscarinic receptor subtype ligand to cerebral cortex synaptosomal membranes. A much higher concentration was needed to inhibit partially 3H-PZ binding to atrial muscarinic receptors. These results support the hypothesis that MTx1 and 2 may be M1 selective muscarinic ligands. Similar activities have been found in Dendroaspis polylepis and D. viridis venoms, but with lower affinities. The Ki obtained from inhibition curves of the binding of 3H-PZ showed that MTx1 has higher affinity for the putative M1 muscarinic receptor subtype, followed by MTx2. DpMTx has lower affinity, while DvMTx seems to have the lowest affinity. All these peptides are devoid of anticholinesterase activity. Dendrotoxin and fasciculin from D. angusticeps venom do not inhibit the binding of muscarinic radioligands to cerebral cortex membranes. The injection of MTxs into dorsal hippocampus of rats immediately after training in an inhibitory avoidance task improves memory consolidation, as does oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Venenos Elapídicos/toxicidade , Agonistas Muscarínicos/toxicidade , Animais , Comportamento Animal/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Técnicas In Vitro , Cinética , Ligantes , Memória/efeitos dos fármacos , N-Metilescopolamina , Pirenzepina/farmacocinética , Ratos , Ratos Wistar , Receptores Colinérgicos/efeitos dos fármacos , Receptores Colinérgicos/metabolismo , Derivados da Escopolamina/metabolismo , Especificidade da Espécie , Técnicas Estereotáxicas , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
The West African green mamba, Dendroaspis angusticeps, has two toxins, fasciculins, that are non-competitive inhibitors of acetylcholinesterase. Arginine residues of fasciculin 2 were modified with 1,2-cyclohexanedione. Two of these residues, Arg24 and Arg37, reacted very slowly or not at all. Modification of Arg28 reduced the activity only by 13%. Arg11 and Arg27 are unique for fasciculins; a comparison of the sequences of 175 snake toxins homologous to fasciculins showed that no other toxin has arginine in the corresponding positions. Modification of the two unique arginines had a large effect and decreased the activity by 73% (Arg11) and 85% (Arg27). This was apparently not due to structural perturbations, since the modification did not change the circular dichroic spectra. The two arginine residues probably participate in the binding to acetylcholinesterase. They are located on the same side of the toxin molecule and the distance between their alpha-carbons is 2.7 nm. This may indicate binding to sites that are far apart and suggests that fasciculin covers a large area of the enzyme.