RESUMO
This work provides evidence that nicotine (1 x 10(-5) M) can cause changes in the intracellular calcium concentration of Trypanosoma cruzi epimastigotes, which can be blocked by alpha-bungarotoxin but not by atropine. Moreover, parasite membranes also bind such nicotinic acetylcholine receptor antagonist as well as agonists such as carbamylcholine (IC(50): 7.6 x 10(-7) M) and nicotine (IC(50): 1 x 10(-7) M). Results suggest that there is a molecular species in the surface of the parasite able to bind nicotinic ligands; therefore, nicotine interaction could lead to the activation of the mechanisms involved in intracellular calcium concentration increase in the parasite.
Assuntos
Cálcio/metabolismo , Agonistas Colinérgicos/metabolismo , Nicotina/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Ligação Competitiva/efeitos dos fármacos , Bungarotoxinas/metabolismo , Sinalização do Cálcio , Carbacol/metabolismo , Carbacol/farmacologia , Relação Dose-Resposta a Droga , Fura-2 , Membranas/efeitos dos fármacos , Membranas/metabolismo , Nicotina/metabolismo , Trypanosoma cruzi/metabolismoRESUMO
We have previously shown that two histidine residues of the nicotinic acetylcholine receptor are relevant for alpha-bungarotoxin binding. This paper studies: (1) the interaction between alpha-bungarotoxin and the peptide alpha173-202--synthesized according to the sequence of the Torpedo californica receptor alpha subunit--and between the toxin and the same peptide containing His186 modified with ethoxyformic anhydride or substituted by Ala; (2) the influence of the presence of Cys192-Cys193 disulfide bridge on such interactions. Solid-phase and in-solution competition assays were performed: ethoxyformylation of His186 or its substitution by Ala led to a significant drop in the toxin binding capacity only for peptides containing the bridge. Circular dichroism and fourth derivate spectra of all peptides were also analyzed. Results strongly indicate the involvement of His186 in the toxin binding to those peptides with the bridge--also present in the native receptor molecules--but not to their reduced forms; on the other hand, they give further support to the already established premise that, though the bridge does not participate directly in receptor-toxin binding, its presence is relevant to define the appropriate conformation of the interaction area.
Assuntos
Bungarotoxinas/farmacocinética , Histidina , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Dicroísmo Circular , Dissulfetos , Órgão Elétrico , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Receptores Nicotínicos/isolamento & purificação , Torpedo , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Human and experimental Chagas' disease causes peripheral nervous system damage involving neuromuscular transmission alterations at the neuromuscular junction. Additionally, autoantibodies directed to peripheral nerves and sarcolemmal proteins of skeletal muscle have been described. In this work, we analyse the ability of serum immunoglobulin factors associated with human chagasic infection to bind the affinity-purified nicotinic acetylcholine receptor (nAChR) from electric organs of Discopyge tschudii and to identify the receptor subunits involved in the interaction. The frequency of serum anti-nAChR reactivity assayed by dot-blot was higher in seropositive chagasic patients than in uninfected subjects. Purified IgG obtained from chagasic patients immunoprecipitated a significantly higher fraction of the solubilized nAChR than normal IgG. Furthermore, immunoblotting assays indicated that alpha and beta are the main subunits involved in the interaction. Chagasic IgG was able to inhibit the binding of alpha-bungarotoxin to the receptor in a concentration-dependent manner, confirming the contribution of the alpha-subunit in the autoantibody-receptor interaction. The presence of anti-nAChR antibodies was detected in 73% of chagasic patients with impairment of neuromuscular transmission in conventional electromyographical studies, indicating a strong association between seropositive reactivity against nAChR and electromyographical abnormalities in chagasic patients. The chronic binding of these autoantibodies to the nAChR could induce a decrease in the population of functional nAChRs at the neuromuscular junction and consequently contribute to the electrophysiological neuromuscular alterations described in the course of chronic Chagas' disease.
Assuntos
Autoanticorpos/sangue , Doença de Chagas/imunologia , Receptores Nicotínicos/imunologia , Autoanticorpos/imunologia , Doença de Chagas/sangue , Humanos , Immunoblotting , Junção Neuromuscular/imunologiaRESUMO
Peptides corresponding to the sequence alpha 124-147 of the Torpedo californica and Homo sapiens nicotinic cholinergic receptors were synthesized. The His residue at position 134 was ethoxyformylated or substituted by Ala. Effects of such modifications were studied by: (a) a toxin blot assay and (b) a competition assay between each peptide and the Discopyge Ischudii receptor for 125I alpha-bungarotoxin, in solution. Apparent Kd values were 0.1 and 0.8 microM for Torpedo californica and Homo sapiens native peptides, respectively, and no binding was observed when the His residue was modified or substituted by Ala. ic50 values for the Torpedo californica and Homo sapiens fragments were 1.0 and 0.8 microM, respectively, and no significant displacement occurred when His 134 was ethoxyformylated or substituted by Ala. Hydroxylamine treatment restored 80-100% of their binding ability. Results strongly support the involvement of His 134 in the binding of alpha-bungarotoxin either to the Torpedo californica or the Homo sapiens receptor.
Assuntos
Bungarotoxinas/metabolismo , Histidina/química , Receptores Nicotínicos/metabolismo , Torpedo/metabolismo , Alanina/química , Sequência de Aminoácidos , Animais , Ligação Competitiva , Humanos , Radioisótopos do Iodo , Modelos Logísticos , Dados de Sequência MolecularRESUMO
Further characterization of an aspartyl protease from Mucor bacilliformis with milk-clotting activity was performed. An extinction coefficient, epsilon 278 cm = 1.61 mL/mg/cm, a molecular mass of 35,400 Da and a pI of 5.2 were determined. Proteolytic activity and kinetic parameters were evaluated by using the hexapeptide Leu-Ser-pNO2-Phe-Nle-Ala-Leu-OMe as the substrate. The effect of pH and temperature on peptide cleavage, as well as protease heat stability, was determined. Such properties, taken as a whole, indicate that the M. bacilliformis protease can be considered a potential substitute for bovine chymosin in cheese manufacture.
Assuntos
Ácido Aspártico Endopeptidases/química , Leite , Mucor/enzimologia , Animais , Ácido Aspártico Endopeptidases/isolamento & purificação , Bovinos , Queijo , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrólise , Ponto Isoelétrico , CinéticaRESUMO
Histidine residues have been shown to be critical for alpha-BgTx binding to the acetylcholine receptor (Lacorazza et al., 1992; Bouzat et al., 1993; Lacorazza et al., 1995). Receptor subunits from Discopyge tschudii were modified with diethylpyrocarbonate (DEP). DEP treatment produces a concentration-dependent decrease of [125I] alpha-BgTx binding to the alpha-subunit. The neurotoxin binding capacity was fully restored by adding the nucleophile hydroxylamine. By proteolytic mapping of the alpha-subunit with V8-protease, we determined that the binding capacity to the fragment alpha V8-19 decreased 80% by DEP treatment. In addition, the [125I] alpha-BgTx binding to the same fragment decreased by 70% when the subunits were reduced and affinity-alkylated. We report the N-terminal sequence of both subunits and V8-fragments (alpha V8-10, alpha V8-13, and alpha V8-18), which constitute a first contribution to the knowledge of the primary structure of the Discopyge tschudii receptor. We propose that the fragment alpha V8-19 contains one or more of the histidine residues involved in the alpha-BgTx binding and probably includes the Cys alpha 192-193 disulfide bond. Only two histidine residues are present in the extracellular sequence of Torpedo californica for such fragments: His alpha 186 and alpha 204.
Assuntos
Bungarotoxinas/química , Histidina/análise , Fragmentos de Peptídeos/química , Receptores Colinérgicos/química , Sequência de Aminoácidos , Animais , Peixe Elétrico , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
In a former paper by Serrani et al. (1990), an overlapping action of ouabain and furosemide on potassium influx was observed. In the present paper we demonstrated that Na, K-ATPase from neonatal red cells ghosts is a target of both drugs. Furthermore the combined action of ouabain and furosemide on potassium (86Rb) influx (in its greatest fraction mediated by primary active transport) was studied by generalized equations for the analysis of inhibitors. The isobologram and the combination index performed from the data obtained point to the existence of synergism or antagonism between both inhibitors according to the fraction affected. Clinical implications of these findings could be postulated.
Assuntos
Membrana Eritrocítica/enzimologia , Sangue Fetal/enzimologia , Furosemida/farmacologia , Ouabaína/farmacologia , Potássio/sangue , ATPase Trocadora de Sódio-Potássio/sangue , Transporte Biológico Ativo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Membrana Eritrocítica/efeitos dos fármacos , Furosemida/administração & dosagem , Humanos , Ouabaína/administração & dosagem , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , SoftwareRESUMO
The potassium influx in human neonatal red blood cells (nRBC) shows an approximately 25% lower value compared to the total potassium influx in adult red blood cells (aRBC). The ouabain-sensitive potassium influx component represents approximately 70-75% of the total potassium influx for both types of cells but with an absolute value significantly lower in nRBC. In nRBC, the half maximum inhibitory effect for ouabain was obtained at a 10(-9) M concentration. The ouabain-insensitive nRBC potassium influx fractions showed two components: (i) a bumetanide-sensitive component, significantly lower than that of aRBC, (ii) a ouabain-bumetanide-insensitive (leak) component with a similar value in both cell types. The sum of the ouabain-sensitive and furosemide-sensitive components amounted in nRBC to a greater value than the total potassium influx. This behaviour could be interpreted as a superposition of the action of the inhibitors on the components affected.