RESUMO
Vitamin B12 acts as a cofactor for various metabolic reactions important in living organisms. The Vitamin B12 biosynthesis is restricted to prokaryotes, which means, all eukaryotic organisms must acquire this molecule through diet. This study presents the investigation of Vitamin B12 metabolism and the characterization of precorrin-4 C(11)-methyltransferase (CobM), an enzyme involved in the biosynthesis of Vitamin B12 in Corynebacterium pseudotuberculosis. The analysis of the C. pseudotuberculosis genome identified two Vitamin B12-dependent pathways, which can be strongly affected by a disrupted vitamin metabolism. Molecular dynamics, circular dichroism, and NMR-STD experiments identified regions in CobM that undergo conformational changes after s-adenosyl-L-methionine binding to promote the interaction of precorrin-4, a Vitamin B12 precursor. The binding of s-adenosyl-L-methionine was examined along with the competitive binding of adenine, dATP, and suramin. Based on fluorescence spectroscopy experiments the dissociation constant for the four ligands and the target protein could be determined; SAM (1.4 ± 0.7 µM), adenine (17.8 ± 1.5 µM), dATP (15.8 ± 2.0 µM), and Suramin (6.3 ± 1.1 µM). The results provide rich information for future investigations of potential drug targets within the C. pseudotuberculosis's Vitamin B12 metabolism and related pathways to reduce the pathogen's virulence in its hosts.
Assuntos
Corynebacterium pseudotuberculosis/metabolismo , Vitamina B 12/metabolismo , Adenina/química , Adenina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cinética , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Secundária de Proteína , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Espectrometria de Fluorescência , Homologia Estrutural de Proteína , Suramina/química , Suramina/metabolismo , Vitamina B 12/biossíntese , Vitamina B 12/químicaRESUMO
Local myonecrosis is the main event resulting from snakebite envenomation by the Bothrops genus and, frequently, it is not efficiently neutralized by antivenom administration. Proteases, phospholipases A2 (PLA2) and PLA2-like toxins are found in venom related to muscle damage. Functional sites responsible for PLA2-like toxins activity have been proposed recently; they consist of a membrane docking-site and a membrane rupture-site. Herein, a combination of functional, biophysical and crystallographic techniques was used to characterize the interaction between suramin and MjTX-I (a PLA2-like toxin from Bothrops moojeni venom). Functional in vitro neuromuscular assays were performed to study the biological effects of the protein-ligand interaction, demonstrating that suramin neutralizes the myotoxic effect of MjTX-I. Calorimetric assays showed two different binding events: (i) inhibitor-protein interactions and (ii) toxin oligomerization processes. These hypotheses were also corroborated with dynamic light and small angle X-ray scattering assays. The crystal structure of the MjTX-I/suramin showed a totally different interaction mode compared to other PLA2-like/suramin complexes. Thus, we suggested a novel myotoxic mechanism for MjTX-I that may be inhibited by suramin. These results can further contribute to the search for inhibitors that will efficiently counteract local myonecrosis in order to be used as an adjuvant of conventional serum therapy.
Assuntos
Fosfolipases A2/metabolismo , Proteínas de Répteis/metabolismo , Suramina/química , Animais , Sítios de Ligação , Bothrops , Venenos de Crotalídeos/metabolismo , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Fosfolipases A2/química , Estrutura Quaternária de Proteína , Proteínas de Répteis/química , Espalhamento a Baixo Ângulo , Suramina/metabolismo , TermodinâmicaRESUMO
Whole-cell patch clamp recordings were used to characterise the physiological and pharmacological properties of P2X receptors of mouse and guinea pig myenteric neurons from the small intestine. ATP application induced a rapid inward current in 95% of recorded neurons of both species when were voltage clamped at -60 mV. Concentration-response curves for ATP (1-3000 microM) yielded EC(50) values of 114 and 115 microM for mouse and guinea pig myenteric neurons, respectively, with a Hill coefficient value of 1.02 and 0.79, respectively, which were not significantly different of unity. alpha,beta-methylene ATP (100 microM) was virtually inactive in both species. Pyridoxalphophate-6-azophenyl-2',4'-disulphonic acid (0.01-30 microM) inhibited the ATP-induced currents (I(ATP)) with a different potency; being the IC(50) 0.6 and 1.8 microM in mouse and guinea pig, respectively. In mouse myenteric neurons, I(ATP) were inhibited by suramin whereas in guinea pig neurons we observed two effects, potentiation and inhibition of these currents. On guinea pig, both effects of suramin had different recovering kinetics and concentration dependency, indicating that they are mediated by at least two different binding sites. Our observations indicate that myenteric P2X receptors in these two species have different pharmacological properties.
Assuntos
Plexo Mientérico , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/metabolismo , Suramina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sítios de Ligação , Fenômenos Biomecânicos , Condutividade Elétrica , Feminino , Cobaias , Masculino , Camundongos , Agonistas do Receptor Purinérgico P2 , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Receptores Purinérgicos P2X , Suramina/farmacologiaRESUMO
Bothropstoxin-II a calcium-dependent enzyme from Bothrops jararacussu venom causes tissue damage and several haemostatic disorders including platelet aggregation. In order to elucidate the structural determinants of its multiple pharmacological activities, we have studied the effects of suramin on Bothropstoxin-II and present details concerning the mode of binding.
Assuntos
Venenos de Crotalídeos/química , Venenos de Crotalídeos/farmacologia , Fosfolipases A2 do Grupo II/química , Fosfolipases A2 do Grupo II/farmacologia , Proteínas de Répteis/química , Proteínas de Répteis/farmacologia , Algoritmos , Animais , Sítios de Ligação , Bothrops , Domínio Catalítico , Simulação por Computador , Creatina Quinase/metabolismo , Venenos de Crotalídeos/metabolismo , Eletroforese em Gel de Poliacrilamida , Fosfolipases A2 do Grupo II/metabolismo , Camundongos , Modelos Moleculares , Conformação Proteica , Proteínas de Répteis/metabolismo , Espalhamento a Baixo Ângulo , Suramina/metabolismoRESUMO
ATP-activated currents were studied in Leydig cells of mice with the patch-clamp technique. Whole cell currents were rapidly activating and slowly desensitizing (55% decrement from the peak value on exposure to 100 microM ATP for 60 s), requiring 3 min of washout to recover 100% of the response. The concentration-response relationships for ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and 2-methylthio-ATP (2-MeS-ATP) were described by the Hill equation with a concentration evoking 50% of maximal ATP response (K(d)) of 44, 110, and 637 microM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP >or= ATPgammaS > 2-MeS-ATP > 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). alphabeta-Methylene-ATP (alphabeta-MeATP), GTP, UTP, cAMP, and adenosine were ineffective. Suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) blocked the responses in a concentration-dependent manner. The ATP-activated currents were dependent on extracellular pH, being maximal at pH 6.5 and decreasing with both acidification and alkalinization (apparent dissociation constant (pK(a)) of 5.9 and 7.4, respectively). The whole cell current-voltage relationship showed inward rectification and reversed near 0 mV. Experiments performed in bi-ionic conditions for measurement of reversal potentials showed that this channel is highly permeable to calcium [permeability (P)(Ca)/P(Na) = 5.32], but not to chloride (P(Cl)/P(Na) = 0.03) or N-methyl-D-glucamine (NMDG) (P(NMDG)/P(Na) = 0.09). Unitary currents recorded in outside-out patches had a chord conductance of 27 pS (between -90 and -50 mV) and were inward rectifying. The average current passing through the excised patch decreased with time [time constant (tau) = 13 s], resembling desensitization of the macroscopic current. These findings indicate that the ATP receptor present in Leydig cells shows properties most similar to those of cloned homomeric P2X(2).
Assuntos
Células Intersticiais do Testículo/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Camundongos , Técnicas de Patch-Clamp , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/metabolismo , Receptores Purinérgicos P2X , Suramina/metabolismoRESUMO
Suramin is a highly charged polysulfonated napthylurea that interferes in a number of physiologically relevant processes such as myotoxicity, blood coagulation and several kinds of cancers. This synthetic compound was complexed with a myotoxic Lys49 PLA(2) from Bothrops asper venom and crystallized by the hanging-drop vapor diffusion method at 18 degrees C. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a=49.05, b=63.84 and c=85.67 angstroms. Diffraction data was collected to 1.78 angstroms.
Assuntos
Bothrops , Venenos de Crotalídeos/química , Fosfolipases A/metabolismo , Fosfolipases A/toxicidade , Suramina/análise , Ureia/análogos & derivados , Ureia/metabolismo , Animais , Venenos de Crotalídeos/enzimologia , Cristalografia por Raios X , Fosfolipases A2 do Grupo II , Lisina/química , Estrutura Molecular , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Suramina/química , Suramina/metabolismo , Difração de Raios XRESUMO
Suramin is a hexasulfonated naphthylurea commonly used as antitrypanosomial drug and more recently for the treatment of malignant tumors. Here we show that suramin binds to human alpha-thrombin inhibiting both the hydrolysis of the synthetic substrate S-2238 (IC50 = 40 microM), and the thrombin-induced fibrinogen clotting (IC50 = 20 microM). The latter is completely reversed by albumin (30 mg mL(-1)) suggesting that, at therapeutic concentrations, suramin is unable to affect alpha-thrombin activity in the plasma. Kinetic analysis showed that suramin acts as a non-competitive inhibitor decreasing Vmax without changing the Km for S-2238 hydrolysis. Calorimetric studies revealed two distinct binding sites for suramin in alpha-thrombin. In addition, circular dichroism studies showed that suramin causes significant changes in alpha-thrombin tertiary structure, without affecting the secondary structure content. Interaction with alpha-thrombin resulted in an increased fluorescence emission of the drug. Complex formation was strongly affected by high ionic strength suggesting the involvement of electrostatic interactions. Altogether our data suggest that part of the biological activities of suramin might be related to alpha-thrombin inhibition at extra-vascular sites.
Assuntos
Suramina/metabolismo , Suramina/farmacologia , Trombina/antagonistas & inibidores , Trombina/metabolismo , Calorimetria , Catálise/efeitos dos fármacos , Dicroísmo Circular , Humanos , Cinética , Estrutura Molecular , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Suramina/química , Trombina/química , TitulometriaRESUMO
In this work we describe the ability of living Crithidia deanei to hydrolyze extracellular ATP. In intact cells at pH 7.2, a low level of ATP hydrolysis was observed in the absence of any divalent metal (0.41+/-0.13 nmol P(i) h(-1) 10(7) cells(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg(2+)-dependent ecto-ATPase activity was 4.05+/-0.17 nmol P(i) h(-1) 10(7) cells(-1). Mg(2+)-dependent ecto-ATPase activity increased linearly with cell density and with time for at least 60 min. The addition of MgCl(2) to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.93+/-0.26 mM MgCl(2). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2), but not CaCl(2) or SrCl(2). The apparent K(m) for Mg-ATP(2-) was 0.26+/-0.03 mM. ATP was the best substrate for this enzyme; other nucleotides, such as ITP, GTP, UTP and CTP, produced lower reaction rates. In the pH range from 6.6 to 8.4, in which the cells were viable, the acid phosphatase activity also present in this cell decreased, while the Mg(2+)-dependent ATPase activity did not change. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, vanadate, molybdate, sodium fluoride and tartrate. To confirm that this Mg(2+)-dependent ATPase was an ecto-ATPase, we used the impermeant inhibitor 4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The cell surface location of the ATP-hydrolyzing site was also confirmed by cytochemical analysis.