RESUMO
The presence of a regulatory site for monovalent cations that affects the conformation of the MgATP-binding pocket leading to enzyme activation has been demonstrated for ribokinases. This site is selective toward the ionic radius of the monovalent cation, accepting those larger than Na(+). Phosphofructokinase-2 (Pfk-2) from Escherichia coli is homologous to ribokinase, but unlike other ribokinase family members, presents an additional site for the nucleotide that negatively regulates its enzymatic activity. In this work, we show the effect of monovalent cations on the kinetic parameters of Pfk-2 together with its three-dimensional structure determined by x-ray diffraction in the presence of K(+) or Cs(+). Kinetic characterization of the enzyme shows that K(+) and Na(+) alter neither the kcat nor the KM values for fructose-6-P or MgATP. However, the presence of K(+) (but not Na(+)) enhances the allosteric inhibition induced by MgATP. Moreover, binding experiments show that K(+) (but not Na(+)) increases the affinity of MgATP in a saturable fashion. In agreement with the biochemical data, the crystal structure of Pfk-2 obtained in the presence of MgATP shows a cation-binding site at the conserved position predicted for the ribokinase family of proteins. This site is adjacent to the MgATP allosteric binding site and is only observed in the presence of Cs(+) or K(+). These results indicate that binding of the monovalent metal ions indirectly influences the allosteric site of Pfk-2 by increasing its affinity for MgATP with no alteration in the conformation of residues present at the catalytic site.
Assuntos
Trifosfato de Adenosina/farmacologia , Sequência Conservada , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Domínio Catalítico , Cátions Monovalentes/metabolismo , Inibidores Enzimáticos/metabolismo , Simulação de Dinâmica Molecular , Especificidade por Substrato , TermodinâmicaRESUMO
Two paralog transcriptional regulators of the MerR family, CueR and GolS, are responsible for monovalent metal ion sensing and resistance in Salmonella enterica. Although similar in sequence and also in their target binding sites, these proteins differ in signal detection and in the set of target genes they control. Recently, we demonstrated that selective promoter recognition depends on the presence of specific bases located at positions 3' and 3 within the operators they interact with. Here, we identify the amino acid residues within the N-terminal DNA-binding domain of these sensor proteins that are directly involved in operator discrimination. We demonstrate that a methionine residue at position 16 of GolS, absolutely conserved among GolS-like proteins but absent in all CueR-like xenologs, is the key to selectively recognize operators that harbor the distinctive GolS-operator signature, whereas the residue at position 19 finely tunes the regulator/operator interaction. Furthermore, swapping these residues switches the set of genes recognized by these transcription factors. These results indicate that co-evolution of a regulator and its cognate operators within the bacterial cell provides the conditions to avoid cross-recognition and guarantees the proper response to metal injury.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metais/metabolismo , Regiões Promotoras Genéticas , Salmonella enterica/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação/genética , Cátions Monovalentes/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Metionina/química , Metionina/genética , Metionina/metabolismo , Modelos Moleculares , Mutação , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Salmonella enterica/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Differential scanning calorimetry (DSC) was applied to ascertain the effect caused by Kâº, Naâº, ATP, detergent, DPPC, DPPE, and subunit γ on the thermostability of Na,K-ATPase. The enthalpy variation (ΔH) for the thermal denaturation of the membrane-bound is twice the ΔH value obtained for solubilized Na,K-ATPase. Denaturation occurs in five steps for membrane-bound against three steps for the solubilized enzyme, therefore a multi-step unfolding process. In the presence of Naâº, the melting temperature is 61.6°C, and the ΔH is lower as compared with the ΔH obtained in the presence or in the absence of Kâº. Addition of ATP does not alter the transition temperatures significantly, but the shape of the curve is modified. Subunit γ probably stabilizes Na,K-ATPase in the beginning of thermal unfolding, and different amounts of detergents in the solubilized sample change the protein stability. Reconstitution of Na,K-ATPase into a liposome shows that lipids exert a protector effect. These results reveal differences on the thermostability depending on the conformation of Na,K-ATPase. They are relevant because it allows a comparison with future studies, e.g. how the composition of the membrane interferes on the stability of Na, K-ATPase, elucidating the importance of the lipid type contained in cell membrane.
Assuntos
Membrana Celular/enzimologia , Potássio/química , Dobramento de Proteína , ATPase Trocadora de Sódio-Potássio/química , Animais , Varredura Diferencial de Calorimetria , Cátions Monovalentes/química , Cátions Monovalentes/metabolismo , Potássio/metabolismo , Desnaturação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Força Próton-Motriz , Coelhos , ATPase Trocadora de Sódio-Potássio/metabolismo , SolubilidadeRESUMO
Potassium ions are non-essential activators of several aldehyde dehydrogenases (ALDHs), whereas a few others require the cation for activity. Two kinds of cation-binding sites, which we named intra-subunit and inter-subunit, have been observed in crystal structures of ALDHs, and based on reported crystallographic data, we here propose the existence of a third kind located in the central cavity of some tetrameric ALDHs. Given the high structural similarity between these enzymes, cation-binding sites may be present in many other members of this superfamily. To explore the prevalence of these sites, we compared 37 known crystal structures from 13 different ALDH families and evaluated the possible existence of a cation on the basis of the number, distance and geometry of its potential interactions, as well as of B-factor values of modeled cations obtained in new refinements of some reported crystal structures. Also, by performing multiple alignments of 855 non-redundant amino acid sequences, we assessed the degree of conservation in their respective families of the amino acid residues putatively relevant for cation binding. Among the ALDH enzymes studied, and according to our analyses, potential intra-subunit cation-binding sites seem to be present in most members of ALDH2, ALDH1L, ALDH4, ALDH5, ALDH7, ALDH10, and ALDH25 families, as well as in the bacterial and fungal members of the ALDH9 family and in a few ALDH1, ALDH6, ALDH11 and ALDH26 enzymes; potential inter-subunit sites in members of ALDH1L, ALDH3, ALDH4 from bacillales, ALDH5, ALDH7, ALDH9, ALDH10, ALDH11 and ALDH25 families; and potential central-cavity sites only in some bacterial and animal ALDH9s and in most members of the ALDH1L family. Because potassium is the most abundant intracellular cation, we propose that these are potassium-binding sites, but the specific structural and/or functional roles of the cation bound to these different sites remain to be investigated.
Assuntos
Aldeído Desidrogenase/química , Aldeído Desidrogenase/metabolismo , Cátions Monovalentes/química , Cátions Monovalentes/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Sítios de Ligação , Cristalografia por Raios X/métodos , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Modelos Moleculares , Alinhamento de Sequência , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismoRESUMO
In this study, we evaluated the photophysical properties of 5,10,15,20-tetrakis[4-(1,4,7,10,13-pentaoxacyclopentadecane-2-aminomethyl)2,3,5,6-(tetrafluoro)-phenyl]-porphyrin (H2C4P) and Zn(II)5,10,15,20-tetrakis[4-(1,4,7,10,13-pentaoxacyclopenta-decane-2-aminomethyl)2,3,5,6-(tetrafluoro)-phenyl]-porphyrinate (ZnC4P). We observed that these porphyrins have unique properties when compared with classical porphyrins. The porphyrins H2C4P and ZnC4P showed efficient transfer energy S1 to T1 by intersystem crossing with high and reasonable yields of triplet excited state and singlet oxygen production. These amphiphilic structures of these porphyrins could improve its localization in the tumor cells due to the presence of the crown ether in its framework. We also believed that the crown ether could modulate the change in ion homeostase (Ca(+2), K+, Na+) as already described by some new phthalocyanine dye. This fact makes us believe that it could be reasonably used as a photosensitizer for PDT purposes.
Assuntos
Porfirinas/química , Radiossensibilizantes/química , Cátions Monovalentes/metabolismo , Coronantes , Indóis/química , Isoindóis , Estrutura Molecular , Fotoquimioterapia/métodosRESUMO
Non-selective cation channels have been identified in the plasma membranes of many different cells. Previous research using fluorescent techniques has demonstrated the presence of cation conductances in membranes from human trophoblast. The purpose of this work was to explore, by electrophysiological methods, a non-selective cation channel in apical membranes from human placenta. Human placental apical membranes were purified by differential centrifugation and reconstituted in giant liposomes. These giant liposomes were then used for electrophysiological studies and were probed for the presence of cation channels by the patch-clamp method. The channel identified had a linear current-potential relationship with a conductance of around 16 pS in symmetrical Na(+) solution. Under asymmetrical conditions the reversal potential was close to the reversal potential for Na(+). The channel was equally permeable to sodium and potassium and the permeability sequence was NH+4>Cs(+) approximately Rb(+)>Na(+) approximately K(+)>Li(+). The channel also showed permeability to calcium and barium. The channel was insensitive to calcium but was blocked by millimolar concentration of Mg(2+). We have demonstrated the presence of a low conductance, non-selective cation channel in placental apical membranes. These channels share some properties with non-selective cation channels previously described in other different cells. The precise role of these channels in placental physiology has yet to be determined.
Assuntos
Canais Iônicos/metabolismo , Placenta/metabolismo , Adulto , Transporte Biológico , Cátions Monovalentes/metabolismo , Condutividade Elétrica , Feminino , Humanos , Lipossomos/metabolismo , Microvilosidades/metabolismo , Técnicas de Patch-Clamp , Gravidez , Vesículas Transportadoras/metabolismoRESUMO
The conduction properties of individual physiologically important cations Na+, K+, Mg2+, and Ca2+ were determined in the slowly activating (SV) channel of sugar beet vacuoles. Current-voltage relationships of the open channel were measured on excised tonoplast patches in a continuous manner by applying a +/-140 mV ramp-wave protocol. Applying KCl gradients of either direction across the patch we have determined that the relative Cl- to K+ permeability was < or =1%. Symmetrical increase of the concentration of tested cation caused an increase of the single channel conductance followed by saturation. Fitting of binding isotherms at zero voltage to the Michaelis-Menten equation resulted in values of maximal conductance of 300, 385, 18, and 13 pS, and of apparent dissociation constants of 64, 103, 0.04, and 0.08 mm for Na+, K+, Mg2+, and Ca2+, respectively. Deviations from the single-ion occupancy mechanism are documented, and alternative models of permeation are discussed. The magnitude of currents carried by divalent cations at low concentrations can be explained by an unrealistically wide (approximately 140 A) radius of the pore entrance. We propose instead a fixed negative charge in the pore vestibules, which concentrates the cations in their proximity. The conduction properties of the SV channel are compared with reported characteristics of voltage-dependent Ca2+-permeable channels, and consequences for a possible reduction of postulated multiplicity of Ca2+ pathways across the tonoplast are drawn.
Assuntos
Cátions Bivalentes/metabolismo , Cátions Monovalentes/metabolismo , Chenopodiaceae/metabolismo , Canais Iônicos/metabolismo , Transporte de Íons , Proteínas de Plantas/metabolismo , Vacúolos/metabolismo , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio/metabolismo , Permeabilidade da Membrana Celular , Cloretos/metabolismo , Ativação do Canal Iônico , Cinética , Magnésio/metabolismo , Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Raízes de Plantas/metabolismo , Potássio/metabolismo , Potássio/farmacologia , Sódio/metabolismo , Sódio/farmacologiaAssuntos
Trifosfato de Adenosina/metabolismo , Ouabaína/farmacologia , Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Cátions Monovalentes/metabolismo , Rim/enzimologia , Cinética , Modelos Químicos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , SuínosRESUMO
System y+L is a broad-scope amino acid transporter which binds and translocates cationic and neutral amino acids. Na+ replacement with K+ does not affect lysine transport, but markedly decreases the affinity of the transporter for L-leucine and L-glutamine. This observation suggests that the specificity of system y+L varies depending on the ionic composition of the medium. Here we have studied the interaction of the carrier with various amino acids in the presence of Na+, K+, Li+ and guanidinium ion. In agreement with the prediction, the specificity of system y+L was altered by the monovalent cations. In the presence of Na+, L-leucine was the neutral amino acid that interacted more powerfully. Elongation of the side chain (glycine - L-norleucine) strengthened binding. In contrast, bulkiness at the level of the beta carbon was detrimental. In K+, the carrier behaved as a cationic amino acid specific carrier, interacting weakly with neutral amino acids. Li+ was found to potentiate neutral amino acid binding and in general the apparent affinities were higher than in Na+; elongation of the nonpolar side chain made a more important contribution to binding and the carrier was more tolerant towards beta carbon substitution. Guanidinium stimulated the interaction of the carrier with neutral amino acids, but the effect was restricted to certain analogues (e.g., L-leucine, L-glutamine, L-methionine). Thus, in the presence of guanidinium, the carrier discriminates sharply among different neutral amino acids. The results suggest that the monovalent cations stabilize different carrier conformations.
Assuntos
Proteínas de Transporte/metabolismo , Cátions Monovalentes/metabolismo , Eritrócitos/metabolismo , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Transporte Biológico/efeitos dos fármacos , Cátions Monovalentes/farmacologia , Membrana Eritrocítica/metabolismo , Humanos , Potássio/metabolismo , Sódio/metabolismoRESUMO
Squid axons display a high activity of Na+/Ca2+ exchange which is largely increased by the presence of external K+, Li+, Rb+ and NH+4. In this work we have investigated whether this effect is associated with the cotransport of the monovalent cation along with Ca2+ ions. 86Rb+ influx and efflux have been measured in dialyzed squid axons during the activation (presence of Ca2+i) of Ca2+o/Na+i and Ca2+i/Ca2+o exchanges, while 86Rb+ uptake was determined in squid optic nerve membrane vesicles under equilibrium Ca2+/Ca2+ exchange conditions. Our results show that although K+o significantly increases Na+i-dependent Ca2+ influx (reverse Na+/Ca2+ exchange) and Rb+i stimulates Ca2+o-dependent Ca2+ efflux (Ca2+/Ca2+ exchange), no sizable transport of rubidium ions is coupled to calcium movement through the exchanger. Moreover, in the isolated membrane preparation no 86Rb+ uptake was associated with Ca2+/Ca2+ exchange. We conclude that in squid axons although monovalent cations activate the Na+/Ca2+ exchange they are not cotransported.