RESUMO
Experiments with multidrug resistance-associated protein 1 (MRP1) showed 10-years ago that transport of vincristine (VCR) by MRP1 could be stimulated by GSH, and transport of GSH by VCR. Since then many examples of stimulated transport have been reported for MRP1, 2, 3, 4 and 8. We discuss here three models to explain stimulated transport. We favour a model in which a large promiscuous binding site can bind more than one ligand, allowing cooperative/competitive interactions between ligands within the binding site. We conclude that there is no unambiguous proof for co-transport of two different ligands by MRPs, but that cross-stimulated transport can explain the published data.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , HumanosRESUMO
The recent discovery of binding protein dependent secondary transporters and the ever-growing family of membrane potential generating secondary transporters emphasize the diversity of transport systems in both the mechanistical and physiological sense. The vast amount of data on the lactose permease is now beginning to crystallize in a model that relates functional events to structural changes of the protein. Evidence has been presented that multidrug transporters pick up their substrates from the membrane, and the binding of a number of substrates to the binding-protein components of ATP-driven transporters is now understood in detail.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Potenciais da MembranaRESUMO
Lactic acid bacteria (LABs) are widely used in the manufacturing of fermented food and are among the best-studied microorganisms. Detailed knowledge of a number of physiological traits has opened new potential applications for these organisms in the food industry, while other traits might be beneficial for human health. Important new developments have been made in the research of LABs in the areas of multidrug resistance, bacteriocins and quorum sensing, osmoregulation, proteolysis, autolysins and bacteriophages. Recently, progress has been made in the construction of food-grade genetically modified LABs.
Assuntos
Microbiologia de Alimentos , Microbiologia Industrial/tendências , Ácido Láctico/metabolismo , Lactobacillus/metabolismo , Bacteriocinas/metabolismo , Fermentação , HumanosRESUMO
In the discovery of some general principles of energy transduction, lactic acid bacteria have played an important role. In this review, the energy transducing processes of lactic acid bacteria are discussed with the emphasis on the major developments of the past 5 years. This work not only includes the biochemistry of the enzymes and the bioenergetics of the processes, but also the genetics of the genes encoding the energy transducing proteins. The progress in the area of carbohydrate transport and metabolism is presented first. Sugar translocation involving ATP-driven transport, ion-linked cotransport, heterologous exchange and group translocation are discussed. The coupling of precursor uptake to product product excretion and the linkage of antiport mechanisms to the deiminase pathways of lactic acid bacteria is dealt with in the second section. The third topic relates to metabolic energy conservation by chemiosmotic processes. There is increasing evidence that precursor/product exchange in combination with precursor decarboxylation allows bacteria to generate additional metabolic energy. In the final section transport of nutrients and ions as well as mechanisms to excrete undesirable (toxic) compounds from the cells are discussed.
Assuntos
Metabolismo Energético , Bactérias Gram-Positivas/metabolismo , Lactatos/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Metabolismo dos Carboidratos , DNA Bacteriano/genética , Bactérias Gram-Positivas/genética , Ácido Láctico , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Dados de Sequência Molecular , Streptococcus/genética , Streptococcus/metabolismoRESUMO
Bacteria can release metabolites into the environment by various mechanisms. Excretion may occur by passive diffusion or by the reversal of the uptake process when the internal concentration of the metabolite exceeds the thermodynamic equilibrium level. In other cases, solutes are excreted against the concentration gradient by special extrusion systems. Their mode of energy coupling is different to that of the well-studied group of uptake systems. A thorough understanding of the transport processes will help to improve the excretion of metabolites of commercial interest, allow a more efficient production of metabolites in bulk quantities, and permit their exploitation to establish new markets.
Assuntos
Bactérias/metabolismo , Bactérias/crescimento & desenvolvimento , Transporte Biológico , DifusãoRESUMO
Drug resistance, mediated by various mechanisms, plays a crucial role in the failure of the drug-based treatment of various infectious diseases. As a result, these infectious diseases re-emerge rapidly and cause many victims every year. Another serious threat is imposed by the development of multidrug resistance (MDR) in eukaryotic (tumor) cells, where many different drugs fail to perform their therapeutic function. One of the causes of the occurrence of MDR in these cells is the action of transmembrane transport proteins that catalyze the active extrusion of a large number of structurally and functionally unrelated compounds out of the cell. The mode of action of these MDR transporters and their apparent lack of substrate specificity is poorly understood and has been subject to many speculations. In this review we will summarize our current knowledge about the occurrence, mechanism and molecular basis of (multi-)drug resistance especially as found in bacteria.
Assuntos
Proteínas de Transporte/fisiologia , Resistência a Múltiplos Medicamentos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/fisiologia , Sequência de Aminoácidos , Transporte Biológico , Humanos , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
The hydrophobin SC3 belongs to a class of small proteins functioning in the growth and development of fungi. Its unique amphipathic property and remarkable surface activity make it interesting not only for biological studies but also for medical and industrial applications. Biophysical studies have revealed that SC3 possesses at least three distinct conformations, named "soluble-state SC3" for the protein in solution, and "alpha-helical-state SC3" and "beta-sheet-state SC3" for the different states of the protein associated at a hydrophobic-water interface. The present fluorescence study shows that the microenvironment of the dansyl-labeled N terminus of soluble-state SC3 is relatively hydrophobic, whereas it is hydrophilic for alpha-helical-state and beta-sheet-state SC3. Fluorescence collisional quenching indicates that the N terminus of soluble-state SC3 is more solvent-accessible than those of alpha-helical-state and beta-sheet-state SC3, with Stern-Volmer constants for acrylamide of 4.63, 0.02, and 0.2 M(-1) for the different states, respectively. Fluorescence resonance energy transfer measurements show that soluble-state SC3 tends to associate in solution but dissociates in TFA. Fluorescence energy transfer was eliminated by conversion of soluble-state SC3 to alpha-helical-state SC3 on a hydrophobic surface, indicating a spatial separation of the molecules in this state. By inducing the beta-sheet state, structural changes were observed, both by CD and by fluorescence, that could be fit to two exponentials with lifetimes of about 10 min and 4 h. Molecules in the beta-sheet state also underwent a slow change in spatial proximity on the hydrophobic surface, as revealed by the reappearance of fluorescence resonance energy transfer in time.
Assuntos
Proteínas Fúngicas/química , Dicroísmo Circular , Transferência de Energia , Proteínas Fúngicas/isolamento & purificação , Cinética , Dobramento de Proteína , Estrutura Secundária de Proteína , Espectrometria de FluorescênciaRESUMO
The nucleotide sequence of the pepN gene from Lactococcus lactis encoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated M(r) of 95,368, which agrees with the apparent M(r) of 95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopeptidase. The amino acid sequence of the aminopeptidase of L. lactis was found to be similar to the corresponding enzymes of human, rat and mouse, with almost 30% of the residues identical. Also, a highly conserved area was identified which has similarity with the active site of thermolysin. A zinc-binding site, as well as the catalytic site for PepN, is predicted to lie within this conserved stretch. Putative promoter regions upstream of PepN were confirmed by primer extension analysis.
Assuntos
Aminopeptidases/genética , Genes Bacterianos , Lactococcus lactis/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias , Sequência de Bases , Western Blotting , Antígenos CD13 , Clonagem Molecular , DNA Bacteriano , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
In this review our knowledge of ATP binding cassette (ABC) transporters specific for peptides is discussed. Besides serving a role in nutrition of the cell, the systems participate in various signaling processes that allow (micro)organisms to monitor the local environment. In bacteria, these include regulation of gene expression, competence development, sporulation, DNA transfer by conjugation, chemotaxis, and virulence development, and the role of ABC transporters in each of these processes is discussed. Particular attention is paid to the specificity determinants of peptide receptors and transporters in relation to their structure and to the mechanisms of peptide binding.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Proteínas de Bactérias/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Bactérias/metabolismo , Bactérias/patogenicidade , Quimiotaxia , Conjugação Genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Transporte Proteico , Homologia de Sequência de Aminoácidos , Esporos Bacterianos , VirulênciaAssuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Transporte Biológico , Proteínas de Transporte/química , Escherichia coli , Klebsiella pneumoniae , Dados de Sequência Molecular , Conformação Proteica , Salmonella typhimuriumAssuntos
Bacillus subtilis/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Membrana Celular/enzimologia , Cromatografia de Afinidade/métodos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Indicadores e Reagentes , Cinética , Lipossomos , Peso Molecular , Fosfolipídeos/farmacologiaAssuntos
Proteínas de Transporte/metabolismo , Dissulfetos/metabolismo , Escherichia coli/metabolismo , Rhodobacter sphaeroides/metabolismo , Tolueno/análogos & derivados , Transporte Biológico Ativo/efeitos dos fármacos , Ditiotreitol/farmacologia , Eletroquímica , Transporte de Elétrons , Ferricianetos/farmacologia , Glucose 1-Desidrogenase , Glucose Desidrogenase/metabolismo , Glutamatos/metabolismo , Ácido Glutâmico , Cinética , Lactose/metabolismo , Maleimidas/farmacologia , Modelos Biológicos , Oxirredução , Consumo de Oxigênio , Cofator PQQ , Prolina/metabolismo , Prótons , Quinolinas/farmacologia , Tolueno/metabolismoRESUMO
Lactococcus lactis, a facultative anaerobic lactic acid bacterium, is known to have an increased growth yield when grown aerobically in the presence of heme. We have now established the presence of a functional, proton motive force-generating electron transfer chain (ETC) in L. lactis under these conditions. Proton motive force generation in whole cells was measured using a fluorescent probe (3',3'-dipropylthiadicarbocyanine), which is sensitive to changes in membrane potential (Delta psi). Wild-type cells, grown aerobically in the presence of heme, generated a Delta psi even in the presence of the F(1)-F(o) ATPase inhibitor N,N'-dicyclohexylcarbodiimide, while a cytochrome bd-negative mutant strain (CydA Delta) did not. We also observed high oxygen consumption rates by membrane vesicles prepared from heme-grown cells, compared to CydA Delta cells, upon the addition of NADH. This demonstrates that NADH is an electron donor for the L. lactis ETC and demonstrates the presence of a membrane-bound NADH-dehydrogenase. Furthermore, we show that the functional respiratory chain is present throughout the exponential and late phases of growth.
Assuntos
Lactococcus lactis/metabolismo , Oxigênio/metabolismo , Aerobiose , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Transporte de Elétrons/genética , Transporte de Elétrons/fisiologia , Fluorescência , Heme/metabolismo , Heme/farmacologia , Lactococcus lactis/genética , Lactococcus lactis/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Modelos Biológicos , Mutação , NAD/metabolismo , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Consumo de Oxigênio , TemperaturaRESUMO
Hydrophobins are a class of small proteins that fulfill a wide spectrum of functions in fungal growth and development. They do so by self-assembling into an amphipathic membrane at hydrophilic-hydrophobic interfaces. The SC3 hydrophobin of Schizophyllum commune is the best-studied hydrophobin. It assembles at the air-water interface into a membrane consisting of functional amyloid fibrils that are called rodlets. Here we examine the dynamics of SC3 assembly at an oil-water and air-water interface and the permeability characteristics of the assembled layer. Hydrophobin assembled at an oil-water interface is a dynamic system capable of emulsifying oil. It accepts soluble-state SC3 oligomers from water in a unidirectional process and sloughs off SC3 vesicles back into the water phase enclosing a portion of the oil phase in their hydrophobic interior. The assembled layer is impermeable to solutes >200 Da from either the water phase or the oil phase; however, due to the emulsification process, oil and the hydrophobic marker molecules in the oil phase can be transferred into the water phase, thus giving the impression that the assembled layer is permeable to the marker molecules. By contrast, the layer assembled at an air-water interface is permeable to water vapor from either the hydrophobic or hydrophilic side.
Assuntos
Biofísica/métodos , Proteínas Fúngicas/química , Membranas/química , Tiazóis/química , Ar , Peptídeos beta-Amiloides/química , Benzotiazóis , Proteínas de Membrana/química , Membranas/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Octoxinol/farmacologia , Óleos/química , Parafina , Permeabilidade , Conformação Proteica , Estrutura Secundária de Proteína , Schizophyllum/metabolismo , Fatores de Tempo , Água/químicaRESUMO
Many microorganisms metabolize their substrates (precursors) only partially and excrete the products of the metabolism into the medium. Although uptake of precursor and exit of product can proceed as two independent steps, there is increasing evidence that these processes are often linked and that transport is facilitated by a single antiport mechanism. Features of antiport mechanisms and advantages for the organism of catalysing precursor/product antiport will be illustrated by discussing a number of well-characterized systems. Based on precursor-product conversion stoichiometries, structural relatedness between precursors and products, and energetic and kinetic considerations, new examples of antiport systems will be proposed.
Assuntos
Bactérias/metabolismo , Bactérias/enzimologia , Transporte Biológico AtivoRESUMO
The kinetic mechanism of the lactose transport system of Streptococcus thermophilus was studied in membrane vesicles fused with cytochrome c oxidase containing liposomes and in proteoliposomes in which cytochrome c oxidase was coreconstituted with the lactose transport protein. Selective manipulation of the components of the proton (and sodium) motive force indicated that both a membrane potential and a pH gradient could drive transport. The galactoside/proton stoichiometry was close to unity. Experiments which discriminate between the effects of internal pH and delta pH as driving force on galactoside/proton symport showed that the carrier is highly activated at alkaline internal pH values, which biases the transport system kinetically toward the pH component of the proton motive force. Galactoside efflux increased with increasing pH with a pKa of about 8, whereas galactoside exchange (and counterflow) exhibited a pH optimum around 7 with pKa values of 6 and 8, respectively. Imposition of delta pH (interior alkaline) retarded the rate of efflux at any pH value tested, whereas the rate of exchange was stimulated by an imposed delta pH at pH 5.8, not affected at pH 7.0, and inhibited at pH 8.0 and 9.0. The results have been evaluated in terms of random and ordered association/dissociation of galactoside and proton on the inner surface of the membrane. Imposition of delta psi (interior negative) decreased the rate of efflux but had no effect on the rate of exchange, indicating that the unloaded transport protein carries a net negative charge and that during exchange and counterflow the carrier recycles in the protonated form.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Lactose/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos , Streptococcus/metabolismo , Simportadores , Transporte Biológico Ativo , Membrana Celular/metabolismo , Galactosídeos/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ionóforos/farmacologia , Cinética , Potenciais da MembranaRESUMO
In their natural habitats, microorganisms are often exposed to osmolality changes in the environment. The osmotic stress must be sensed and converted into an activity change of specific enzymes and transport proteins and/or it must trigger their synthesis such that the osmotic imbalance can be rapidly restored. On the basis of the available literature, we conclude that representative gram-negative and gram-positive bacteria use different strategies to respond to osmotic stress. The main focus of this paper is on the initial response of bacteria to hyper- and hypo-osmotic conditions, and in particular the osmosensing devices that allow the cell to rapidly activate and/or to synthesize the transport systems necessary for uptake and excretion of compatible solutes. The experimental data allow us to discriminate the transport systems by the physicochemical parameter that is sensed, which can be a change in external osmotic pressure, turgor pressure, membrane strain, internal osmolality and/or concentration of specific signal molecule. We also evaluate the molecular basis for osmosensing by reviewing the unique structural features of known osmoregulated transport systems.
Assuntos
Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Concentração Osmolar , Aquaporinas/metabolismo , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Pressão Osmótica , Potássio/metabolismoRESUMO
Microorganisms react upon hyperosmotic stress by accumulating compatible solutes. Here we report that Lactococcus lactis uses a transport system for glycine betaine that, contrary to earlier observations (D. Molenaar et al., J. Bacteriol. 175:5438-5444, 1993), is osmotically regulated at the levels of both expression and transport activity.
Assuntos
Betaína/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Osmose , Translocação Genética , Betaína/farmacocinética , Transporte Biológico , Meios de Cultura , Lactococcus lactis/fisiologia , Pressão OsmóticaRESUMO
A carbohydrate-transport assay was developed that does not require isotopically labelled substrates, but allows transport reactions to be followed spectrophotometrically. It makes use of a membrane system (hybrid membranes or proteoliposomes) bearing the transport system of interest, and a pyrroloquinoline quinone-dependent aldose dehydrogenase [soluble glucose dehydrogenase (sGDH)] and the electron acceptor 2,6-dichloroindophenol (Cl2Ind) enclosed in the vesicle lumen. After transport across the vesicular membrane, the sugar is oxidized by sGDH. The accompanying reduction of Cl2Ind results in a decrease in A600. The assay was developed and optimized for the lactose carrier (LacS) of Streptococcus thermophilus, and both solute/H+ symport and exchange types of transport could be measured with high sensitivity in crude membranes as well as in proteoliposomes. To observe exchange transport, the membranes were preloaded with a nonoxidizable substrate analogue and diluted in assay buffer containing an oxidizable sugar. Transport rates measured with this assay are comparable with those obtained with the conventional assay using isotopically labelled substrates. The method is particularly suited for determining transport reactions that are not coupled to any form of metabolic energy such as uniport reactions, or for characterizing mutant proteins with a defective energy-coupling mechanism or systems with high-affinity constants for sugars.