RESUMEN
ATP synthases are unique rotatory molecular machines that supply biochemical reactions with adenosine triphosphate (ATP)-the universal "currency", which cells use for synthesis of vital molecules and sustaining life. ATP synthases of F-type (FOF1) are found embedded in bacterial cellular membrane, in thylakoid membranes of chloroplasts, and in mitochondrial inner membranes in eukaryotes. The main functions of ATP synthases are control of the ATP synthesis and transmembrane potential. Although the key subunits of the enzyme remain highly conserved, subunit composition and structural organization of ATP synthases and their assemblies are significantly different. In addition, there are hypotheses that the enzyme might be involved in the formation of the mitochondrial permeability transition pore and play a role in regulation of the cell death processes. Dysfunctions of this enzyme lead to numerous severe disorders with high fatality levels. In our review, we focus on FOF1-structure-based approach towards development of new therapies by using FOF1 structural features inherited by the representatives of this enzyme family from different taxonomy groups. We analyzed and systematized the most relevant information about the structural organization of FOF1 to discuss how this approach might help in the development of new therapies targeting ATP synthases and design tools for cellular bioenergetics control.
Asunto(s)
Diseño de Fármacos , ATPasas de Translocación de Protón/metabolismo , Adenosina Trifosfato/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/metabolismo , Cloroplastos/metabolismo , Eucariontes/metabolismo , Filogenia , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/clasificación , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
In plants and fungi the plasma membrane proton pump generates a large proton-motive force that performs essential functions in many processes, including solute transport and the control of cell elongation. Previous studies in yeast and higher plants have indicated that phosphorylation of an auto-inhibitory domain is involved in regulating pump activity. In this report we examine the Medicago truncatula plasma membrane proton pump gene family, and in particular MtAHA5. Yeast complementation assays with phosphomimetic mutations at six candidate sites support a phosphoregulatory role for two residues, suggesting a molecular model to explain early Nod factor-induced changes in the plasma membrane proton-motive force of legume root cells.
Asunto(s)
Membrana Celular/enzimología , Medicago truncatula/enzimología , Proteínas de Plantas/metabolismo , Raíces de Plantas/enzimología , ATPasas de Translocación de Protón/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Western Blotting , Análisis por Conglomerados , Regulación Enzimológica de la Expresión Génica , Prueba de Complementación Genética , Interacciones Huésped-Patógeno , Medicago truncatula/genética , Medicago truncatula/microbiología , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Fosforilación , Filogenia , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Raíces de Plantas/genética , Raíces de Plantas/microbiología , ATPasas de Translocación de Protón/clasificación , ATPasas de Translocación de Protón/genética , Rhizobium/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Serina/genética , Serina/metabolismo , Transducción de Señal , Simbiosis , Treonina/genética , Treonina/metabolismoRESUMEN
BACKGROUND AND AIMS: Recently, a non-M2-related mitochondrial 60 kDa protein found to be recognized by antimitochondrial antibody (AMA) negative sera from patients with primary biliary cirrhosis (PBC) has been shown to contain parts of the five F(1)-ATPase subunits α, ß, γ, δ and ε. In this study, we examined whether this enzyme is, indeed, a target antigen in PBC. METHODS: Analysed were 60 AMA-positive/anti-M2-negative and 103 anti-M2-positive PBC patients, 46 patients with autoimmune hepatitis (AIH), 35 patients with primary sclerosing cholangitis (PSC), 110 patients with viral hepatitis, 40 patients with inflammatory bowel diseases (IBD), 33 patients with connective tissue diseases (systemic lupus erythematosus, mixed connective tissue disease, Sjögren disease, systemic sclerosis) and 25 blood donors. The F(1)-ATPase-subunits α-δ were recombinantly expressed in Escherichia coli, purified and applied to ELISA and Western blotting. RESULTS: In all, 40 of the 60 AMA-positive/anti-M2-negative (67%) and 44 (43%) of the 103 anti-M2-positive PBC-sera reacted with at least one of the F(1)-subunits α-δ. The ß- and γ-subunits were preferentially recognized. However, also up to 57% of patients with AIH and 34% of patients with PSC had anti-ß- or γ-antibodies, while patients with viral hepatitis had these antibodies in up to 13%. Patients with IBD had anti-ß and anti-γ-antibodies in up to 20 and 5% respectively. None of the patients with connective tissue diseases had antibodies to the ß- and only 6% to the γ-subunit. Sera from healthy blood donors were negative. CONCLUSIONS: Antibodies to the ß- and γ-subunits of F(1)-ATPase are further AMAs in PBC but occur also in other autoimmune liver disorders; they may be, therefore, indicators for a general autoimmune process of the liver.
Asunto(s)
Autoanticuerpos/sangre , Autoantígenos/inmunología , Hepatitis Autoinmune/inmunología , Membranas Mitocondriales/enzimología , ATPasas de Translocación de Protón/inmunología , Adolescente , Adulto , Anciano , Niño , Colangitis Esclerosante/sangre , Colangitis Esclerosante/enzimología , Colangitis Esclerosante/inmunología , Enfermedades del Tejido Conjuntivo/sangre , Enfermedades del Tejido Conjuntivo/enzimología , Enfermedades del Tejido Conjuntivo/inmunología , Femenino , Hepatitis Autoinmune/sangre , Hepatitis Autoinmune/enzimología , Hepatitis Viral Humana/sangre , Hepatitis Viral Humana/enzimología , Hepatitis Viral Humana/inmunología , Humanos , Enfermedades Inflamatorias del Intestino/sangre , Enfermedades Inflamatorias del Intestino/enzimología , Enfermedades Inflamatorias del Intestino/inmunología , Hígado/patología , Masculino , Persona de Mediana Edad , Subunidades de Proteína/genética , Subunidades de Proteína/inmunología , ATPasas de Translocación de Protón/clasificación , ATPasas de Translocación de Protón/genética , Adulto JovenRESUMEN
P-type ATPases are a superfamily of membrane proteins involved in many physiological processes that are fundamental for all living organisms. Using ATP, they can transport a variety of ions and other substances across all types of cell membranes against a concentration electrochemical gradient. P-type ATPases form a phosphorylated intermediate and are sensitive to vanadate. Based on evolutionary relations and sequence homology, P-type ATPases are divided into five major families. All P-type ATPases share a simple structure and mechanism, but also possess domains characteristic for each family, which are crucial for substrate specificity. These proteins usually have a single subunit with eight to twelve transmembrane segments, a large central cytoplasmic domain with the conservative ATP binding site along with N and C termini exposed to the cytoplasm. Because of variety of proteins that belong to P-type ATPase superfamily, in this review the comparison of functional and structure properties of plant cells P-type ATPases is presented, as well as their important role in adaptation to environmental stress.
Asunto(s)
Plantas/enzimología , ATPasas de Translocación de Protón/metabolismo , Adaptación Fisiológica , ATPasas de Translocación de Protón de Cloroplastos/metabolismo , Fenómenos Fisiológicos de las Plantas , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/clasificación , Especificidad por SustratoRESUMEN
To date, the nomenclature of mammalian genes encoding the numerous subunits and their many isoforms that comprise the family of vacuolar H(+)-ATPases has not been systematic, resulting in confusion both in the literature and among investigators. We present the official new system for these genes, approved by both Human and Mouse Gene Nomenclature Committees.
Asunto(s)
Mamíferos/genética , ATPasas de Translocación de Protón/clasificación , ATPasas de Translocación de Protón/genética , Terminología como Asunto , Animales , Humanos , Isoenzimas/clasificación , Isoenzimas/genética , Modelos Moleculares , Subunidades de Proteína/clasificación , Subunidades de Proteína/genéticaRESUMEN
The mechanism of proton translocation by P-type proton ATPases is poorly defined. Asp684 in transmembrane segment M6 of the Arabidopsis thaliana AHA2 plasma membrane P-type proton pump is suggested to act as an essential proton acceptor during proton translocation. Arg655 in transmembrane segment M5 seems to be involved in this proton translocation too, but in contrast to Asp684, is not essential for transport. Asp684 may participate in defining the E(1) proton-binding site, which could possibly exist as a hydronium ion coordination center. A model of proton translocation of AHA2 involving the side chains of amino acids Asp684 and Arg655 is discussed.
Asunto(s)
Arabidopsis/enzimología , Membrana Celular/enzimología , ATPasas de Translocación de Protón/metabolismo , Proteínas de Arabidopsis/metabolismo , Arginina , Sitios de Unión , Transporte Biológico , Concentración de Iones de Hidrógeno , Modelos Moleculares , Neurospora crassa/enzimología , Conformación Proteica , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/clasificación , ProtonesRESUMEN
The plant plasma membrane H(+)-ATPase is a proton pump which plays a central role in physiological functions such as nutrient uptake and intracellular pH regulation. This pump belongs to the P(3)-type ATPase family and creates an electrochemical gradient across the plasma membrane. The generation of this gradient has a major role in providing the energy for secondary active transport across the plasma membrane. The activity of the proton pump is regulated by the transcriptional and post-translational levels and by membrane environmental factors such as membrane lipids. Several reviews have appeared during the last few years concerning the regulatory mechanism at transcriptional and post-translational levels. The plasma membrane H(+)-ATPase requires lipids for activity. This lipid dependency suggests a possible mode of regulation of the H(+)-ATPase via modification of its lipid environment. This review focuses on the regulation of plasma membrane H(+)-ATPase by membrane lipids surrounding H(+)-ATPase molecules.
Asunto(s)
Membrana Celular/enzimología , Oryza/enzimología , ATPasas de Translocación de Protón/metabolismo , Secuencia de Aminoácidos , Membrana Celular/fisiología , Secuencia Conservada , Electroquímica , Activación Enzimática/efectos de los fármacos , Liposomas/metabolismo , Modelos Moleculares , Oryza/genética , Fosfolípidos/farmacología , Conformación Proteica , ATPasas de Translocación de Protón/clasificación , ATPasas de Translocación de Protón/genéticaRESUMEN
Here we show a complete list of the P-type ATPase genes in Caenorhabditis elegans and Drosophila melanogaster. A detailed comparison of the deduced amino-acid sequences in combination with phylogenetic and chromosomal analyses has revealed the following: (1) The diversity of this gene family has been achieved by two major evolutionary steps; the establishment of the major P-type ATPase subgroups with distinct substrate (ion) specificities in a common ancestor of vertebrate and invertebrate, followed by the evolution of multiple isoforms occurring independently in vertebrate and invertebrate phyla. (2) Pairs of genes that have intimate phylogenetic relationship are frequently found in proximity on the same chromosome. (3) Some of the Na,K- and H,K-ATPase isoforms in D. melanogaster and C. elegans lack motifs shown to be important for alpha/beta-subunit assembly, suggesting that such alpha- and beta-subunits might exist by themselves (lonely subunits). The mutation rates for these subunits are much faster than those for the subunits with recognizable assembly domains. (4) The lonely alpha-subunits also lack the major site for ouabain binding that apparently arose before the separation of vertebrates and invertebrates and thus well before the separation of vertebrate Na,K-ATPases and H,K-ATPases. These findings support the idea that a relaxation of functional constraints would increase the rate of evolution and provide clues for identifying the origins of inhibitor sensitivity, subunit assembly, and separation of Na,K- and H,K-ATPases.
Asunto(s)
Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Evolución Molecular , Perfilación de la Expresión Génica/métodos , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/química , Caenorhabditis elegans/enzimología , Análisis Mutacional de ADN/métodos , Drosophila melanogaster/química , Drosophila melanogaster/enzimología , ATPasa Intercambiadora de Hidrógeno-Potásio/química , ATPasa Intercambiadora de Hidrógeno-Potásio/clasificación , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Datos de Secuencia Molecular , Fenotipo , Subunidades de Proteína , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/clasificación , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Alineación de Secuencia/métodos , Análisis de Secuencia de Proteína/métodos , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/clasificación , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Especificidad de la EspecieRESUMEN
Proton-translocating, vacuolar-type ATPases, well known energizers of eukaryotic, vacuolar membranes, now emerge as energizers of many plasma membranes. Just as Na(+) gradients, imposed by Na(+)/K(+) ATPases, energize basolateral plasma membranes of epithelia, so voltage gradients, imposed by H(+) V-ATPases, energize apical plasma membranes. The energized membranes acidify or alkalinize compartments, absorb or secrete ions and fluids, and underwrite cellular homeostasis. V-ATPases acidify extracellular spaces of single cells such as phagocytes and osteoclasts and of polarized epithelia, such as vertebrate kidney and epididymis. They alkalinize extracellular spaces of lepidopteran midgut. V-ATPases energize fluid secretion by insect Malpighian tubules and fluid absorption by insect oocytes. They hyperpolarize external plasma membranes for Na(+) uptake by amphibian skin and fish gills. Indeed, it is likely that ion uptake by osmotically active membranes of all fresh water organisms is energized by V-ATPases. Awareness of plasma membrane energization by V-ATPases provides new perspectives for basic science and presents new opportunities for medicine and agriculture.
Asunto(s)
Membrana Celular/enzimología , ATPasas de Translocación de Protón/metabolismo , ATPasas de Translocación de Protón Vacuolares , Animales , Membrana Celular/metabolismo , Sistema Digestivo/enzimología , Metabolismo Energético , Riñón/enzimología , Masculino , Osteoclastos/metabolismo , Fagocitos/metabolismo , Bombas de Protones/metabolismo , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/clasificación , Sodio/metabolismo , Espermatozoides/enzimologíaRESUMEN
Frogs are faced with various osmoregulatory problems, such as compensation of salt and water loss or metabolic acidification. Being exposed both to air and to pond water of low salinity in their natural habitat, the epithelium of the frog skin serves as one of the major organs for body fluid homeostasis. For years, the frog skin has been the guiding model for ion transport processes in animal cells energized by a Na(+)-motive force. Meanwhile, however, it was demonstrated that under natural conditions Na+ uptake is electrically coupled to active H+ secretion, mediated by an electrogenic H+ pump. A proton-motive force generated at the apical membrane of the mitochondria-rich cells (MR cells) energizes Na+ entry via apical Na+ channels. The basolateral Na+/K+ P-ATPase then pumps Na+ out of the cell into the body fluid. Thus, there are two pumps functioning in series, both involved in transepithelial Na+ transport. Our recent investigations provided conclusive evidence that the H+ pump of the frog skin is an H+ V-ATPase. In transport studies, Na+ absorption and H+ secretion were blocked by micromolar concentrations of bafilomycin A1 or concanamycin A, two highly specific inhibitors of H(+)-V-ATPases. Using immunofluorescence microscopy, H(+)-V-ATPase-like immunoreactivity was found in MR cells in the region of their apical membrane foldings and intracellularly in the apical portion of the cell at so far unidentified locations. Besides the definition of its molecular nature, these results also confirmed the localization of the H+ pump in the apical membrane of the MR cells. These cells were already candidates for H(+)-V-ATPase localization mostly from correlations between their morphological features and their epithelial H+ secretion capacity. So far, there is evidence for only one type of MR cell serving both H+ and HCO3- secretion through an apical Cl-/HCO3- antiporter. H(+)-V-ATPase-mediated H+ secretion and thus Na+ absorption can be modulated by complementary mechanisms. Changes in intracellular H+ concentration linked to the animal's acid-base status will directly influence H+ V-ATPase activity. Acute acidification increases H+ current, probably as a result of the insertion of H(+)-V-ATPase-bearing vesicles by exocytotic processes, while alkalization causes the reverse effects. Chronic metabolic acidosis induces an increase in MR cell number in response to hormonal signals.
Asunto(s)
Equilibrio Ácido-Base/fisiología , Anuros/fisiología , Bombas de Protones/fisiología , Fuerza Protón-Motriz , ATPasas de Translocación de Protón/fisiología , Piel/metabolismo , Sodio/metabolismo , ATPasas de Translocación de Protón Vacuolares , Equilibrio Hidroelectrolítico/fisiología , Aerobiosis , Animales , Polaridad Celular , Electroquímica , Epitelio/enzimología , Homeostasis/fisiología , Potenciales de la Membrana , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/clasificaciónRESUMEN
More than 11 different P-type H(+)-ATPases have been identified in Arabidopsis by DNA cloning. The subcellular localization for individual members of this proton pump family has not been previously determined. We show by membrane fractionation and immunocytology that a subfamily of immunologically related P-type H(+)-ATPases, including isoforms AHA2 and AHA3, are primarily localized to the plasma membrane. To verify that AHA2 and AHA3 are both targeted to the plasma membrane, we added epitope tags to their C-terminal ends and expressed them in transgenic plants. Both tagged isoforms localized to the plasma membrane, as indicated by aqueous two-phase partitioning and sucrose density gradients. In contrast, a truncated AHA2 (residues 1-193) did not, indicating that the first two transmembrane domains alone are not sufficient for plasma membrane localization. Two epitope tags were evaluated: c-myc, a short, 11-amino acid sequence, and beta-glucuronidase (GUS), a 68-kD protein. The c-myc tag is recommended for its sensitivity and specific immunodetection. GUS worked well as an epitope tag when transgenes were expressed at relatively high levels (e.g. with AHA2-GUS944); however, evidence suggests that GUS activity may be inhibited when a GUS domain is tethered to an H(+)-ATPase complex. Nevertheless, the apparent ability to localize a GUS protein to the plasma membrane indicates that a P-type H(+)-ATPase can be used as a delivery vehicle to target large, soluble proteins to the plasma membrane.
Asunto(s)
Arabidopsis/enzimología , Compartimento Celular , Membrana Celular/enzimología , Isoenzimas/aislamiento & purificación , ATPasas de Translocación de Protón/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Epítopos , Isoenzimas/clasificación , Isoenzimas/genética , Isoenzimas/inmunología , Microscopía Inmunoelectrónica , Microsomas/enzimología , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Plantas Modificadas Genéticamente , ATPasas de Translocación de Protón/clasificación , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Fracciones Subcelulares/enzimología , beta-Glucosidasa/genética , beta-Glucosidasa/aislamiento & purificaciónRESUMEN
Sequences of the three integral membrane subunits (subunits a, b and c) of the F0 sector of the proton-translocating F-type (F0F1-) ATPases of bacteria, chloroplasts and mitochondria have been analysed. All homologous-sequenced proteins of these subunits, comprising three distinct families, have been identified by database searches, and the homologous protein sequences have been aligned and analysed for phylogenetic relatedness. The results serve to define the relationships of the members of each of these three families of proteins, to identify regions of relative conservation, and to define relative rates of evolutionary divergence. Of these three subunits, c-subunits exhibited the slowest rate of evolutionary divergence, b-subunits exhibited the most rapid rate of evolutionary divergence, and a-subunits exhibited an intermediate rate of evolutionary divergence. The results allow definition of the relative times of occurrence of specific events during evolutionary history, such as the intragenic duplication event that gave rise to large c-subunits in eukaryotic vacuolar-type ATPases after eukaryotes diverged from archaea, and the extragenic duplication of F-type ATPase b-subunits that occurred in blue-green bacteria before the advent of chloroplasts. The results generally show that the three F0 subunits evolved as a unit from a primordial set of genes without appreciable horizontal transmission of the encoding genetic information although a few possible exceptions were noted.
Asunto(s)
Bacterias/genética , Cloroplastos/genética , Mitocondrias/genética , Filogenia , ATPasas de Translocación de Protón/genética , Secuencia de Aminoácidos , Bacterias/clasificación , Bacterias/enzimología , Cloroplastos/clasificación , Cloroplastos/enzimología , Sustancias Macromoleculares , Mitocondrias/clasificación , Mitocondrias/enzimología , Datos de Secuencia Molecular , Conformación Proteica , ATPasas de Translocación de Protón/clasificación , Alineación de Secuencia/métodos , Homología de Secuencia de AminoácidoRESUMEN
Lemon fruit vacuoles acidify their lumens to pH 2.5, 3 pH units lower than typical plant vacuoles. To study the mechanism of hyperacidification, the kinetics of ATP-driven proton pumping by tonoplast vesicles from lemon fruits and epicotyls were compared. Fruit vacuolar membranes. H+ pumping by epicotyl membranes was chloride-dependent, stimulated by sulfate, and inhibited by the classical vacuolar ATPase (V-ATPase) inhibitors nitrate, bafilomycin, N-ethylmaleimide, and N,N'-dicyclohexylcarbodiimide. In addition, the epicotyl H+ pumping activity was inactivated by oxidation was reversed by dithiothreitol. Cold inactivation of the epicotyl V-ATPase by nitrate ( > or = 100 mM) was correlated with the release of V1 complexes from the membrane. In contrast, H+ pumping by the fruit tonoplast-enriched membranes was chloride-independent, largely insensitive to the V-ATPase inhibitors, and resistant to oxidation. Unlike the epicotyl inhibitors, and resistant to oxidation. Unlike the epicotyl H(+)-ATPase, the fruit H(+)-ATPase activity was partially inhibited by 200 microM vanadate. Cold inactivation treatment failed to inhibit H+ pumping activity of the fruit membranes, even though immunoblasts showed that V1 complexes were released from the membrane. However, cold inactivation doubled the percent inhibition by 200 microM vanadate from 30% to 60%. These results suggest the presence of two H(+)-ATPases in the fruit preparation: a V-ATPase and an unidentified vanadate-sensitive H(+)-ATPase. Attempts to separate the two activities in their native membranes on linear sucrose density density gradients were unsuccessful. However, following detergent-solubilization and centrifugation on a glycerol density gradient, the two ATPase activities were resolved: a nitrate-sensitive V-type ATPase that is also partially inhibited by 200 microM vanadate, and an apparently novel vanadate-sensitive ATPase that is also partially inhibited by nitrate.
Asunto(s)
Citrus/fisiología , ATPasas de Translocación de Protón/metabolismo , Adenosina Difosfato/metabolismo , Transporte Biológico Activo , Cloruros/fisiología , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Membranas Intracelulares/fisiología , Cinética , Nitratos/farmacología , Oxidación-Reducción , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/clasificación , Solubilidad , Vacuolas/fisiología , Vanadatos/farmacologíaRESUMEN
H(+)-ATPase cDNAs were identified in a potato leaf library using an Arabidopsis gene as a probe. Based on their sequences, the clones could be grouped into at least two classes. A similar classification was obtained from the analysis of sequence data from four tobacco genes. Both potato genes are expressed in all tissues analysed, higher levels of expression were found in leaves and stem than in roots and tubers. For both genes, no significant differences in level of expression could be detected under a variety of conditions such as cold treatment, anaerobiosis, sucrose induction or treatment with a synthetic cytokinin. Only 2,4-D and prolonged periods of darkness lead to a slight reduction in mRNA levels. The reduction in darkness was compensated after transfer of the plants back into the light. Expression of the ATPase genes remained constant in transgenic plants which are inhibited in phloem loading due to antisense inhibition of the sucrose transporter. On the other hand, expression of the sucrose transporter is inducible by auxin and cytokinin but not by sucrose. Taken together, these data suggest that at least the two plasma membrane H(+)-ATPase genes analysed are rather constant in their expression and that either other genes respond to external stimuli or that most of the regulation occurs at the posttranscriptional level.
Asunto(s)
Genes de Plantas/genética , ATPasas de Translocación de Protón/genética , Solanum tuberosum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Northern Blotting , Southern Blotting , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/genética , Expresión Génica , Biblioteca de Genes , Datos de Secuencia Molecular , Hojas de la Planta/metabolismo , ATPasas de Translocación de Protón/biosíntesis , ATPasas de Translocación de Protón/clasificación , Selección Genética , Homología de Secuencia de Aminoácido , Sacarosa/metabolismo , Distribución TisularRESUMEN
NIH-3T3 cells transfected with yeast H(+)-ATPases (RN1a cells) are tumorigenic (Perona and Serrano, 1988, Nature, 334:438). We have previously shown that RN1a cells maintain a chronically high intracellular pH (pHin) under physiological conditions. We have also shown that RN1a cells are serum-independent for growth, maintain a higher intracellular Ca2+ ([Ca2+]in), and glycolyze more rapidly than their non-transformed counterparts (Gillies et al., Proc. Natl. Acad. Sci., 1990, 87:7414; Gillies et al., Cell. Physiol. Biochem., 1992, 2:159). The present study was aimed to understand the interrelationships between glycolysis, pHin, and [Ca2+]in in RN1a cells and their non-transformed counterparts, NIH-3T3 cells. Our data show that the higher rate of glycolysis observed in RN1a cells is due to the presence of low affinity glucose transporters. Consequently, the higher rate of glycolysis is exacerbated at high glucose concentration in RN1a cells. Moreover, the maximal velocity (Vmax) for glucose utilization is up to sixfold higher in RN1a cells than in the NIH-3T3 cells, suggesting that the number of glucose transporters is higher in RN1a than NIH-3T3 cells. Glucose addition to NIH-3T3 cells results in modest decreases in both pHin and [Ca2+]in. In contrast, RN1a cells respond to glucose with a large decrease in pHin, followed by a large decrease in [Ca2+]in. The decrease in [Ca2+]in observed upon glucose addition is likely due to activation of Ca(2+)-ATPase by glycolysis, since the Ca2+ decrease is abolished by the Ca2+ ATPase inhibitors thapsigargin and cyclopiazonic acid. Glucose addition to ATP-depleted cells results in a decrease in [Ca2+]in, suggesting that ATP furnished by glycolysis is utilized by this pump.
Asunto(s)
Calcio/metabolismo , Glucosa/farmacología , ATPasas de Translocación de Protón/genética , Transfección , Levaduras/genética , Células 3T3 , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Línea Celular Transformada , Glucólisis , Concentración de Iones de Hidrógeno , Membranas Intracelulares/metabolismo , Ratones , Concentración Osmolar , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/clasificación , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Levaduras/enzimologíaRESUMEN
A gene (aha9) for a plasma membrane H(+)-ATPase was isolated from an Arabidopsis thaliana genomic library and sequenced. Comparison of the aha9 predicted amino acid sequence with those of aha1, 2 and 3 and analogous genes from other species indicated the existence of at least two aha gene subfamilies whose divergence precedes that of the Nicotiana and Arabidopsis species. Transcript analysis in various organs revealed expression of aha9 in flower tissues only. Introduction of aha9 into Nicotiana tabacum by genetic transformation gave rise to transgenic plants which also express aha9 in flower tissues. A more detailed analysis showed that aha9 expression was restricted to anther tissues.
Asunto(s)
Arabidopsis/genética , Membrana Celular/enzimología , Genes de Plantas/genética , ATPasas de Translocación de Protón/genética , Secuencia de Aminoácidos , Arabidopsis/enzimología , Secuencia de Bases , Biblioteca Genómica , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Plantas Tóxicas , ATPasas de Translocación de Protón/clasificación , ARN Mensajero/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Nicotiana/genéticaRESUMEN
Intrahepatic bile duct epithelial cells contribute to bile formation by hormone-dependently secreting HCO3- to bile and H+ to periductular fluid. The present study was undertaken to determine whether the secretin-induced H+ secretion is due to activation of a H(+)-ATPase or Na(+)-H+ exchange. H+ secretion was estimated from the rate of intracellular pH (pHi) recovery after acid loading (24 mM NH4Cl) of microdissected bile ductules from pig liver mounted in a flow-through chamber on the stage of a microscope. pHi was measured from an estimated average of 10-15 epithelial cells using the fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein and dual-wavelength excitation of fluorescence. The ducts were superfused with HCO3(-)-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffers. We found that secretin induced net H+ secretion of 4.53 +/- 0.7 mumol.ml cell volume-1 x min-1. This H+ secretion was blocked by 10(-6) M bafilomycin A1 but was unaffected by Na+ substitution with choline in the superfusion buffer. The experiments also showed that bafilomycin A1 did not block Na(+)-H+ exchange. The secretin-induced H+ secretion is probably caused by a vacuolar-type H(+)-ATPase and may constitute an important element of the cellular mechanisms causing secretin-dependent ductular HCO3- secretion into bile.
Asunto(s)
Conductos Biliares Intrahepáticos/metabolismo , Macrólidos , ATPasas de Translocación de Protón/metabolismo , Protones , Secretina/farmacología , Vacuolas/enzimología , Amilorida/farmacología , Cloruro de Amonio/farmacología , Animales , Antibacterianos/farmacología , Concentración de Iones de Hidrógeno , ATPasas de Translocación de Protón/clasificación , Descanso , Sodio/farmacología , PorcinosRESUMEN
A cDNA clone was isolated for a fourth pma gene encoding a putative plasma membrane H(+)-ATPase of Nicotiana plumbaginifolia. The sequence of the predicted 952 residue PMA4 polypeptide was compared with those of other known plant PMAs, revealing a higher identity with the Arabidopsis thaliana proteins (86-89%) than with the other three N. plumbaginifolia PMA proteins (80-82%). This supports the view that there are two pma subfamilies which probably arose from a gene duplication predating the separation of the Dilleniidae and Asteridae plant subclasses. Measured pma4 transcript levels indicate that pma4 is similarly expressed in root, stem, leaf, and flower tissues, contrary to the pmal-3 subfamily whose members displayed differential expression according to the organ.
Asunto(s)
Genes de Plantas , Familia de Multigenes , Nicotiana/genética , Plantas Tóxicas , ATPasas de Translocación de Protón/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Membrana Celular/enzimología , ADN , Datos de Secuencia Molecular , Filogenia , ATPasas de Translocación de Protón/clasificación , Nicotiana/enzimología , Transcripción GenéticaRESUMEN
Two types of proton-translocating ATPases, H-ATPase and H-K-ATPase, are found in the renal tubular cells. H-ATPase is present in both endocytic vesicles and apical membranes in almost all nephron segments. On the other hand, H-K-ATPase is present only in the connecting tubule and collecting duct. There is evidence to suggest that H-ATPase may be involved in H secretion in almost all nephron segments. H-K-ATPase is involved not only in H secretion but also in K absorption in the collecting duct segments. Aldosterone administration and metabolic acidosis stimulate the activity of H-ATPase in all collecting duct segments, whereas hypokalemia has only a limited effect on H-ATPase activity. On the other hand, hypokalemia, as well as metabolic acidosis, stimulates H-K-ATPase activity in the collecting duct segments, whereas aldosterone administration alone plays a minor role in the regulation of this enzyme. The physiological role and regulation of H-ATPase in the proximal tubule has not been established.