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Sample records for atpase pfserca gene

  1. Geographic structuring of the Plasmodium falciparum sarco(endoplasmic reticulum Ca2+ ATPase (PfSERCA gene diversity.

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    Ronan Jambou

    Full Text Available Artemisinin, a thapsigargin-like sesquiterpene has been shown to inhibit the Plasmodium falciparum sarco/endoplasmic reticulum calcium-ATPase PfSERCA. To collect baseline pfserca sequence information before field deployment of Artemisinin-based Combination therapies that may select mutant parasites, we conducted a sequence analysis of 100 isolates from multiple sites in Africa, Asia and South America. Coding sequence diversity was large, with 29 mutated codons, including 32 SNPs (average of one SNP/115 bp, of which 19 were novel mutations. Most SNP detected in this study were clustered within a region in the cytosolic head of the protein. The PfSERCA functional domains were very well conserved, with non synonymous mutations located outside the functional domains, except for the S769N mutation associated in French Guiana with elevated IC(50 for artemether. The S769N mutation is located close to the hinge of the headpiece, which in other species modulates calcium affinity and in consequence efficacy of inhibitors, possibly linking calcium homeostasis to drug resistance. Genetic diversity was highest in Senegal, Brazil and French Guiana, and few mutations were identified in Asia. Population genetic analysis was conducted for a partial fragment of the gene encompassing nucleotide coordinates 87-2862 (unambiguous sequence available for 96 isolates. This supported a geographic clustering, with a separation between Old and New World samples and one dominant ancestral haplotype. Genetic drift alone cannot explain the observed polymorphism, suggesting that other evolutionary mechanisms are operating. One possible contributor could be the frequency of haemoglobinopathies that are associated with calcium dysregulation in the erythrocyte.

  2. Calcium-ATPases: Gene disorders and dysregulation in cancer.

    Science.gov (United States)

    Dang, Donna; Rao, Rajini

    2016-06-01

    Ca(2+)-ATPases belonging to the superfamily of P-type pumps play an important role in maintaining low, nanomolar cytoplasmic Ca(2+) levels at rest and priming organellar stores, including the endoplasmic reticulum, Golgi, and secretory vesicles with high levels of Ca(2+) for a wide range of signaling functions. In this review, we introduce the distinct subtypes of Ca(2+)-ATPases and their isoforms and splice variants and provide an overview of their specific cellular roles as they relate to genetic disorders and cancer, with a particular emphasis on recent findings on the secretory pathway Ca(2+)-ATPases (SPCA). Mutations in human ATP2A2, ATP2C1 genes, encoding housekeeping isoforms of the endoplasmic reticulum (SERCA2) and secretory pathway (SPCA1) pumps, respectively, confer autosomal dominant disorders of the skin, whereas mutations in other isoforms underlie various muscular, neurological, or developmental disorders. Emerging evidence points to an important function of dysregulated Ca(2+)-ATPase expression in cancers of the colon, lung, and breast where they may serve as markers of differentiation or novel targets for therapeutic intervention. We review the mechanisms underlying the link between calcium homeostasis and cancer and discuss the potential clinical relevance of these observations. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene

    National Research Council Canada - National Science Library

    Thakur, Nidhi; Upadhyay, Santosh Kumar; Verma, Praveen C; Chandrashekar, Krishnappa; Tuli, Rakesh; Singh, Pradhyumna K

    2014-01-01

    .... The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation...

  4. Enhanced Whitefly Resistance in Transgenic Tobacco Plants Expressing Double Stranded RNA of v-ATPase A Gene: e87235

    National Research Council Canada - National Science Library

    Nidhi Thakur; Santosh Kumar Upadhyay; Praveen C Verma; Krishnappa Chandrashekar; Rakesh Tuli; Pradhyumna K Singh

    2014-01-01

    .... The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation...

  5. A plasma membrane H + ATPase gene is germinationinduced in ...

    African Journals Online (AJOL)

    The expression pattern of a germination specific plasma membrane H+-ATPase was analyzed by RTPCR and in situ RNA hybridization methods. RT-PCR results revealed that germination specific plasma membrane H+-ATPase accumulation was detectable in all organs and tissues of germinating wheat embryos.

  6. A comparative study of ATPase subunit 9 (Atp9) gene between ...

    African Journals Online (AJOL)

    ATPase subunit 9 gene (Atp9) is an important functional gene in mitochondria, and is closely related with energy supply. RNA editing of atp9 gene was associated with male sterility in plants. In this study, the atp9 gene in soybeans was cloned from a soybean cytoplasmic male sterile line NJCMS2A and its maintainer line ...

  7. A plasma membrane H ATPase gene is germination- induced in ...

    African Journals Online (AJOL)

    ONOS

    2010-01-18

    Jan 18, 2010 ... Cereal embryo development results in the formation of a fully differentiated, immature embryo within 10-12 ... quent root, coleoptile and leaf development. Although the transition from the dormant embryo ... cally stressed cultured Arabidopsis thaliana cells respon- ded by activating H+-ATPase-mediated H+ ...

  8. RNAi-based silencing of genes encoding the vacuolar- ATPase ...

    African Journals Online (AJOL)

    2016-11-09

    Nov 9, 2016 ... Searching the PROSITE database revealed no common motifs for both subunits sequences. The phylogenetic tree analysis shows that the V-ATPase subunit a of both. P. gossypiella and Tribolium castaneum are located on the same branch as Bactrocera dorsalis with 94% bootstrap support (Figure 1a).

  9. Compact tomato seedlings and plants upon overexpression of a tomato chromatin remodelling ATPase gene

    NARCIS (Netherlands)

    Folta, A.; Bargsten, J.W.; Bisseling, T.; Nap, J.P.H.; Mlynarova, L.

    2016-01-01

    Control of plant growth is an important aspect of crop productivity and yield in agriculture. Overexpression of the AtCHR12/23 genes in Arabidopsis thaliana reduced growth habit without other morphological changes. These two genes encode Snf2 chromatin remodelling ATPases. Here, we translate this

  10. Aspects of gene structure and functional regulation of the isozymes of Na,K-ATPase

    DEFF Research Database (Denmark)

    Jorgensen, P.L.

    2001-01-01

    Gaps in our understanding of the complex regulated expression of isozymes of Na,K-ATPase and the diverse systems for posttranslational modification and short term regulation of active Na,K-transport in animals and humans are the main problems in comprehensive Na,K-pump physiology. In mammalian...... genomes, the genes of four alpha-subunit and at least three beta-subunit isoforms of Na,K-ATPase are identified and two gamma-subunits are expressed in kidney. The isoforms combine in a number of Na,K-ATPase isozymes that are expressed in a tissue and cell specific manner. Models of the molecular...... mechanism of regulation of these isozymes have become more reliable due to progress in understanding the three-dimensional protein structure and conformational transitions mediating transfer of energy from the P-domain to intramembrane Na+ and K+ binding sites....

  11. A putative P-type Cu(2+)-transporting ATPase gene on chromosome II of Saccharomyces cerevisiae.

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    Rad, M R; Kirchrath, L; Hollenberg, C P

    1994-09-01

    We have sequenced a gene on the right arm near the telomere of chromosome II of Saccharomyces cerevisiae which codes for a putative P-type cation-transporting ATPase (PCA1). The gene codes for a 1216 amino acids protein. The PCA1 gene expresses a 3.5 kb message in both haploid and diploid cells when grown in glucose-based rich medium YPD. The gene product is most similar at the C-terminal region to a human copper-transporting ATPase and Enterococcus hirae copper-transporting ATPases and also an N-terminal dithiol region that was proposed to be a 'metal-binding motif'. Cells lacking PCA1 display no obvious phenotype when tested under standard conditions: whereas they cease growth much earlier than the isogenic wild-type cells in a minimal medium with high copper concentration. Overexpression of PCA1 under GAL1/10 promoter in yeast cells causes poor growth. We also show that yeast strains carrying PCA1 in multiple copies grow slower than isogenic wild-type strains in a minimal synthetic medium containing 0.3 mM-CuSO4.

  12. Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

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    Bartlett Marilyn S

    2001-06-01

    Full Text Available Abstract Background Pneumocystis carinii causes pneumonia in immunocompromised patients with a high morbidity and mortality rate, but the interaction between this organism and the host cell is not well understood. The purpose of this research was to study the response of host cells to P. carinii infection on a molecular level. Results The technique of mRNA differential display was used to detect genes whose expression may be affected by P. carinii infection. The nucleotide sequence of one differentially displayed DNA fragment was found to be identical to that of the rat mitochondrial ATPase 6 gene, which is a subunit of the F0F1-ATP synthase complex. A four-fold increase in expression of this gene was verified by Northern blot analysis of total RNA extracted from P. carinii-infected rat lung versus that from mock-infected rat lung. Localization of the cells containing ATPase 6 mRNA was accomplished by in situ hybridization. In sections of non-infected rat lung, these cells were found lining the distal parts of the respiratory tree and in apical areas of the alveoli. Histological location of these cells suggested that they were Clara cells and type II pneumocytes. This hypothesis was confirmed by co-localizing the mRNAs for ATPase 6 and surfactant protein B (SP-B to the same cells by two-color fluorescent in situ hybridization. Conclusions The ATPase 6 gene is over expressed during P. carinii infection, and type II pneumocytes and Clara cells are the cell types responsible for this over-expression.

  13. Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

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    Nidhi Thakur

    Full Text Available BACKGROUND: Expression of double strand RNA (dsRNA designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi, thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci upon oral feeding. The v-ATPase subunit A (v-ATPaseA coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. METHODOLOGY/PRINCIPAL FINDINGS: Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. CONCLUSIONS/SIGNIFICANCE: Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.

  14. Expression of genes encoding F-1-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis

    DEFF Research Database (Denmark)

    Købmann, Brian Jensen; Solem, Christian; Pedersen, M.B.

    2002-01-01

    of the genes encoding F-1-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F-1-ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase...... threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions...

  15. Compact tomato seedlings and plants upon overexpression of a tomato chromatin remodelling ATPase gene.

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    Folta, Adam; Bargsten, Joachim W; Bisseling, Ton; Nap, Jan-Peter; Mlynarova, Ludmila

    2016-02-01

    Control of plant growth is an important aspect of crop productivity and yield in agriculture. Overexpression of the AtCHR12/23 genes in Arabidopsis thaliana reduced growth habit without other morphological changes. These two genes encode Snf2 chromatin remodelling ATPases. Here, we translate this approach to the horticultural crop tomato (Solanum lycopersicum). We identified and cloned the single tomato ortholog of the two Arabidopsis Snf2 genes, designated SlCHR1. Transgenic tomato plants (cv. Micro-Tom) that constitutively overexpress the coding sequence of SlCHR1 show reduced growth in all developmental stages of tomato. This confirms that SlCHR1 combines the functions of both Arabidopsis genes in tomato. Compared to the wild type, the transgenic seedlings of tomato have significantly shorter roots, hypocotyls and reduced cotyledon size. Transgenic plants have a much more compact growth habit with markedly reduced plant height, severely compacted reproductive structures with smaller flowers and smaller fruits. The results indicate that either GMO-based or non-GMO-based approaches to modulate the expression of chromatin remodelling ATPase genes could develop into methods to control plant growth, for example to replace the use of chemical growth retardants. This approach is likely to be applicable and attractive for any crop for which growth habit reduction has added value. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  16. Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

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    Thabet, Rahma; Rouault, J-D; Ayadi, Habib; Leignel, Vincent

    2016-01-01

    The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, β and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. An Arabidopsis ATPase gene involved in nematode-induced syncytium development and abiotic stress responses

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    Ali, Muhammad Amjad; Plattner, Stephan; Radakovic, Zoran; Wieczorek, Krzysztof; Elashry, Abdelnaser; Grundler, Florian MW; Ammelburg, Moritz; Siddique, Shahid; Bohlmann, Holger

    2013-01-01

    The beet cyst nematode Heterodera schachtii induces syncytia in the roots of Arabidopsis thaliana, which are its only nutrient source. One gene, At1g64110, that is strongly up-regulated in syncytia as shown by RT-PCR, quantitative RT-PCR, in situ RT-PCR and promoter::GUS lines, encodes an AAA+-type ATPase. Expression of two related genes in syncytia, At4g28000 and At5g52882, was not detected or not different from control root segments. Using amiRNA lines and T-DNA mutants, we show that At1g64110 is important for syncytium and nematode development. At1g64110 was also inducible by wounding, jasmonic acid, salicylic acid, heat and cold, as well as drought, sodium chloride, abscisic acid and mannitol, indicating involvement of this gene in abiotic stress responses. We confirmed this using two T-DNA mutants that were more sensitive to abscisic acid and sodium chloride during seed germination and root growth. These mutants also developed significantly smaller roots in response to abscisic acid and sodium chloride. An in silico analysis showed that ATPase At1g64110 (and also At4g28000 and At5g52882) belong to the ‘meiotic clade’ of AAA proteins that includes proteins such as Vps4, katanin, spastin and MSP1. PMID:23480402

  18. Analysis of Canis mitochondrial DNA demonstrates high concordance between the control region and ATPase genes.

    Science.gov (United States)

    Rutledge, Linda Y; Patterson, Brent R; White, Bradley N

    2010-07-16

    Phylogenetic studies of wild Canis species have relied heavily on the mitochondrial DNA control region (mtDNA CR) to infer species relationships and evolutionary lineages. Previous analyses of the CR provided evidence for a North American evolved eastern wolf (C. lycaon), that is more closely related to red wolves (C. rufus) and coyotes (C. latrans) than grey wolves (C. lupus). Eastern wolf origins, however, continue to be questioned. Therefore, we analyzed mtDNA from 89 wolves and coyotes across North America and Eurasia at 347 base pairs (bp) of the CR and 1067 bp that included the ATPase6 and ATPase8 genes. Phylogenies and divergence estimates were used to clarify the evolutionary history of eastern wolves, and regional comparisons of nonsynonomous to synonomous substitutions (dN/dS) at the ATPase6 and ATPase8 genes were used to elucidate the potential role of selection in shaping mtDNA geographic distribution. We found high concordance across analyses between the mtDNA regions studied. Both had a high percentage of variable sites (CR = 14.6%; ATP = 9.7%) and both phylogenies clustered eastern wolf haplotypes monophyletically within a North American evolved lineage apart from coyotes. Divergence estimates suggest the putative red wolf sequence is more closely related to coyotes (DxyCR = 0.01982 +/- 0.00494 SD; DxyATP = 0.00332 +/- 0.00097 SD) than the eastern wolf sequences (DxyCR = 0.03047 +/- 0.00664 SD; DxyATP = 0.00931 +/- 0.00205 SD). Neutrality tests on both genes were indicative of the population expansion of coyotes across eastern North America, and dN/dS ratios suggest a possible role for purifying selection in the evolution of North American lineages. dN/dS ratios were higher in European evolved lineages from northern climates compared to North American evolved lineages from temperate regions, but these differences were not statistically significant. These results demonstrate high concordance between coding and non-coding regions of mtDNA, and provide

  19. Analysis of Canis mitochondrial DNA demonstrates high concordance between the control region and ATPase genes

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    White Bradley N

    2010-07-01

    Full Text Available Abstract Background Phylogenetic studies of wild Canis species have relied heavily on the mitochondrial DNA control region (mtDNA CR to infer species relationships and evolutionary lineages. Previous analyses of the CR provided evidence for a North American evolved eastern wolf (C. lycaon, that is more closely related to red wolves (C. rufus and coyotes (C. latrans than grey wolves (C. lupus. Eastern wolf origins, however, continue to be questioned. Therefore, we analyzed mtDNA from 89 wolves and coyotes across North America and Eurasia at 347 base pairs (bp of the CR and 1067 bp that included the ATPase6 and ATPase8 genes. Phylogenies and divergence estimates were used to clarify the evolutionary history of eastern wolves, and regional comparisons of nonsynonomous to synonomous substitutions (dN/dS at the ATPase6 and ATPase8 genes were used to elucidate the potential role of selection in shaping mtDNA geographic distribution. Results We found high concordance across analyses between the mtDNA regions studied. Both had a high percentage of variable sites (CR = 14.6%; ATP = 9.7% and both phylogenies clustered eastern wolf haplotypes monophyletically within a North American evolved lineage apart from coyotes. Divergence estimates suggest the putative red wolf sequence is more closely related to coyotes (DxyCR = 0.01982 ± 0.00494 SD; DxyATP = 0.00332 ± 0.00097 SD than the eastern wolf sequences (DxyCR = 0.03047 ± 0.00664 SD; DxyATP = 0.00931 ± 0.00205 SD. Neutrality tests on both genes were indicative of the population expansion of coyotes across eastern North America, and dN/dS ratios suggest a possible role for purifying selection in the evolution of North American lineages. dN/dS ratios were higher in European evolved lineages from northern climates compared to North American evolved lineages from temperate regions, but these differences were not statistically significant. Conclusions These results demonstrate high concordance between coding

  20. Apoptosis Gene Hunting Using Retroviral Expression Cloning: Identification of Vacuolar ATPase Subunit E

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    Claire L. Anderson

    2003-01-01

    Full Text Available Over the past 10-15 years there has been an explosion of interest in apoptosis. The delayed realisation that cell death is an essential part of life for any multicellular organism has meant that, despite the recent and rapid developments of the last decade, the precise biochemical pathways involved in apoptosis remain incomplete and potentially novel genes may, as yet, remain undiscovered. The hunt is therefore on to bridge the remaining gaps in our knowledge. Our contribution to this research effort utilises a functional cloning approach to isolate important regulatory genes involved in apoptosis. This mini-review focuses on the use and advantages of a retroviral expression cloning strategy and describes the isolation and identification of one such potential apoptosis regulatory gene, namely that encoding vacuolar ATPase subunit E.

  1. First case of compound heterozygosity in Na,K-ATPase gene ATP1A2 in familial hemiplegic migraine.

    Science.gov (United States)

    Vanmolkot, Kaate R J; Stam, Anine H; Raman, Ashok; Koenderink, Jan B; de Vries, Boukje; van den Boogerd, Eelke H; van Vark, Judith; van den Heuvel, Jeroen J M W; Bajaj, Nin; Terwindt, Gisela M; Haan, Joost; Frants, Rune R; Ferrari, Michel D; van den Maagdenberg, Arn M J M

    2007-08-01

    Familial hemiplegic migraine (FHM) is a rare autosomal-dominant subtype of migraine with aura, associated with hemiparesis during the aura. Here we describe a unique FHM family in which two novel allelic missense mutations in the Na,K-ATPase gene ATP1A2 segregate in the proband with hemiplegic migraine. Both mutations show reduced penetrance in family members of the proband. Cellular survival assays revealed Na,K-ATPase dysfunction for both ATP1A2 mutants, indicating that both mutations are disease causative. This is the first case of compound heterozygosity for any of the known FHM genes.

  2. Population genetic structure of Macrobrachium rosenbergii (Palaemonidae) from Indian waters using mitochondrial ATPase 6/8 gene.

    Science.gov (United States)

    Kumar, Raj; Gopalakrishnan, A; Divya, P R; Basheer, V S; Singh, Rajeev K; Mohindra, Vindya; Lal, Kuldeep K; Jena, J K

    2017-07-01

    Macrobrachium rosenbergii, giant freshwater prawn, is one of the most commercially important crustaceans. In the present study, primers for ATPase 6/8 region of mt-DNA were designed and successfully amplified (827 bp) in the species. The nucleotide variation in ATPase 6/8 gene revealed the population structuring in natural populations of M. rosenbergii in Indian waters. A total of 35 haplotypes were observed in 93 individuals collected from different locations. Low nucleotide diversity and high haplotype diversity were noticed for the ATPase 6/8 gene. Significant pairwise FST and, haplotype network indicated occurrence of distinct populations. Observed mismatch distribution and Tajima's D test suggested demographical stability of giant freshwater prawn. The genetic stock structure revealed in this study will be helpful for conservation and management of stocks of M. rosenbergii in Indian waters.

  3. Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro.

    Science.gov (United States)

    Camargo, Luiz Sergio Almeida; Boite, Mariana Cortes; Wohlres-Viana, Sabine; Mota, Gustavo Bruno; Serapiao, Raquel Varela; Sa, Wanderlei Ferreira; Viana, Joao Henrique Moreira; Nogueira, Luiz Altamiro Garcia

    2011-12-01

    The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900 mOsm) for 5 min following 120 min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (PEmbryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72 h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (Pembryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (Pembryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Gene silencing reveals multiple functions of Na+/K+-ATPase in the salmon louse (Lepeophtheirus salmonis).

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    Komisarczuk, Anna Z; Kongshaug, Heidi; Nilsen, Frank

    2018-01-12

    Na+/K+-ATPase has a key function in a variety of physiological processes including membrane excitability, osmoregulation, regulation of cell volume, and transport of nutrients. While knowledge about Na+/K+-ATPase function in osmoregulation in crustaceans is extensive, the role of this enzyme in other physiological and developmental processes is scarce. Here, we report characterization, transcriptional distribution and likely functions of the newly identified L. salmonis Na+/K+-ATPase (LsalNa+/K+-ATPase) α subunit in various developmental stages. The complete mRNA sequence was identified, with 3003 bp open reading frame encoding a putative protein of 1001 amino acids. Putative protein sequence of LsalNa+/K+-ATPase revealed all typical features of Na+/K+-ATPase and demonstrated high sequence identity to other invertebrate and vertebrate species. Quantitative RT-PCR analysis revealed higher LsalNa+/K+-ATPase transcript level in free-living stages in comparison to parasitic stages. In situ hybridization analysis of copepodids and adult lice revealed LsalNa+/K+-ATPase transcript localization in a wide variety of tissues such as nervous system, intestine, reproductive system, and subcuticular and glandular tissue. RNAi mediated knock-down of LsalNa+/K+-ATPase caused locomotion impairment, and affected reproduction and feeding. Morphological analysis of dsRNA treated animals revealed muscle degeneration in larval stages, severe changes in the oocyte formation and maturation in females and abnormalities in tegmental glands. Thus, the study represents an important foundation for further functional investigation and identification of physiological pathways in which Na+/K+-ATPase is directly or indirectly involved. Copyright © 2018. Published by Elsevier Inc.

  5. An extended nomenclature for mammalian V-ATPase subunit genes and splice variants.

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    Kevin C Miranda

    2010-03-01

    Full Text Available The vacuolar-type H(+-ATPase (V-ATPase is a multisubunit proton pump that is involved in both intra- and extracellular acidification processes throughout the body. Multiple homologs and splice variants of V-ATPase subunits are thought to explain its varied spatial and temporal expression pattern in different cell types. Recently subunit nomenclature was standardized with a total of 22 subunit variants identified. However this standardization did not accommodate the existence of splice variants and is therefore incomplete. Thus, we propose here an extension of subunit nomenclature along with a literature and sequence database scan for additional V-ATPase subunits. An additional 17 variants were pulled from a literature search while 4 uncharacterized potential subunit variants were found in sequence databases. These findings have been integrated with the current V-ATPase knowledge base to create a new V-ATPase subunit catalogue. It is envisioned this catalogue will form a new platform on which future studies into tissue- and organelle-specific V-ATPase expression, localization and function can be based.

  6. Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells.

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    Pauw, P G; Johnson, M D; Moore, P; Morgan, M; Fineman, R M; Kalka, T; Ash, J F

    1986-01-01

    Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17. Images PMID:3023874

  7. Characterization of a heavy metal translocating P-type ATPase gene from an environmental heavy metal resistance Enterobacter sp. isolate.

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    Chien, Chih-Ching; Huang, Chia-Hsuan; Lin, Yi-Wei

    2013-03-01

    Heavy metals are common contaminants found in polluted areas. We have identified a heavy metal translocating P-type ATPase gene (hmtp) via fosmid library and in vitro transposon mutagenesis from an Enterobacter sp. isolate. This gene is believed to participate in the bacterium's heavy metal resistance traits. The complete gene was identified, cloned, and expressed in a suitable Escherichia coli host cell. E. coli W3110, RW3110 (zntA::Km), GG48 (ΔzitB::Cm zntA::Km), and GG51 (ΔzitB::Cm) were used to study the possible effects of this gene for heavy metal (cadmium and zinc in particular) resistance. Among the E. coli strains tested, RW3110 and GG48 showed more sensitivity to cadmium and zinc compared to the wild-type E. coli W3110 and strain GG51. Therefore, strains RW3110 and GG48 were chosen for the reference hosts for further evaluation of the gene's effect. The results showed that expression of this heavy metal translocating P-type ATPase gene could increase the ability for zinc and cadmium resistance in the tested microorganisms.

  8. Sarco(endoplasmic Reticulum Ca2+-ATPase-2 Gene: Structure and Transcriptional Regulation of the Human Gene

    Directory of Open Access Journals (Sweden)

    Angel Zarain-Herzberg

    2002-01-01

    Full Text Available The sarco(endoplasmic reticulum Ca2+-ATPases (SERCAs belong to a family of active calcium transport enzymes encoded by the SERCA1, 2, and 3 genes. In this study, we describe the complete structure of the human SERCA2 gene and its 5’ -regulatory region. The hSERCA2 gene is located in chromosome 12 position q24.1 in Contig NT_009770.8, spans 70 kb, and is organized in 21 exons intervened by 20 introns. The last two exons of the pre-mRNA produce by alternatively splicing the cardiac/slow-twitch muscle-specific SERCA2a isoform and the ubiquitous SERCA2b isoform. The sequence of the proximal 225-bp regulatory region of the SERCA2 genes is 80% G+C-rich and is conserved among human, rabbit, rat, and mouse species. It contains a TATA-like-box, an E-box/USF sequence, a CAAT-box, four Sp1 binding sites, and a thyroid hormone responsive element (TRE. There are two other conserved regulatory regions located between positions -410 to -661 bp and from -919 to -1410 bp. Among the DNA cis-elements present in these two regulatory regions there are potential binding sites for: GATA-4, -5, -6, Nkx-2.5/Csx, OTF-1, USF, MEF-2, SRF, PPAR/RXR, AP-2, and TREs. Upstream from position -1.5 kb, there is no significant homology among the SERCA2 genes cloned. In addition, the human gene has several repeated sequences mainly of the Alu and L2 type located upstream from position -1.7 kb, spanning in a continuous fashion for more than 40 kb. In this study, we report the cloning of 2.4 kb of 5’-regulatory region and demonstrate that the proximal promoter region is sufficient for expression in cardiac myocytes, and the region from -225 to -1232 bp contains regulatory DNA elements which down-regulate the expression of the SERCA2 gene in neonatal cardiomyocytes.

  9. Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness

    NARCIS (Netherlands)

    Karet, FE; Finberg, KE; Nelson, RD; Nayir, A; Mocan, H; Sanjad, SA; Rodriguez-Soriano, J; Santos, F; Cremers, CWRJ; di Pietro, A; Hoffbrand, BI; Winiarski, J; Bakkaloglu, A; Ozen, S; Dusunsel, R; Goodyer, P; Hulton, SA; Wu, DK; Skvorak, AB; Morton, CC; Cunningham, MJ; Jha, [No Value; Lifton, RP

    H+-ATPases are ubiquitous in nature; V-ATPases pump protons against an electrochemical gradient, whereas F-ATPases reverse the process, synthesizing ATP. We demonstrate here that mutations in ATP6B1, encoding the B-subunit of the apical proton pump mediating distal nephron acid secretion, cause

  10. Effects of oestrogen on sarcoplasmic reticulum Ca2+-ATPase activity and gene expression in genioglossus in chronic intermittent hypoxia rat.

    Science.gov (United States)

    Liu, Yue-Hua; Li, Wen; Song, Wei-Hua

    2009-04-01

    This study was designed to investigate the effects of oestrogen on sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity and gene expression in ovariectomised rats under the condition of chronic intermittent hypoxia (CIH). Thirty-two female Sprague-Dawley rats were randomly divided into four groups: the normal control group (NC), the CIH group (CIH), the CIH-ovariectomised group (CIH+OVX), and the group of CIH-ovariectomised rats receiving estradiol replacement (CIH+OVX+E(2)). Rats in the latter three groups were exposed to CIH for 5 weeks. The animals were killed before genioglossus (GG) was rapidly excised, and their body and uterus mass were determined. Estradiol level was detected by radioimmunoassay. SR Ca(2+)-ATPase (SERCA) activity was observed by detecting inorganic phosphorus ion, and the SERCA mRNA level was measured using real-time quantitative polymerase chain reaction (real-time PCR). It was found that, compared with the NC group, the SERCA activity and mRNA level were remarkably reduced (pSERCA activity and mRNA level were also significantly reduced (pSERCA activity and mRNA level significantly increased (pSERCA activity and mRNA expression, and oestrogen-deficiency could exacerbate this effect; whilst estradiol replacement can partially reverse the effect of CIH in ovariectomised rats.

  11. Regulatory interplay of Cockayne syndrome B ATPase and stress-response gene ATF3 following genotoxic stress.

    Science.gov (United States)

    Kristensen, Ulrik; Epanchintsev, Alexey; Rauschendorf, Marc-Alexander; Laugel, Vincent; Stevnsner, Tinna; Bohr, Vilhelm A; Coin, Frédéric; Egly, Jean-Marc

    2013-06-18

    Cockayne syndrome type B ATPase (CSB) belongs to the SwItch/Sucrose nonfermentable family. Its mutations are linked to Cockayne syndrome phenotypes and classically are thought to be caused by defects in transcription-coupled repair, a subtype of DNA repair. Here we show that after UV-C irradiation, immediate early genes such as activating transcription factor 3 (ATF3) are overexpressed. Although the ATF3 target genes, including dihydrofolate reductase (DHFR), were unable to recover RNA synthesis in CSB-deficient cells, transcription was restored rapidly in normal cells. There the synthesis of DHFR mRNA restarts on the arrival of RNA polymerase II and CSB and the subsequent release of ATF3 from its cAMP response element/ATF target site. In CSB-deficient cells ATF3 remains bound to the promoter, thereby preventing the arrival of polymerase II and the restart of transcription. Silencing of ATF3, as well as stable introduction of wild-type CSB, restores RNA synthesis in UV-irradiated CSB cells, suggesting that, in addition to its role in DNA repair, CSB activity likely is involved in the reversal of inhibitory properties on a gene-promoter region. We present strong experimental data supporting our view that the transcriptional defects observed in UV-irradiated CSB cells are largely the result of a permanent transcriptional repression of a certain set of genes in addition to some defect in DNA repair.

  12. Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Gogarten Johann

    2002-04-01

    Full Text Available Abstract Background Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene. Results Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene. Conclusions Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2–4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation.

  13. Functional characterization of the copper-transporting P-type ATPase gene of Penicillium janthinellum strain GXCR.

    Science.gov (United States)

    Lai, Hongmin; Sun, Changbin; Tang, Huaying; Fan, Xianwei; Ma, Yili; Li, Youzhi

    2009-12-01

    Copper (Cu)-transporting P-type ATPase (CTPA) genes have been documented to play an important role in resistance to heavy metals. However, our understanding of roles of CTPA genes of the filamentous fungi was based only on sequence similarity prediction before. In a previous study, we isolated a Penicillum janthinellum strain GXCR of higher tolerance to Cu (200 mM). In this study, we cloned the partial cDNA of CTPA gene, named PcpA, from the strain GXCR. Sequence alignment indicated that the cloned cDNA sequence has the highest identity (94.4%) with a predictive CTPA gene of Aspergillus clavatus. The PcpA-encoded protein, termed PcpA, has classical functional domains of CTPAs, and shows differences from reported CTPAs in some specific sequence motifs and transmembrane regions. Expression of the PcpA was induced by extracellular Cu, cadmium (Cd), and silver (Ag). PcpA RNA interference (RNAi) mutants with a reduced level of PcpA mRNA were more sensitive to Cu, iron, Cd, and Ag than the wild-type (WT) strain GXCR. When grown in the presence of Cu, iron, and Cd, intracellular Cu and iron contents in the PcpA RNAi mutant were significantly (P<0.05) lower than those in the WT; However, intracellular Cd content in the mutant was significantly (P<0.05) higher than that in the WT. Taken together, it can be concluded that the PcpA functions in Cu uptake and homeostasis, iron uptake, and Cd export from the cytosol to the extracytosol.

  14. Cloning and expression analysis of GhDET3, a vacuolar H+ -ATPase subunit C gene, from cotton.

    Science.gov (United States)

    Xiao, Zhongyi; Tan, Kunling; Hu, Mingyu; Liao, Peng; Chen, Kuijun; Luo, Ming

    2008-05-01

    Vacuolar H(+)-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an important role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To analyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5'-upstream sequence, and the 3'-RACE technique was used to clone the 3'-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5'-UTR, an ORF of 1,134 bp, and a 196 bp 3'-UTR. This cDNA sequence encoded a polypeptide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA of GhDET3 was also cloned by PCR. The deduced amino acid sequence had a high homology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expression pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.

  15. Rotary ATPases

    Science.gov (United States)

    Stewart, Alastair G.; Sobti, Meghna; Harvey, Richard P.; Stock, Daniela

    2013-01-01

    Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual “machine elements” to the requirement of the right “fuel” and “oil” for different types of motors. PMID:23369889

  16. Electroporation-mediated in vivo gene delivery of the Na+/K+-ATPase pump reduced lung injury in a mouse model of lung contusion.

    Science.gov (United States)

    Machado-Aranda, David A; Suresh, M V; Yu, Bi; Raghavendran, Krishnan

    2012-01-01

    Lung contusion (LC) is an independent risk factor for acute respiratory distress syndrome. The final common pathway in ARDS involves accumulation of fluid in the alveoli. In this study, we demonstrate the application of a potential gene therapy approach by delivering the Na+/K+-ATPase pump subunits in a murine model of LC. We hypothesized that restoring the activity of the pump will result in removal of excess alveolar fluid and additionally reduce inflammation. Under anesthesia, C57/BL6 mice were struck along the right posterior axillary line 1 cm above the costal margin with a cortical contusion impactor. Immediately afterward, 100 μg of plasmid DNA coding for the α,β of the Na+/K+-ATPase pump were instilled into the lungs (LC-electroporation-pump group). Contusion only (LC-only) and a sham saline instillation group after contusion were used as controls (LC-electroporation-sham). By using a BTX 830 electroporator, eight electrical pulses of 200 V/cm field strength were applied transthoracically. Mice were killed at 24 hours, 48 hours, and 72 hours after delivery. Bronchial alveolar lavage was recollected to measure albumin and cytokines by enzyme-linked immunosorbent assay. Pulmonary compliance was measured, and lungs were subject to histopathologic analysis. After the electroporation and delivery of genes coding for the α,β subunits of the Na+/K+-ATPase pump, there was a significant mitigation of acute lung injury as evidenced by reduction in bronchial alveolar lavage levels of albumin, improved pressure volume curves, and reduced inflammation seen on histology. Electroporation-mediated gene transfer of the subunits of the Na+/K+-ATPase pump enhanced recovery from acute inflammatory lung injury after LC.

  17. Sodium, potassium-atpases in algae and oomycetes.

    Science.gov (United States)

    Barrero-Gil, Javier; Garciadeblás, Blanca; Benito, Begoña

    2005-08-01

    We have investigated the presence of K(+)-transporting ATPases that belong to the phylogenetic group of animal Na(+),K(+)-ATPases in the Pythium aphanidermatum Stramenopile oomycete, the Porphyra yezoensis red alga, and the Udotea petiolata green alga, by molecular cloning and expression in heterologous systems. PCR amplification and search in EST databases allowed one gene to be identified in each species that could encode ATPases of this type. Phylogenetic analysis of the sequences of these ATPases revealed that they cluster with ATPases of animal origin, and that the algal ATPases are closer to animal ATPases than the oomycete ATPase is. The P. yezoensis and P. aphanidermatum ATPases were functionally expressed in Saccharomyces cerevisiae and Escherichia coli alkali cation transport mutants. The aforementioned cloning and complementary searches in silicio for H(+)- and Na(+),K(+)-ATPases revealed a great diversity of strategies for plasma membrane energization in eukaryotic cells different from typical animal, plant, and fungal cells.

  18. Evolution of plant P-type ATPases

    Directory of Open Access Journals (Sweden)

    Christian N.S. Pedersen

    2012-02-01

    Full Text Available Five organisms having completely sequenced genomes and belonging to all major branches of green plants (Viridiplantae were analyzed with respect to their content of P-type ATPases encoding genes. These were the chlorophytes Ostreococcus tauria and Chlamydomonas reinhardtii, and the streptophytes Physcomitrella patens (a moss, Selaginella moellendorffii (a primitive vascular plant, and Arabidopsis thaliana (a model flowering plant. Each organism contained sequences for all five subfamilies of P-type ATPases. Our analysis demonstrates when specific subgroups of P-type ATPases disappeared in the evolution of Angiosperms. Na/K-pump related P2C ATPases were lost with the evolution of streptophytes whereas Na+ or K+ pumping P2D ATPases and secretory pathway Ca2+-ATPases remained until mosses. An N-terminally located calmodulin binding domain in P2B ATPases can only be detected in pumps from Streptophytae, whereas, like in animals, a C-terminally localized calmodulin binding domain might be present in chlorophyte P2B Ca2+-ATPases. Chlorophyte genomes encode P3A ATPases resembling protist plasma membrane H+-ATPases and a C-terminal regulatory domain is missing. The complete inventory of P-type ATPases in the major branches of Viridiplantae is an important starting point for elucidating the evolution in plants of these important pumps.

  19. Resistência a mosca branca (Bemisia tabaci) em plantas transgênicas expressando siRNA do gene de uma v-ATPase

    OpenAIRE

    Ibrahim, Abdulrazak Baba

    2016-01-01

    RNA de interferência (RNAi) é um processo bioquímico potente e específico de silenciamento de genes, que ocorre em uma variedade de organismos como mamíferos, fungos e plantas. O mecanismo atua a nível pós-transcricional, e pode resultar na degradação ou não tradução de RNAs mensageiros (mRNA). Esse mecanismo foi utilizado com o objetivo de silenciar o gene v-ATPase da mosca branca (Bemisia tabaci), que é considerada uma importante peste da agricultura em regiões tropicais e sub-tropicais em ...

  20. The Plasma Membrane H+-ATPase from the Biotrophic Rust Fungus Uromyces fabae : molecular Characterization of the Gene (PMA1) and Functional Expression of the Enzyme in Yeast

    OpenAIRE

    Struck, Christine; Siebels, Claudia; Rommel, Oliver; Wernitz, Marcus; Hahn, Matthias

    1998-01-01

    To study the molecular basis of biotrophic nutrient uptake by plant parasitic rust fungi, the gene (Uf-PMA1) encoding the plasma membrane H+-ATPase from Uromyces fabae was isolated. Uf-PMA1 exists probably as a single gene. However, two nearly identical sequences were identified; the similarity apparently is due to two Uf-PMA1 alleles in the dikaryotic hyphae. Multiple Uf-PMA1 transcripts were observed during early rust development, and reduced amounts of a single Uf-PMA1 mRNA were observed i...

  1. Helicobacter pylori-induced posttranscriptional regulation of H-K-ATPase α-subunit gene expression by miRNA.

    Science.gov (United States)

    Zhang, Yong-Mei; Noto, Jennifer M; Hammond, Charles E; Barth, Jeremy L; Argraves, W Scott; Backert, Steffen; Peek, Richard M; Smolka, Adam J

    2014-04-01

    Acute Helicobacter pylori infection of gastric epithelial cells induces CagA oncoprotein- and peptidoglycan (SLT)-dependent mobilization of NF-κB p50 homodimers that bind to H-K-ATPase α-subunit (HKα) promoter and repress HKα gene transcription. This process may facilitate gastric H. pylori colonization by induction of transient hypochlorhydria. We hypothesized that H. pylori also regulates HKα expression posttranscriptionally by miRNA interaction with HKα mRNA. In silico analysis of the HKα 3' untranslated region (UTR) identified miR-1289 as a highly conserved putative HKα-regulatory miRNA. H. pylori infection of AGS cells transfected with HKα 3' UTR-Luc reporter construct repressed luciferase activity by 70%, whereas ΔcagA or Δslt H. pylori infections partially abrogated repression. Transfection of AGS cells expressing HKα 3' UTR-Luc construct with an oligoribonucleotide mimetic of miR-1289 induced maximal repression (54%) of UTR activity within 30 min; UTR activity was unchanged by nontargeting siRNA transfection. Gastric biopsies from patients infected with cagA(+) H. pylori showed a significant increase in miR-1289 expression compared with uninfected patients or those infected with cagA(-) H. pylori. Finally, miR-1289 expression was necessary and sufficient to attenuate biopsy HKα protein expression in the absence of infection. Taken together, these data indicate that miR-1289 is upregulated by H. pylori in a CagA- and SLT-dependent manner and targets HKα 3' UTR, affecting HKα mRNA translation. The sensitivity of HKα mRNA 3' UTR to binding of miR-1289 identifies a novel regulatory mechanism of gastric acid secretion and offers new insights into mechanisms underlying transient H. pylori-induced hypochlorhydria.

  2. Cloning and regulation of a stress-regulated Pennisetum glaucum vacuolar ATPase c gene and characterization of its promoter that is expressed in shoot hairs and floral organs.

    Science.gov (United States)

    Tyagi, Wricha; Rajagopal, Divya; Singla-Pareek, Sneh Lata; Reddy, Malireddy K; Sopory, Sudhir K

    2005-08-01

    We have cloned and characterized the cDNA, genomic clone and upstream promoter region of a vacuolar ATPase (V-ATPase) c subunit (PgVHA-c1) from Pennisetum glaucum. The deduced amino acid sequence shows 98-71% sequence identity with V-ATPase from rice and Arabidopsis, and is a highly hydrophobic protein with four transmembrane regions. PgVHA-c1-GFP fusion protein is expressed in BY2 cells on the endo-membranes surrounding vacuoles; however, PgVHA-c1 could not functionally complement V-ATPase-c deletion mutants of yeast. The sequence analysis of the genomic clone revealed the presence of two introns in the coding region, and the splice junctions followed the typical canonical GU-AG consensus sequence. The transcript analysis showed that the expression of PgVHA-c1 was stimulated more in response to salinity stress and very marginally in response to drought and low temperature stress. Exogenous application of abscisic acid, salicylic acid and calcium stimulated the transcript level in the absence of stress. We have cloned the 5'-flanking regions of PgVHA-c1 and mapped its transcript start site at 78 bp upstream of ATG. Transgenic tobacco with promoter::GUS constructs showed that the region -288/+78 was sufficient for GUS expression. The expression of the reporter gene even with the full-length promoter was limited to shoot hairs and to male and female reproductive organs. The dehydration-responsive element (DRE) and ABA-responsive element (ABRE) in the promoter did not show consensus flanking regions; however, gel mobility shift assays showed that Pennisetum has specific transacting factors that showed binding to the core DRE, ABRE and TCA elements.

  3. Early salt stress effects on the differential expression of vacuolar H(+)-ATPase genes in roots and leaves of Mesembryanthemum crystallinum.

    Science.gov (United States)

    Löw, R; Rockel, B; Kirsch, M; Ratajczak, R; Hörtensteiner, S; Martinoia, E; Lüttge, U; Rausch, T

    1996-01-01

    In Mesembryanthemum crystallinum, the salt stress-induced metabolic switch from C3 photosynthesis to Crassulacean acid metabolism is accompanied by major changes in gene expression. However, early effects of salt exposure (i.e. prior to Crassulacean acid metabolism induction) on genes coding for vacuolar transport functions have not yet been studied. Therefore, the expression of vacuolar H(+)-ATPase genes was analyzed in different organs of 4-week-old plants stressed with 400 mM NaCl for 3, 8, or 24 h. Partial cDNAs for the subunits A, B, and c were cloned and used as homologous probes for northern blot analysis. In control plants, the mRNA levels for the different subunits showed organ-specific differences. In fully expanded leaves, subunit c mRNA was very low but increased transiently during the light period. Plant organs also differed in their salt-stress response. In roots and young leaves, mRNA levels for all three subunits increased about 2-fold compared to control plants, whereas in fully expanded leaves only subunit c mRNA responded to salt. The results indicate that the expression of vacuolar H(+)-ATPase genes does not always involve a fixed stoichiometry of mRNAs for the different subunits and that the mRNA level for subunit c is particularly sensitive to developmental and environmental changes.

  4. Regulatory interplay of Cockayne syndrome B ATPase and stress-response gene ATF3 following genotoxic stress

    DEFF Research Database (Denmark)

    Kristensen, Hans-Ulrik Svejstrup; Epanchintsev, Alexey; Rauschendorf, Marc-Alexander

    2013-01-01

    Cockayne syndrome type B ATPase (CSB) belongs to the SwItch/Sucrose nonfermentable family. Its mutations are linked to Cockayne syndrome phenotypes and classically are thought to be caused by defects in transcription-coupled repair, a subtype of DNA repair. Here we show that after UV-C irradiation...

  5. Plant regeneration and Agrobacterium-mediated transformation of Vacuolar H+-ATPase c Subunit Gene in hybrid poplar populus davidiana Dode × P. bollena Lauche

    Directory of Open Access Journals (Sweden)

    Han Xue

    2017-01-01

    Full Text Available An efficient regeneration and transformation system was developed for hybrid poplar populus davidiana Dode × P. bollena Lauche. Several factors, such as a competent regeneration protocol, antibiotics, bacterial concentration, infection time have been shown to affect transformation efficiency. It is the first report describing an efficient protocol for Vacuolar H+-ATPase c Subunit Gene from salt tolerant plant Puccinellia chinampoensis, which transformed into populus. Stable transgene integration was confirmed by the expression of GFP fusion proteins in leaves, stem and root for populus.

  6. Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

    Energy Technology Data Exchange (ETDEWEB)

    Pestov, Nikolay B. [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997 (Russian Federation); Zhao, Hao [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Basrur, Venkatesha [Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

    2011-09-09

    Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

  7. Cloning of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) gene from white shrimp, Litopenaeus vannamei and its expression level analysis under salinity stress.

    Science.gov (United States)

    Wang, Yanhong; Luo, Peng; Zhang, Lvping; Hu, Chaoqu; Ren, Chunhua; Xia, Jianjun

    2013-11-01

    Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is an intracellular membrane bound enzyme that utilizes the free energy of ATP to transport Ca(2+) against a concentration gradient. In the present study, a new SERCA gene (LvSERCA) from white shrimp (Litopenaeus vannamei) was cloned using suppression subtractive hybridization and rapid amplification of cDNA ends. The full-length cDNA of LvSERCA contained an open reading frame of 3,009 bp coding for 1,002 amino acids with a calculated molecular weight of approximately 109.8 kDa. The identity analysis of the amino acid sequence of LvSERCA showed that it is highly conserved with 10 transmembrane α-helices, one P-domain, one A-domain and one N-domain. The phylogenetic analysis revealed that LvSERCA is similar to other Arthropoda SERCA proteins. The mRNA levels of LvSERCA under salinity stress (3 and 40 g L(-1)) were analyzed by reverse transcription PCR and quantitative real-time PCR. The results showed that LvSERCA was expressed in all tissues detected. LvSERCA mRNA levels were significantly higher under hyper-salinity than hypo-salinity. These results highlight that Ga(2+)-ATPase plays an essential role in adjustment salinity stress, which may be useful for selective breeding of L. vannamei.

  8. Transcription of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase type 3 gene, ATP2A3, is regulated by the calcineurin/NFAT pathway in endothelial cells.

    Science.gov (United States)

    Hadri, Lahouaria; Pavoine, Catherine; Lipskaia, Larissa; Yacoubi, Sabrina; Lompré, Anne-Marie

    2006-02-15

    Histamine, known to induce Ca2+ oscillations in endothelial cells, was used to alter Ca2+ cycling. Treatment of HUVEC (human umbilical-vein endothelial cell)-derived EA.hy926 cells with histamine for 1-3 days increased the levels of SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 3, but not of SERCA 2b, transcripts and proteins. Promoter-reporter gene assays demonstrated that this increase in expression was due to activation of SERCA 3 gene transcription. The effect of histamine was abolished by mepyramine, but not by cimetidine, indicating that the H1 receptor, but not the H2 receptor, was involved. The histamine-induced up-regulation of SERCA 3 was abolished by cyclosporin A and by VIVIT, a peptide that prevents calcineurin and NFAT (nuclear factor of activated T-cells) from interacting, indicating involvement of the calcineurin/NFAT pathway. Histamine also induced the nuclear translocation of NFAT. NFAT did not directly bind to the SERCA 3 promoter, but activated Ets-1 (E twenty-six-1), which drives the expression of the SERCA 3 gene. Finally, cells treated with histamine and loaded with fura 2 exhibited an improved capacity in eliminating high cytosolic Ca2+ concentrations, in accordance with an increase in activity of a low-affinity Ca2+-ATPase, like SERCA 3. Thus chronic treatment of endothelial cells with histamine up-regulates SERCA 3 transcription. The effect of histamine is mediated by the H1R (histamine 1 receptor) and involves activation of the calcineurin/NFAT pathway. By increasing the rate of Ca2+ sequestration, up-regulation of SERCA 3 counteracts the cytosolic increase in Ca2+ concentration.

  9. Slowed relaxation of diaphragm in septic rats is associated with reduced expression of sarco-endoplasmic reticulum CA2+ -ATPase genes SERCA1 and SERCA2.

    Science.gov (United States)

    Wu, Jin; Zhang, Jian You; Gong, Yuan; Li, Shi Tong

    2016-12-01

    The aim of this study was to study the effects of sepsis on diaphragm relaxation properties and the associated expression of sarco-endoplasmic reticulum Ca2+ -ATPase genes SERCA1 and SERCA2. Rats were randomized to undergo either sham surgery or cecal ligation and puncture (CLP). Diaphragm isometric relaxation parameters were measured after 24 h. The mRNA expression and protein content of SERCA1 and SERCA2 in diaphragm muscles were determined. Both diaphragm maximal twitch and tetanus relaxation rates were reduced. Twitch half-relaxation time was prolonged after normalization to half of peak twitch tension. The mRNA expression and protein content of SERCA1 and SERCA2 were decreased. Slowed relaxation of the diaphragm in septic rats was associated with reduced expression of SERCA1 and SERCA2. Muscle Nerve 54: 1108-1113, 2016. © 2016 Wiley Periodicals, Inc.

  10. Reduced expressions of calmodulin genes and protein and reduced ability of calmodulin to activate plasma membrane Ca(2+)-ATPase in the brain of protein undernourished rats: modulatory roles of selenium and zinc supplementation.

    Science.gov (United States)

    Adebayo, Olusegun L; Khera, Alka; Sandhir, Rajat; Adenuga, Gbenga A

    2016-03-01

    The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca(2+)-ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein-undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l(-1), respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca(2+)-ATPase (PMCA) activity, Ca(2+)/CaM activated EGM Ca(2+) ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca(2+)/CaM to activate EGM Ca(2+)-ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Arabidopsis plasma membrane H+-ATPase genes AHA2 and AHA7 have distinct and overlapping roles in the modulation of root tip H+ efflux in response to low-phosphorus stress.

    Science.gov (United States)

    Yuan, Wei; Zhang, Dongping; Song, Tao; Xu, Feiyun; Lin, Sheng; Xu, Weifeng; Li, Qianfeng; Zhu, Yiyong; Liang, Jiansheng; Zhang, Jianhua

    2017-03-01

    Phosphorus deficiency in soil is one of the major limiting factors for plant growth. Plasma membrane H+-ATPase (PM H+-ATPase) plays an important role in the plant response to low-phosphorus stress (LP). However, few details are known regarding the action of PM H+-ATPase in mediating root proton (H+) flux and root growth under LP. In this study, we investigated the involvement and function of different Arabidopsis PM H+-ATPase genes in root H+ flux in response to LP. First, we examined the expressions of all Arabidopsis PM H+-ATPase gene family members (AHA1-AHA11) under LP. Expression of AHA2 and AHA7 in roots was enhanced under this condition. When the two genes were deficient in their respective Arabidopsis mutant plants, root growth and responses of the mutants to LP were highly inhibited compared with the wild-type plant. AHA2-deficient plants exhibited reduced primary root elongation and lower H+ efflux in the root elongation zone. AHA7-deficient plants exhibited reduced root hair density and lower H+ efflux in the root hair zone. The modulation of H+ efflux by AHA2 or AHA7 was affected by the action of 14-3-3 proteins and/or auxin regulatory pathways in the context of root growth and response to LP. Our results suggest that under LP conditions, AHA2 acts mainly to modulate primary root elongation by mediating H+ efflux in the root elongation zone, whereas AHA7 plays an important role in root hair formation by mediating H+ efflux in the root hair zone. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  12. Analysis of tomato plasma membrane H(+)-ATPase gene family suggests a mycorrhiza-mediated regulatory mechanism conserved in diverse plant species.

    Science.gov (United States)

    Liu, Junli; Liu, Jianjian; Chen, Aiqun; Ji, Minjie; Chen, Jiadong; Yang, Xiaofeng; Gu, Mian; Qu, Hongye; Xu, Guohua

    2016-10-01

    In plants, the plasma membrane H(+)-ATPase (HA) is considered to play a crucial role in regulating plant growth and respoding to environment stresses. Multiple paralogous genes encoding different isozymes of HA have been identified and characterized in several model plants, while limited information of the HA gene family is available to date for tomato. Here, we describe the molecular and expression features of eight HA-encoding genes (SlHA1-8) from tomato. All these genes are interrupted by multiple introns with conserved positions. SlHA1, 2, and 4 were widely expressed in all tissues, while SlHA5, 6, and 7 were almost only expressed in flowers. SlHA8, the transcripts of which were barely detectable under normal or nutrient-/salt-stress growth conditions, was strongly activated in arbuscular mycorrhizal (AM) fungal-colonized roots. Extreme lack of SlHA8 expression in M161, a mutant defective to AM fungal colonization, provided genetic evidence towards the dependence of its expression on AM symbiosis. A 1521-bp SlHA8 promoter could direct the GUS reporter expression specifically in colonized cells of transgenic tobacco, soybean, and rice mycorrhizal roots. Promoter deletion assay revealed a 223-bp promoter fragment of SlHA8 containing a variant of AM-specific cis-element MYCS (vMYCS) sufficient to confer the AM-induced activity. Targeted deletion of this motif in the corresponding promoter region causes complete abolishment of GUS staining in mycorrhizal roots. Together, these results lend cogent evidence towards the evolutionary conservation of a potential regulatory mechanism mediating the activation of AM-responsive HA genes in diverse mycorrhizal plant species.

  13. Effects of anthropogenic sound on digging behavior, metabolism, Ca2+/Mg2+ ATPase activity, and metabolism-related gene expression of the bivalve Sinonovacula constricta

    Science.gov (United States)

    Peng, Chao; Zhao, Xinguo; Liu, Saixi; Shi, Wei; Han, Yu; Guo, Cheng; Jiang, Jingang; Wan, Haibo; Shen, Tiedong; Liu, Guangxu

    2016-01-01

    Anthropogenic sound has increased significantly in the past decade. However, only a few studies to date have investigated its effects on marine bivalves, with little known about the underlying physiological and molecular mechanisms. In the present study, the effects of different types, frequencies, and intensities of anthropogenic sounds on the digging behavior of razor clams (Sinonovacula constricta) were investigated. The results showed that variations in sound intensity induced deeper digging. Furthermore, anthropogenic sound exposure led to an alteration in the O:N ratios and the expression of ten metabolism-related genes from the glycolysis, fatty acid biosynthesis, tryptophan metabolism, and Tricarboxylic Acid Cycle (TCA cycle) pathways. Expression of all genes under investigation was induced upon exposure to anthropogenic sound at ~80 dB re 1 μPa and repressed at ~100 dB re 1 μPa sound. In addition, the activity of Ca2+/Mg2+-ATPase in the feet tissues, which is directly related to muscular contraction and subsequently to digging behavior, was also found to be affected by anthropogenic sound intensity. The findings suggest that sound may be perceived by bivalves as changes in the water particle motion and lead to the subsequent reactions detected in razor clams. PMID:27063002

  14. Regulation of ENA1 Na+-ATPase Gene Expression by the Ppz1 Protein Phosphatase Is Mediated by the Calcineurin Pathway

    Science.gov (United States)

    Ruiz, Amparo; Yenush, Lynne; Ariño, Joaquín

    2003-01-01

    Saccharomyces cerevisiae strains lacking the Ppz1 protein phosphatase are salt tolerant and display increased expression of the ENA1 Na+-ATPase gene, a major determinant for sodium extrusion, while cells devoid of the similar Ppz2 protein do not show these phenotypes. However, a ppz1 ppz2 mutant displays higher levels of ENA1 expression than the ppz1 strain. We show here that the increased activity of the ENA1 promoter in a ppz1 ppz2 mutant maps to two regions: one region located at −751 to −667, containing a calcineurin-dependent response element (CDRE), and one downstream region (−573 to −490) whose activity responds to intracellular alkalinization. In contrast, the increased ENA1 expression in a ppz1 mutant is mediated solely by an intact calcineurin/Crz1 signaling pathway, on the basis that (i) this effect maps to a single region that contains the CDRE and (ii) it is blocked by the calcineurin inhibitor FK506, as well as by deletion of the CNB1 or CRZ1 gene. The calcineurin dependence of the increased ENA1 expression of a ppz1 mutant would suggest that Ppz1 could negatively regulate calcineurin activity. In agreement with this notion, a ppz1 strain is calcium sensitive, and this mutation does not result in a decrease in the calcium hypertolerance of a cnb1 mutant. It has been shown that ENA1 can be induced by alkalinization of the medium and that a ppz1 ppz2 strain has a higher intracellular pH. However, we present several lines of evidence that show that the gene expression profile of a ppz1 mutant does not involve an alkalinization effect. In conclusion, we have identified a novel role for calcineurin, but not alkalinization, in the control of ENA1 expression in ppz1 mutants. PMID:14555476

  15. Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes.

    Science.gov (United States)

    Seth, M; Sumbilla, C; Mullen, S P; Lewis, D; Klein, M G; Hussain, A; Soboloff, J; Gill, D L; Inesi, G

    2004-11-23

    Transient elevations of cytosolic Ca2+ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) plays an important role in terminating Ca2+ transients by returning cytosolic Ca2+ to intracellular stores. Stored Ca2+ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca2+ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na+/Ca2+ exchanger, indicating that intracellular store deficiency was compensated for by Ca2+ fluxes through the plasma membrane. In fact, SERCA silencing was followed by increased transcription of Na+/Ca2+ exchanger, TRPC4, TRPC5, and related transcriptional factors, such as stimulating protein 1, myocyte enhancer factor 2, and nuclear factor of activated cells 4, through activation of calcineurin. This finding demonstrates that the observed compensation occurs through transcriptional crosstalk and the remodeling of Ca2+ signaling pathways. The wide significance of this regulatory mechanism is related to its general involvement in Ca2+ signaling dynamics and in cardiac development and hypertrophy.

  16. Effects of cortisol and salinity acclimation on Na+/K+/2Cl–- cotransporter gene expression and Na+, K+-ATPase activity in the gill of Persian sturgeon, Acipenser persicus, fry

    Directory of Open Access Journals (Sweden)

    Saber Khodabandeh

    2009-10-01

    Full Text Available Na+, K+-ATPase activity and Na+/K+/2Cl–- cotransporter (NKCC gene expression in the gills of Persian sturgeon, Acipenser persicus, fry (2-3 g, 3.30-8.12 cm total body length in freshwater (control group, diluted Caspian Sea water (5 ppt and after treatment with cortisol in freshwater were studied. Na+, K+-ATPase activity was lower in the 5 ppt-acclimated fish (1.07±0.05 _mol Pi/mg protein/h than in the control fish (1.19±0.05 μmol Pi/mg protein/h but this difference was not significant. nKCC gene expression in the 5 ppt-acclimated fish (1.6±0.07 was significantly higher than in the control fish (0.8±0.00. In the cortisol treated fish, Na+, K+-ATPase activity (1.91±0.05 μmol Pi/mg protein/h and NKCC gene expression (3.2±0.1 were significantly higher than in the control group. our results show that Persian sturgeon fry (2-3 g can tolerate 5 ppt salinity by changing their enzymatic content and activity, and that exogenous cortisol application can increase the osmoregulatory capacity of fry before release into brackish water and can reduce their mortality.

  17. P4-ATPases

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Theorin, Lisa; Palmgren, Michael Broberg

    2014-01-01

    ) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4......Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Among these ATP-driven transporters, the P4 subfamily of P-type ATPases (P4-ATPases...

  18. Induction of cell differentiation activates transcription of the Sarco/Endoplasmic Reticulum calcium-ATPase 3 gene (ATP2A3) in gastric and colon cancer cells.

    Science.gov (United States)

    Flores-Peredo, Lucía; Rodríguez, Gabriela; Zarain-Herzberg, Angel

    2017-02-01

    The Sarco/Endoplasmic Reticulum Ca2+ -ATPases (SERCAs), pump Ca2+ into the endoplasmic reticulum lumen modulating cytosolic Ca2+ concentrations to regulate various cellular processes including cell growth. Previous studies have reported a downregulation of SERCA3 protein expression in gastric and colon cancer cell lines and showed that in vitro cell differentiation increases its expression. However, little is known about the transcriptional mechanisms and transcription factors that regulate SERCA3 expression in epithelial cancer cells. In this work, we demonstrate that SERCA3 mRNA is upregulated up to 45-fold in two epithelial cancer cell lines, KATO-III and Caco-2, induced to differentiate with histone deacetylase inhibitors (HDACi) and by cell confluence, respectively. To evaluate the transcriptional elements responding to the differentiation stimuli, we cloned the human ATP2A3 promoter, generated deletion constructs and transfected them into KATO-III cells. Basal and differentiation responsive DNA elements were located by functional analysis within the first -135 bp of the promoter region. Using site-directed mutagenesis and DNA-protein binding assays we found that Sp1, Sp3, and Klf-4 transcription factors bind to ATP2A3 proximal promoter elements and regulate basal gene expression. We showed that these factors participated in the increase of ATP2A3 expression during cancer cell differentiation. This study provides evidence for the first time that Sp1, Sp3, and Klf-4 transcriptionally modulate the expression of SERCA3 during induction of epithelial cancer cell differentiation. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Studies of Ca2+ ATPase (SERCA) inhibition.

    Science.gov (United States)

    Inesi, Giuseppe; Hua, Suming; Xu, Cheng; Ma, Hailun; Seth, Malini; Prasad, Anand M; Sumbilla, Carlota

    2005-12-01

    The Ca(2+) transport ATPase of intracellular membranes (SERCA) can be inhibited by a series of chemical compounds such as Thapsigargin (TG), 2,5-di(tert-butyl)hydroquinone (DBHQ) and 1,3-dibromo-2,4,6-tris (methyl-isothio-uronium) benzene (Br(2)-TITU). These compounds have specific binding sites in the ATPase protein, and different mechanisms of inhibition. On the other hand, SERCA gene silencing offers a convenient and specific method for suppression of SERCA activity in cells. The physiological and pharmacological implications of SERCA inhibition are discussed.

  20. Citrus PH5-like H+-ATPase genes: identification and transcript analysis to investigate their possible relationship with citrate accumulation in fruits

    Directory of Open Access Journals (Sweden)

    Cai-Yun eShi

    2015-03-01

    Full Text Available PH5 is a petunia gene that encodes a plasma membrane H+-ATPase and determines the vacuolar pH. The citrate content of fruit cell vacuoles influences citrus organoleptic qualities. Although citrus could have PH5-like homologs that are involved in citrate accumulation, the details are still unknown. In this study, extensive data-mining with the PH5 sequence and PCR amplification confirmed that there are at least eight PH5-like genes (CsPH1-8 in the citrus genome. CsPHs have a molecular mass of approximately 100 kDa, and they have high similarity to PhPH5, AtAHA10 or AtAHA2 (from 64.6% to 80.9%. They contain 13-21 exons and 12-20 introns and were evenly distributed into four subgroups of the P3A-subfamily (CsPH1, CsPH2, and CsPH3 in Group I, CsPH4 and CsPH5 in Group II, CsPH6 in Group IV, and CsPH7 and CsPH8 in Group III together with PhPH5. A transcript analysis showed that CsPH1, 3, and 4 were predominantly expressed in mature leaves, whereas CsPH2 and 7 were predominantly expressed in roots, CsPH5 and 6 were predominantly expressed in flowers, and CsPH8 was predominantly expressed in fruit juice sacs. Moreover, the CsPH transcript profiles differed between orange and pummelo, as well as between high-acid and low-acid cultivars. The low-acid orange ‘Honganliu’ exhibits low transcript levels of CsPH3, CsPH4, CsPH5, and CsPH8, whereas the acid-free pummelo has only a low transcript level of CsPH8. In addition, ABA injection increased the citrate content significantly, which was accompanied by the obvious induction of CsPH2, 6, 7, and 8 transcript levels. Taken together, we suggest that CsPH8 seems likely to regulate citrate accumulation in the citrus fruit vacuole.

  1. The Whitish Inner Mantle of the Giant Clam, Tridacna squamosa, Expresses an Apical Plasma Membrane Ca2+-ATPase (PMCA Which Displays Light-Dependent Gene and Protein Expressions

    Directory of Open Access Journals (Sweden)

    Yuen K. Ip

    2017-10-01

    Full Text Available Giant clams live in symbiosis with extracellular zooxanthellae and display high rates of growth and shell formation (calcification in light. Light-enhanced calcification requires an increase in the supply of Ca2+ to, and simultaneously an augmented removal of H+ from, the extrapallial fluid where shell formation occurs. We have obtained the complete coding cDNA sequence of Plasma Membrane Ca2+-ATPase (PMCA from the thin and whitish inner mantle, which is in touch with the extrapallial fluid, of the giant clam Tridacna squamosa. The deduced PMCA sequence consisted of an apical targeting element. Immunofluorescence microscopy confirmed that PMCA had an apical localization in the shell-facing epithelium of the inner mantle, whereby it can actively secrete Ca2+ in exchange for H+. More importantly, the apical PMCA-immunofluorescence of the shell-facing epithelium of the inner mantle increased significantly after 12 h of exposure to light. The transcript and protein levels of PMCA/PMCA also increased significantly in the inner mantle after 6 or 12 h of light exposure. These results offer insights into a light-dependable mechanism of shell formation in T. squamosa and a novel explanation of light-enhanced calcification in general. As the inner mantle normally lacks light sensitive pigments, our results support a previous proposition that symbiotic zooxanthellae, particularly those in the colorful and extensible outer mantle, may act as light-sensing elements for the host clam.

  2. The Whitish Inner Mantle of the Giant Clam, Tridacna squamosa, Expresses an Apical Plasma Membrane Ca2+-ATPase (PMCA) Which Displays Light-Dependent Gene and Protein Expressions.

    Science.gov (United States)

    Ip, Yuen K; Hiong, Kum C; Goh, Enan J K; Boo, Mel V; Choo, Celine Y L; Ching, Biyun; Wong, Wai P; Chew, Shit F

    2017-01-01

    Giant clams live in symbiosis with extracellular zooxanthellae and display high rates of growth and shell formation (calcification) in light. Light-enhanced calcification requires an increase in the supply of Ca2+ to, and simultaneously an augmented removal of H+ from, the extrapallial fluid where shell formation occurs. We have obtained the complete coding cDNA sequence of Plasma Membrane Ca2+-ATPase (PMCA) from the thin and whitish inner mantle, which is in touch with the extrapallial fluid, of the giant clam Tridacna squamosa. The deduced PMCA sequence consisted of an apical targeting element. Immunofluorescence microscopy confirmed that PMCA had an apical localization in the shell-facing epithelium of the inner mantle, whereby it can actively secrete Ca2+ in exchange for H+. More importantly, the apical PMCA-immunofluorescence of the shell-facing epithelium of the inner mantle increased significantly after 12 h of exposure to light. The transcript and protein levels of PMCA/PMCA also increased significantly in the inner mantle after 6 or 12 h of light exposure. These results offer insights into a light-dependable mechanism of shell formation in T. squamosa and a novel explanation of light-enhanced calcification in general. As the inner mantle normally lacks light sensitive pigments, our results support a previous proposition that symbiotic zooxanthellae, particularly those in the colorful and extensible outer mantle, may act as light-sensing elements for the host clam.

  3. Differential regulation of the fiber type-specific gene expression of the sarcoplasmic reticulum calcium-ATPase isoforms induced by exercise training.

    Science.gov (United States)

    Morissette, Marc P; Susser, Shanel E; Stammers, Andrew N; O'Hara, Kimberley A; Gardiner, Phillip F; Sheppard, Patricia; Moffatt, Teri L; Duhamel, Todd A

    2014-09-01

    The regulatory role of adenosine monophosphate-activated protein kinase (AMPK)-α2 on sarcoplasmic reticulum calcium-ATPase (SERCA) 1a and SERCA2a in different skeletal muscle fiber types has yet to be elucidated. Sedentary (Sed) or exercise-trained (Ex) wild-type (WT) and AMPKα2-kinase dead (KD) transgenic mice, which overexpress a mutated and inactivated AMPKα2 subunit, were utilized to characterize how genotype or exercise training influenced the regulation of SERCA isoforms in gastrocnemius. As expected, both Sed and Ex KD mice had >40% lower AMPK phosphorylation and 30% lower SERCA1a protein than WT mice (P SERCA2a protein was not different among KD and WT mice. Exercise increased SERCA1a and SERCA2a protein content among WT and KD mice, compared with their Sed counterparts. Maximal SERCA activity was lower in KD mice, compared with WT. Total phospholamban protein was higher in KD mice than in WT and lower in Ex compared with Sed mice. Exercise training increased phospholamban Ser(16) phosphorylation in WT mice. Laser capture microdissection and quantitative PCR indicated that SERCA1a mRNA expression among type I fibers was not altered by genotype or exercise, but SERCA2a mRNA was increased 30-fold in WT+Ex, compared with WT+Sed. In contrast, the exercise-stimulated increase for SERCA2a mRNA was blunted in KD mice. Exercise upregulated SERCA1a and SERCA2a mRNA among type II fibers, but was not altered by genotype. Collectively, these data suggest that exercise differentially influences SERCA isoform expression in type I and type II fibers. Additionally, AMPKα2 influences the regulation of SERCA2a mRNA in type I skeletal muscle fibers following exercise training. Copyright © 2014 the American Physiological Society.

  4. Chronic inhibition of endoplasmic reticulum calcium-release channels and calcium-ATPase lengthens the period of hepatic clock gene Per1

    Directory of Open Access Journals (Sweden)

    Díaz-Muñoz Mauricio

    2011-07-01

    Full Text Available Abstract Background The role played by calcium as a regulator of circadian rhythms is not well understood. The effect of the pharmacological inhibition of the ryanodine receptor (RyR, inositol 1,4,5-trisphosphate receptor (IP3R, and endoplasmic-reticulum Ca2+-ATPase (SERCA, as well as the intracellular Ca2+-chelator BAPTA-AM was explored on the 24-h rhythmicity of the liver-clock protein PER1 in an experimental model of circadian synchronization by light and restricted-feeding schedules. Methods Liver explants from Period1-luciferase (Per1-luc transgenic rats with either free food access or with a restricted meal schedule were treated for several days with drugs to inhibit the activity of IP3Rs (2-APB, RyRs (ryanodine, or SERCA (thapsigargin as well as to suppress intracellular calcium fluctuations (BAPTA-AM. The period of Per1-luc expression was measured during and after drug administration. Results Liver explants from rats fed ad libitum showed a lengthened period in response to all the drugs tested. The pharmacological treatments of the explants from meal-entrained rats induced the same pattern, with the exception of the ryanodine treatment which, unexpectedly, did not modify the Per1-luc period. All effects associated with drug application were reversed after washout, indicating that none of the pharmacological treatments was toxic to the liver cultures. Conclusions Our data suggest that Ca2+ mobilized from internal deposits modulates the molecular circadian clock in the liver of rats entrained by light and by restricted meal access.

  5. Regulation of the synthesis and assembly of the plant vacuolar H sup + -ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Taiz, L.

    1992-01-01

    During the past three years we have focused on four main areas: the characterization of the 5{prime}-upstream sequence of the gene for the V-ATPase 70 kDa (A) subunit gene, the generation of V-ATPase-deficient mutants using antisense constructs of the A subunit cDNA, analysis of V-ATPase ultrastructure by negative staining and the characterization of organelle-specific isoforms of the A subunit of carrot. In addition we have extended our studies on the cellular distribution of the V-ATPase and we have continued our investigation of the evolution of the V-ATPases by characterizing the A and B subunits of two species of the archaebacterium, Methanococcus.

  6. Identification of calcium-transporting ATPases of Entamoeba histolytica and cellular localization of the putative SERCA.

    Science.gov (United States)

    Martinez-Higuera, Aarón; Salas-Casas, Andrés; Calixto-Gálvez, Mercedes; Chávez-Munguía, Bibiana; Pérez-Ishiwara, D Guillermo; Ximénez, Cecilia; Rodríguez, Mario A

    2013-09-01

    Calcium has an important role on signaling of different cellular processes in the protozoa parasite Entamoeba histolytica, including development and pathogenesis. However, the systems that control calcium responses in this parasite are incompletely understood. Calcium-ATPases (Ca(2+)-ATPases) are proteins that play an important role in calcium homeostasis by catalyzing the active efflux of this ion from cytoplasm and are essential to the correct functioning of the cell machinery. Here, we reported the identification of five E. histolytica genes encoding putative Ca(2+)-ATPases, three related to PMCA, and two related to organellar ATPases. RT-PCR assays showed that all those genes are expressed in trophozoites and specific antibodies against the SERCA-like member located this protein in a continuous cytoplasmic network, supporting the hypothesis that it corresponds to the Ca(2+)-ATPase responsible to sequester calcium in the endoplasmic reticulum of this parasite. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. SMC6 is an essential gene in mice, but a hypomorphic mutant in the ATPase domain has a mild phenotype with a range of subtle abnormalities.

    Science.gov (United States)

    Ju, Limei; Wing, Jonathan; Taylor, Elaine; Brandt, Renata; Slijepcevic, Predrag; Horsch, Marion; Rathkolb, Birgit; Rácz, Ildikó; Becker, Lore; Hans, Wolfgang; Adler, Thure; Beckers, Johannes; Rozman, Jan; Klingenspor, Martin; Wolf, Eckhard; Zimmer, Andreas; Klopstock, Thomas; Busch, Dirk H; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; van der Horst, Gilbertus; Lehmann, Alan R

    2013-05-01

    Smc5-6 is a highly conserved protein complex related to cohesin and condensin involved in the structural maintenance of chromosomes. In yeasts the Smc5-6 complex is essential for proliferation and is involved in DNA repair and homologous recombination. siRNA depletion of genes involved in the Smc5-6 complex in cultured mammalian cells results in sensitivity to some DNA damaging agents. In order to gain further insight into its role in mammals we have generated mice mutated in the Smc6 gene. A complete knockout resulted in early embryonic lethality, demonstrating that this gene is essential in mammals. However, mutation of the highly conserved serine-994 to alanine in the ATP hydrolysis motif in the SMC6 C-terminal domain, resulted in mice with a surprisingly mild phenotype. With the neo gene selection marker in the intron following the mutation, resulting in reduced expression of the SMC6 gene, the mice were reduced in size, but fertile and had normal lifespans. When the neo gene was removed, the mice had normal size, but detailed phenotypic analysis revealed minor abnormalities in glucose tolerance, haematopoiesis, nociception and global gene expression patterns. Embryonic fibroblasts derived from the ser994 mutant mice were not sensitive to killing by a range of DNA damaging agents, but they were sensitive to the induction of sister chromatid exchanges induced by ultraviolet light or mitomycin C. They also accumulated more oxidative damage than wild-type cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Retinoschisin is linked to retinal Na/K-ATPase signaling and localization.

    Science.gov (United States)

    Plössl, Karolina; Royer, Melanie; Bernklau, Sarah; Tavraz, Neslihan N; Friedrich, Thomas; Wild, Jens; Weber, Bernhard H F; Friedrich, Ulrike

    2017-08-01

    Mutations in the RS1 gene cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy. We recently showed that retinoschisin, the protein encoded by RS1 , regulates ERK signaling and apoptosis in retinal cells. In this study, we explored an influence of retinoschisin on the functionality of the Na/K-ATPase, its interaction partner at retinal plasma membranes. We show that retinoschisin binding requires the β2-subunit of the Na/K-ATPase, whereas the α-subunit is exchangeable. Our investigations revealed no effect of retinoschisin on Na/K-ATPase-mediated ATP hydrolysis and ion transport. However, we identified an influence of retinoschisin on Na/K-ATPase-regulated signaling cascades and Na/K-ATPase localization. In addition to the known ERK deactivation, retinoschisin treatment of retinoschisin-deficient ( Rs1h -/Y ) murine retinal explants decreased activation of Src, an initial transmitter in Na/K-ATPase signal transduction, and of Ca 2+ signaling marker Camk2. Immunohistochemistry on murine retinae revealed an overlap of the retinoschisin-Na/K-ATPase complex with proteins involved in Na/K-ATPase signaling, such as caveolin, phospholipase C, Src, and the IP3 receptor. Finally, retinoschisin treatment altered Na/K-ATPase localization in photoreceptors of Rs1h -/Y retinae. Taken together, our results suggest a regulatory effect of retinoschisin on Na/K-ATPase signaling and localization, whereas Na/K-ATPase-dysregulation caused by retinoschisin deficiency could represent an initial step in XLRS pathogenesis. © 2017 Plössl et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  9. TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

    National Research Council Canada - National Science Library

    Appelhagen, I; Nordholt, N; Seidel, T; Spelt, K; Koes, R; Quattrochio, F; Sagasser, M; Weisshaar, B

    2015-01-01

    ...) is caused by disruption of the gene encoding the P3A -ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants...

  10. Identification of the F1-ATPase at the cell surface of colonic epithelial cells: role in mediating cell proliferation.

    Science.gov (United States)

    Kowalski-Chauvel, Aline; Najib, Souad; Tikhonova, Irina G; Huc, Laurence; Lopez, Fredéric; Martinez, Laurent O; Cohen-Jonathan-Moyal, Elizabeth; Ferrand, Audrey; Seva, Catherine

    2012-11-30

    F1 domain of F(1)F(o)-ATPase was initially believed to be strictly expressed in the mitochondrial membrane. Interestingly, recent reports have shown that the F1 complex can serve as a cell surface receptor for apparently unrelated ligands. Here we show for the first time the presence of the F(1)-ATPase at the cell surface of normal or cancerous colonic epithelial cells. Using surface plasmon resonance technology and mass spectrometry, we identified a peptide hormone product of the gastrin gene (glycine-extended gastrin (G-gly)) as a new ligand for the F(1)-ATPase. By molecular modeling, we identified the motif in the peptide sequence (E(E/D)XY), that directly interacts with the F(1)-ATPase and the amino acids in the F(1)-ATPase that bind this motif. Replacement of the Glu-9 residue by an alanine in the E(E/D)XY motif resulted in a strong decrease of G-gly binding to the F(1)-ATPase and the loss of its biological activity. In addition we demonstrated that F(1)-ATPase mediates the growth effects of the peptide. Indeed, blocking F(1)-ATPase activity decreases G-gly-induced cell growth. The mechanism likely involves ADP production by the membrane F(1)-ATPase, which is induced by G-gly. These results suggest an important contribution of cell surface F(1)-ATPase in the pro-proliferative action of this gastrointestinal peptide.

  11. Gene delivery of l-caldesmon protects cytoskeletal cell membrane integrity against adenovirus infection independently of myosin ATPase and actin assembly.

    Science.gov (United States)

    Haxhinasto, Kari; Kamath, Anant; Blackwell, Ken; Bodmer, James; Van Heukelom, Jon; English, Anthony; Bai, Er-Wei; Moy, Alan B

    2004-10-01

    The cytoskeleton is critical to the viral life cycle. Agents like cytochalasin inhibit viral infections but cannot be used for antiviral therapy because of their toxicity. We report the efficacy, safety, and mechanisms by which gene delivery of human wild-type low-molecular-weight caldesmon (l-CaD) protects cell membrane integrity from adenovirus infection in a DF-1 cell line, an immortalized avian fibroblast that is null for l-CaD. Transfection with an adenovirus (Ad)-controlled construct mediated a dose-dependent decline in transcellular resistance. In accordance with a computational model of cytoskeletal membrane properties, Ad disturbed cell-cell and cell-matrix adhesion and membrane capacitance. Transfection with the Ad-l-CaD construct attenuated adenovirus-mediated loss in transcellular resistance. Quantitation of vinculin-stained plaques revealed an increase in total focal contact mass in monolayers transfected with the Ad-l-CaD construct. Expression of l-CaD protected transcellular resistance through primary effects on membrane capacitance and independently of actin solubility and effects on pre-stress, as measured by the decline in isometric tension in response to cytochalasin D. Expression of l-CaD exhibited less Trypan blue cell toxicity than cytochalasin, and, unlike cytochalasin, it did not interfere with wound closure or adversely effect transcellular resistance. These findings demonstrate the gene delivery of wild-type human l-CaD as a potentially efficacious and safe agent that inhibits some of the cytopathic effects of adenovirus.

  12. α3Na+/K+-ATPase deficiency causes brain ventricle dilation and abrupt embryonic motility in zebrafish

    DEFF Research Database (Denmark)

    Doganli, Canan; Beck, Hans Christian; Ribera, Angeles B

    2013-01-01

    Na+/K+-ATPases are transmembrane ion pumps that maintain ion gradients across the basolateral plasma membrane in all animal cells to facilitate essential biological functions. Mutations in the Na+/K+-ATPase α3 subunit gene (ATP1A3) cause rapid-onset dystonia-parkinsonism, a rare movement disorder...

  13. Silicon mitigates heavy metal stress by regulating P-type heavy metal ATPases, Oryza sativa low silicon genes, and endogenous phytohormones.

    Science.gov (United States)

    Kim, Yoon-Ha; Khan, Abdul Latif; Kim, Duk-Hwan; Lee, Seung-Yeol; Kim, Kyung-Min; Waqas, Muhammad; Jung, Hee-Young; Shin, Jae-Ho; Kim, Jong-Guk; Lee, In-Jung

    2014-01-09

    Silicon (Si) application has been known to enhance the tolerance of plants against abiotic stresses. However, the protective mechanism of Si under heavy metals contamination is poorly understood. The aim of this study was to assess the role of Si in counteracting toxicity due to cadmium (Cd) and copper (Cu) in rice plants (Oryza sativa). Si significantly improved the growth and biomass of rice plants and reduced the toxic effects of Cd/Cu after different stress periods. Si treatment ameliorated root function and structure compared with non-treated rice plants, which suffered severe root damage. In the presence of Si, the Cd/Cu concentration was significantly lower in rice plants, and there was also a reduction in lipid peroxidation and fatty acid desaturation in plant tissues. The reduced uptake of metals in the roots modulated the signaling of phytohormones involved in responses to stress and host defense, such as abscisic acid, jasmonic acid, and salicylic acid. Furthermore, the low concentration of metals significantly down regulated the mRNA expression of enzymes encoding heavy metal transporters (OsHMA2 and OsHMA3) in Si-metal-treated rice plants. Genes responsible for Si transport (OsLSi1 and OsLSi2), showed a significant up-regulation of mRNA expression with Si treatment in rice plants. The present study supports the active role of Si in the regulation of stresses from heavy metal exposure through changes in root morphology.

  14. Characterization of Bovine Brain ATPase

    Science.gov (United States)

    1988-07-01

    with a complex structure (presence of galactose, vannose and sialic acid ). This structure raises the possibility of immobilizing via immobilized...SYNAPIIC The (Ca2+ + M9*2)- dependent ATPase-Calcitu Channel Complex is found at the synaptic termini of neurons ( 1). With the exception of Mojave toxin...for 60% inhibition. We examined the effects of various phospholipids on catalytic activity (Table IX). With the exception of phosphatidic acid , all

  15. Polymorphism of PfATPase in Niger: detection of three new point mutations

    Directory of Open Access Journals (Sweden)

    Adam Hassane

    2009-02-01

    Full Text Available Abstract Background Plasmodium falciparum resistance to drugs remains a major public health issue in Niger. The therapeutic failure index for chloroquine and sulphadoxine-pyrimethamine are, respectively 20% and 21.9%. In December 2005, the National Malaria Control Programme promoted the use of artemisinin combination therapy (ACT as first-line treatment of the uncomplicated malaria cases. Recently, studies have shown a relationship between the SERCA PfATPase6 gene and artemisinin efficacy, and pointed it out as a potential molecular marker for resistance. The goal of this work was to describe the baseline polymorphism of PfATPase6 gene in Niger, at a time when the national implementation of the ACT policy had just begun. Materials and methods The DNA polymorphism of the PfATPase6 gene of 87 P. falciparum samples from Niger was analysed by sequencing. The links between the mutation occurrence and environment and human host factors were tested by bivariate analysis. Results The P. falciparum PfATPase6 gene presented polymorphisms at codons 537, 561, 569, 630, 639, 716 levels. All the mutations found were rare, except the PfATPaseN569K found in 17.2% of samples. No associated factor has been observed. Conclusion The P. falciparum PfATPase gene is polymorphic at the 569 codon. As ACT is getting more and more used, the PfATPase6 gene polymorphism needs to be monitored in association with phenotypic – in vivo and/or in vitro – drug efficacy tests.

  16. Immunocytolocalization of Plasma Membrane H-ATPase.

    Science.gov (United States)

    Parets-Soler, A; Pardo, J M; Serrano, R

    1990-08-01

    The localization of plasma membrane H(+)-ATPase has been studied at the optical microscope level utilizing frozen and paraffin sections of Avena sativa and Pisum sativum, specific anti-ATPase polyclonal antibody, and second antibody coupled to alkaline phosphatase. In leaves and stems the ATPase is concentrated at the phloem, supporting the notion that it generates the driving force for phloem loading. In roots the ATPase is concentrated at both the periphery (rootcap and epidermis) and at the central cylinder, including endodermis and vascular cells. This supports a ;two-pump' mechanism for ion absorption, involving active uptake at the epidermis, symplast transport across the cortex, and active efflux at the xylem. The low ATPase content of root meristem and elongation zone may explain the observed transorgan H(+) currents, which leave nongrowing parts and enter growing tips.

  17. The basidiomycete Ustilago maydis has two plasma membrane H⁺-ATPases related to fungi and plants.

    Science.gov (United States)

    Robles-Martínez, Leobarda; Pardo, Juan Pablo; Miranda, Manuel; Mendez, Tavis L; Matus-Ortega, Macario Genaro; Mendoza-Hernández, Guillermo; Guerra-Sánchez, Guadalupe

    2013-10-01

    The fungal and plant plasma membrane H⁺-ATPases play critical roles in the physiology of yeast, plant and protozoa cells. We identified two genes encoding two plasma membrane H⁺-ATPases in the basidiomycete Ustilago maydis, one protein with higher identity to fungal (um02581) and the other to plant (um01205) H⁺-ATPases. Proton pumping activity was 5-fold higher when cells were grown in minimal medium with ethanol compared to cells cultured in rich YPD medium, but total vanadate-sensitive ATPase activity was the same in both conditions. In contrast, the activity in cells cultured in minimal medium with glucose was 2-fold higher than in YPD or ethanol, implicating mechanisms for the regulation of the plasma membrane ATPase activity in U. maydis. Analysis of gene expression of the H⁺-ATPases from cells grown under different conditions, showed that the transcript expression of um01205 (plant-type) was higher than that of um02581 (fungal-type). The translation of the two proteins was confirmed by mass spectrometry analysis. Unlike baker's yeast and plant H⁺-ATPases, where the activity is increased by a short incubation with glucose or sucrose, respectively, U. maydis H⁺-ATPase activity did not change in response to these sugars. Sequence analysis of the two U. maydis H⁺-ATPases revealed the lack of canonical threonine and serine residues which are targets of protein kinases in Saccharomyces cerevisiae and Arabidopsis thaliana plasma membrane H⁺-ATPases, suggesting that phosphorylation of the U. maydis enzymes occurs at different amino acid residues.

  18. Oryza sativa H+-ATPase (OSA) is Involved in the Regulation of Dumbbell-Shaped Guard Cells of Rice.

    Science.gov (United States)

    Toda, Yosuke; Wang, Yin; Takahashi, Akira; Kawai, Yuya; Tada, Yasuomi; Yamaji, Naoki; Feng Ma, Jian; Ashikari, Motoyuki; Kinoshita, Toshinori

    2016-06-01

    The stomatal apparatus consists of a pair of guard cells and regulates gas exchange between the leaf and atmosphere. In guard cells, blue light (BL) activates H(+)-ATPase in the plasma membrane through the phosphorylation of its penultimate threonine, mediating stomatal opening. Although this regulation is thought to be widely adopted among kidney-shaped guard cells in dicots, the molecular basis underlying that of dumbbell-shaped guard cells in monocots remains unclear. Here, we show that H(+)-ATPases are involved in the regulation of dumbbell-shaped guard cells. Stomatal opening of rice was promoted by the H(+)-ATPase activator fusicoccin and by BL, and the latter was suppressed by the H(+)-ATPase inhibitor vanadate. Using H(+)-ATPase antibodies, we showed the presence of phosphoregulation of the penultimate threonine in Oryza sativa H(+)-ATPases (OSAs) and localization of OSAs in the plasma membrane of guard cells. Interestingly, we identified one H(+)-ATPase isoform, OSA7, that is preferentially expressed among the OSA genes in guard cells, and found that loss of function of OSA7 resulted in partial insensitivity to BL. We conclude that H(+)-ATPase is involved in BL-induced stomatal opening of dumbbell-shaped guard cells in monocotyledon species. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  19. Arbuscular mycorrhizae improve low temperature tolerance in cucumber via alterations in H2O2 accumulation and ATPase activity.

    Science.gov (United States)

    Liu, Airong; Chen, Shuangchen; Chang, Rui; Liu, Dilin; Chen, Haoran; Ahammed, Golam Jalal; Lin, Xiaomin; He, Chaoxing

    2014-11-01

    The combined effects of arbuscular mycorrhizal fungi (AMF) and low temperature (LT) on cucumber plants were investigated with respect to biomass production, H2O2 accumulation, NADPH oxidase, ATPase activity and related gene expression. Mycorrhizal colonization ratio was gradually increased after AMF-inoculation. However, LT significantly decreased mycorrhizal colonization ability and mycorrhizal dependency. Regardless of temperature, the total fresh and dry mass, and root activity of AMF-inoculated plants were significantly higher than that of the non-AMF control. The H2O2 accumulation in AMF-inoculated roots was decreased by 42.44% compared with the control under LT. H2O2 predominantly accumulated on the cell walls of apoplast but was hardly detectable in the cytosol or organelles of roots. Again, NADPH oxidase activity involved in H2O2 production was significantly reduced by AMF inoculation under LT. AMF-inoculation remarkably increased the activities of P-type H(+)-ATPase, P-Ca(2+)-ATPase, V-type H(+)-ATPase, total ATPase activity, ATP concentration and plasma membrane protein content in the roots under LT. Additionally, ATP concentration and expression of plasma membrane ATPase genes were increased by AMF-inoculation. These results indicate that NADPH oxidase and ATPase might play an important role in AMF-mediated tolerance to chilling stress, thereby maintaining a lower H2O2 accumulation in the roots of cucumber.

  20. [ATPase and phosphatase activity of drone brood].

    Science.gov (United States)

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae.

  1. Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state

    Science.gov (United States)

    Morito, Daisuke; Nishikawa, Kouki; Hoseki, Jun; Kitamura, Akira; Kotani, Yuri; Kiso, Kazumi; Kinjo, Masataka; Fujiyoshi, Yoshinori; Nagata, Kazuhiro

    2014-03-01

    Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell.

  2. Genetic and biochemical characterization of the F-ATPase operon from Streptococcus sanguis 10904.

    Science.gov (United States)

    Kuhnert, Wendi L; Quivey Jr, Robert G

    2003-03-01

    Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species Streptococcus sanguis and Streptococcus mutans. Differences in the pH optima of the respective ATPases appears to be the main reason that S. mutans is more tolerant of low pH values than S. sanguis and hence pathogenic. We have recently reported the genetic arrangement for the S. mutans operon. For purposes of comparative structural biology we have also investigated the F-ATPase from S. sanguis. Here, we report the genetic characterization and expression in Escherichia coli of the S. sanguis ATPase operon. Sequence analysis showed a gene order of atpEBFHAGDC and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both S. sanguis and S. mutans; however, it is not induced to the same extent in the nonpathogenic S. sanguis. Expression studies with an atpD deletion strain of E. coli showed that S. sanguis-E. coli hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.

  3. Phylogenetic analysis of P5 P-type ATPases, a eukaryotic lineage of secretory pathway pumps

    DEFF Research Database (Denmark)

    Møller, Annette B; Asp, Torben; Holm, Preben Bach

    2008-01-01

    Eukaryotes encompass a remarkable variety of organisms and unresolved lineages. Different phylogenetic analyses have lead to conflicting conclusions as to the origin and associations between lineages and species. In this work, we investigated evolutionary relationship of a family of cation pumps ...... far, while P5B ATPases appear to be lost in three eukaryotic lineages; excavates, entamoebas and land plants. A lineage-specific gene expansion of up to four different P5B ATPases is seen in animals....

  4. Regulation of the synthesis and assembly of the plant vacuolar H{sup +}-ATPase. Progress report, [April 1, 1991--March 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Taiz, L.

    1992-04-01

    During the past three years we have focused on four main areas: the characterization of the 5{prime}-upstream sequence of the gene for the V-ATPase 70 kDa (A) subunit gene, the generation of V-ATPase-deficient mutants using antisense constructs of the A subunit cDNA, analysis of V-ATPase ultrastructure by negative staining and the characterization of organelle-specific isoforms of the A subunit of carrot. In addition we have extended our studies on the cellular distribution of the V-ATPase and we have continued our investigation of the evolution of the V-ATPases by characterizing the A and B subunits of two species of the archaebacterium, Methanococcus.

  5. Alternating Hemiplegia of Childhood mutations have a differential effect on Na(+),K(+)-ATPase activity and ouabain binding.

    Science.gov (United States)

    Weigand, Karl M; Messchaert, Muriël; Swarts, Herman G P; Russel, Frans G M; Koenderink, Jan B

    2014-07-01

    De novo mutations in ATP1A3, the gene encoding the α3-subunit of Na(+),K(+)-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N, D801N, E815K, and G947R) associated with AHC. Wild type and mutant Na(+),K(+)-ATPases were expressed in Sf9 insect cells using the baculovirus expression system. Ouabain binding, ATPase activity, and phosphorylation were absent in mutants I274N, E815K and G947R. Mutants S137Y and D801N were able to bind ouabain, although these mutants lacked ATPase activity, phosphorylation, and the K(+)/ouabain antagonism indicative of modifications in the cation binding site. Mutant D220N showed similar ouabain binding, ATPase activity, and phosphorylation to wild type Na(+),K(+)-ATPase. Functional impairment of Na(+),K(+)-ATPase in mutants S137Y, I274N, D801N, E815K, and G947R might explain why patients having these mutations suffer from AHC. Moreover, mutant D801N is able to bind ouabain, whereas mutant E815K shows a complete loss of function, possibly explaining the different phenotypes for these mutations. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Immunohistochemical localization of C1 subunit of V-ATPase (ATPase C1) in oral squamous cell cancer and normal oral mucosa.

    Science.gov (United States)

    García-García, A; Pérez-Sayáns García, M; Rodríguez, M J; Antúnez-López, J; Barros-Angueira, F; Somoza-Martín, M; Gándara-Rey, J M; Aguirre-Urízar, J M

    2012-02-01

    The ATP6V1C1 gene encodes the C1 subunit of the vacuolar-ATPase (V-ATPase) proton pump. This gene is over-expressed in oral squamous cell carcinoma (OSCC) as determined by real-time quantitative polymerase chain reaction. The aim of our study was to perform an immunohistochemical study of the distribution of the C1 subunit in normal epithelium of the oral cavity and in OSCC. We analyzed the expression of the C1 subunit in eight OSCC samples and two normal oral mucosa samples using polyclonal V-ATPase C1 antibody (clone H-300). In the normal oral mucosa samples, C1 subunit staining was observed in the basal and intermediate layers of the epithelium. No staining was visible in the keratinized superficial layers. More intense staining was observed in the OSCC samples, with the predominant expression at the periphery of tumor nests and absence of expression in dyskeratotic areas. C1 subunit expression in tumor cells was predominantly cytoplasmic, although there was perinuclear and nuclear expression in some samples. These findings demonstrate that V-ATPase is necessary for proper epithelial functioning and show its importance in the development of OSCC as evidenced by the over-expression of ATP6V1C1 in OSCC.

  7. Experimental determination of control by the H+-ATPase in Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole; Westerhoff, H. V.

    1995-01-01

    Strains carrying deletions in the atp genes, encoding the H+-ATPase, were unable to grow on nonfermentable substrates such as succinate, whereas with glucose as the substrate the growth rate of an atp deletion mutant was surprisingly high (some 75-80% of wild-type growth rate). The rate of glucos...

  8. Molecular cloning of a plasma membrane Ca²⁺ ATPase (PMCA) from Y-organs of the blue crab (Callinectes sapidus), and determination of spatial and temporal patterns of PMCA gene expression.

    Science.gov (United States)

    Chen, Hsiang-Yin; Roer, Robert D; Watson, R Douglas

    2013-06-10

    Existing data indicate that a stage-specific increase in intracellular free Ca(2+) stimulates ecdysteroid production by crustacean molting glands (Y-organs). The concentration of Ca(2+) in cytosol is controlled mainly by proteins intrinsic to the plasma membrane and to the membranes of organelles. Several families of proteins are involved, including Ca(2+) channels, Ca(2+) pumps (ATPases), and Ca(2+) exchangers. The family of Ca(2+) pumps includes plasma membrane calcium ATPases (PMCAs). As a step toward understanding the involvement of calcium signaling in regulation of ecdysteroidogenesis, we used a PCR-based cloning strategy (RT-PCR followed by 3'- and 5'-RACE) to clone from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative PMCA. The 4292 base pair (bp) cDNA includes a 3510 bp open reading frame encoding a 1170-residue protein (Cas-PMCA). The conceptually translated protein has a relative molecular mass of 128.8×10(3) and contains all signature domains of an authentic PMCA, including ten transmembrane domains and a calmodulin binding site. The predicted membrane topography of Cas-PMCA is as expected for an authentic PMCA protein. A phylogenetic analysis of nonredundant amino acid sequences of PMCA proteins from different species showed Cas-PMCA clusters with other arthropod PMCA proteins. An assessment of tissue distribution showed the Cas-PMCA transcript to be broadly distributed in both neural and non-neural tissues. Studies using quantitative real-time PCR revealed stage-specific changes in Cas-PMCA abundance during the molting cycle, with peak expression occurring during premolt stage D2, a pattern consistent with the hypothesis that Cas-PMCA functions to maintain cellular Ca(2+) homeostasis in Y-organs. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. The F-ATPase operon from the oral streptococci S. mutans and S. sanguis: How structure relates to function

    Science.gov (United States)

    Kuhnert, Wendi Lee

    1999-10-01

    The oral microbe, Streptococcus mutans is known to be a primary contributor to the most common infection in humans, dental caries. In the plaque environment, resident bacteria metabolize dietary sucrose which results in the production of organic acids and a decrease in plaque pH. The proton-translocating ATPase (F-ATPase) protects the bacteria from acidification by extruding protons, at the expense of ATP, to maintain an internal pH which is more neutral than the external environment. Examination of this enzyme will help us to gain insight regarding its contribution to the aciduricity characteristics of oral bacteria. In this work, our goal was to begin the molecular dissection of the mechanism by which streptococcal ATPases are regulated and function enzymatically. Sequence analysis of the F-ATPase from the non-pathogenic S. sanguis revealed that the structural genes are homologous to S. mutans as well as other sequenced F-ATPases. Cloned subunits were functionally similar as shown by complementing E. coli ATPase mutants. S. sanguis/E. coli hybrid enzymes hydrolyzed ATP, but proton conduction was uncoupled as demonstrated with inhibition studies. Transcriptional regulation of the F-ATPase operon from S. mutans was examined using chloramphenicol acetyltransferase gene fusions. Fusions containing 136 bp of DNA upstream of the promoter showed higher levels of expression as compared to those with only 16 bp. Similar to ATPase enzymatic activity, CAT expression also increased during growth at low pH. Analysis of RNA demonstrated that ATPase mRNA levels were higher at low pH, which supported the CAT activity data. Therefore, the F-ATPase from S. mutans was regulated, at least partially, by both the DNA located upstream of the promoter as well as by pH. Examination of structural models of the F-ATPase from the pathogenic oral organisms S. mutans and Lactobacillus casei and the non- pathogenic S. sanguis showed that the differences noted in the sequence of the catalytic

  10. [Na+, K(+)-ATPase, endogenous cardiotonic steroids and their transducing role].

    Science.gov (United States)

    Tsymbaliuk, O V; Kosterin, S O

    2012-01-01

    Na+, K(+)-ATPase--a protein complex of plasmatic membrane, which performs the dual function: firstly, it supports the Na+ and K+ homeostasis, and also transmembrane potential gradient, secondly, it serves as the transducer of signals and as the regulator of the expression of many key genes. Endogenous cardiotonic steroids, which are synthesized in the adrenal glands and hypothalamus, serve as the signal molecules. New concepts about the mechanisms of the realization of the Na+, K(+)-ATPase signal function and their connection with cellular functions, apoptosis, and with pathologies of cardiovascular system and water-salt homeostasis are described in the survey.

  11. Retinoschisin is linked to retinal Na/K-ATPase signaling and localization

    OpenAIRE

    Plössl, Karolina; Royer, Melanie; Bernklau, Sarah; Tavraz, Neslihan N.; Friedrich, Thomas; Wild, Jens; Weber, Bernhard H. F.; Friedrich, Ulrike

    2017-01-01

    Mutations in the RS1 gene cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy. We recently showed that retinoschisin, the protein encoded by RS1, regulates ERK signaling and apoptosis in retinal cells. In this study, we explored an influence of retinoschisin on the functionality of the Na/K-ATPase, its interaction partner at retinal plasma membranes. We show that retinoschisin binding requires the β2-subunit of the Na/K-ATPase, whereas the α-subunit is exchangeable. O...

  12. The evolutionary history of sarco(endoplasmic calcium ATPase (SERCA.

    Directory of Open Access Journals (Sweden)

    Ianina Altshuler

    Full Text Available Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na(+/K(+ transporters, H(+/K(+ transporters, and plasma membrane Ca(2+ pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endoplasmic reticulum calcium ATPase (SERCA, which maintains calcium homeostasis in the cell by actively pumping Ca(2+ into the sarco(endoplasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech Helobdella robusta. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain.

  13. Cold stress affects H(+)-ATPase and phospholipase D activity in Arabidopsis.

    Science.gov (United States)

    Muzi, Carlo; Camoni, Lorenzo; Visconti, Sabina; Aducci, Patrizia

    2016-11-01

    Low temperature is an environmental stress that greatly influences plant performance and distribution. Plants exposed to cold stress exhibit modifications of plasma membrane physical properties that can affect their functionality. Here it is reported the effect of low temperature exposure of Arabidopsis plants on the activity of phospholipase D and H(+)-ATPase, the master enzyme located at the plasma membrane. The H(+)-ATPase activity was differently affected, depending on the length of cold stress imposed. In particular, an exposure to 4 °C for 6 h determined the strong inhibition of the H(+)-ATPase activity, that correlates with a reduced association with the regulatory 14-3-3 proteins. A longer exposure first caused the full recovery of the enzymatic activity followed by a significant activation, in accordance with both the increased association with 14-3-3 proteins and induction of H(+)-ATPase gene transcription. Different time lengths of cold stress treatment were also shown to strongly stimulate the phospholipase D activity and affect the phosphatidic acid levels of the plasma membranes. Our results suggest a functional correlation between the activity of phospholipase D and H(+)-ATPase mediated by phosphatidic acid release during the cold stress response. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  14. The dynamic stator stalk of rotary ATPases

    Science.gov (United States)

    Stewart, Alastair G.; Lee, Lawrence K.; Donohoe, Mhairi; Chaston, Jessica J.; Stock, Daniela

    2012-01-01

    Rotary ATPases couple ATP hydrolysis/synthesis with proton translocation across biological membranes and so are central components of the biological energy conversion machinery. Their peripheral stalks are essential components that counteract torque generated by rotation of the central stalk during ATP synthesis or hydrolysis. Here we present a 2.25-Å resolution crystal structure of the peripheral stalk from Thermus thermophilus A-type ATPase/synthase. We identify bending and twisting motions inherent within the structure that accommodate and complement a radial wobbling of the ATPase headgroup as it progresses through its catalytic cycles, while still retaining azimuthal stiffness necessary to counteract rotation of the central stalk. The conformational freedom of the peripheral stalk is dictated by its unusual right-handed coiled-coil architecture, which is in principle conserved across all rotary ATPases. In context of the intact enzyme, the dynamics of the peripheral stalks provides a potential mechanism for cooperativity between distant parts of rotary ATPases. PMID:22353718

  15. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

      The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded H+-ATPases extrude protons from cells...... of plants and fungi to generate electrochemical proton gradients. A recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Together with biochemical and structural data presented in this thesis we are now able...... to describe the basic molecular components that allow the plasma membrane proton H+-ATPase to carry out proton transport against large membrane potentials. Moreover, a completely new paradigm for post-translational activation of these proteins is presented. The talk will focus on the following themes...

  16. ABCG2/V-ATPase was associated with the drug resistance and tumor metastasis of esophageal squamous cancer cells

    Directory of Open Access Journals (Sweden)

    Huang Lijun

    2012-12-01

    Full Text Available Abstract Background ATP-binding cassette sub-family G member 2 (ABCG2 is a protein that in humans is encoded by the ABCG2 gene. ABCG2 participates in efflux of many chemotherapeutic agents. ABCG2 is often expressed in hematopoietic progenitor or stem cells. Vacuolar-H + −ATPase (V-ATPase plays a key role in adjusting and maintaining intracellular pH and in regulating the drug tolerance of cells. The TNM Classification of Malignant Tumours (TNM is a cancer staging system that describes the extent of cancer in a patient’s body. In this study, the expression of ABCG2 and V-ATPase in esophageal squamous cancer cells was detected. Methods Immunohistochemistry staining and Immunofluorescence double staining were used to detect the expression of ABCG2 and V-ATPase in in 66 cases of esophageal squamous cancer cells. Associations and differences in expression of ABCG2 with that of V-ATPase were analyzed. Results Positive staining patterns for both ABCG2 (66.67% and V-ATPase (68.18% were located mainly in the plasma membrane and cytoplasm. Marked differences in expression were also shown (P  Conclusions Both ABCG2 and V-ATPase were over-expressed in esophageal squamous cancer cells. Their expression was associated with pathological grade, TNM stage and tumor metastasis in esophageal squamous cancer cells, suggesting interaction relationship between them. ABCG2 and V-ATPase expression may be strongly associated with drug resistance and tumor metastasis. Virtual slides The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3823783918433897

  17. The V-ATPase subunit A is essential for salt tolerance through participating in vacuolar Na+ compartmentalization in Salicornia europaea.

    Science.gov (United States)

    Lv, Sulian; Jiang, Ping; Tai, Fang; Wang, Duoliya; Feng, Juanjuan; Fan, Pengxiang; Bao, Hexigeduleng; Li, Yinxin

    2017-12-01

    The V-ATPase subunit A participates in vacuolar Na + compartmentalization in Salicornia europaea regulating V-ATPase and V-PPase activities. Na+ sequestration into the vacuole is an efficient strategy in response to salinity in many halophytes. However, it is not yet fully understood how this process is achieved. Particularly, the role of vacuolar H+-ATPase (V-ATPase) in this process is controversial. Our previous proteomic investigation in the euhalophyte Salicornia europaea L. found a significant increase of the abundance of V-ATPase subunit A under salinity. Here, the gene encoding this subunit named SeVHA-A was characterized, and its role in salt tolerance was demonstrated by RNAi directed downregulation in suspension-cultured cells of S. europaea. The transcripts of genes encoding vacuolar H+-PPase (V-PPase) and vacuolar Na+/H+ antiporter (SeNHX1) also decreased significantly in the RNAi cells. Knockdown of SeVHA-A resulted in a reduction in both V-ATPase and vacuolar H+-PPase (V-PPase) activities. Accordingly, the SeVHA-A-RNAi cells showed increased vacuolar pH and decreased cell viability under different NaCl concentrations. Further Na+ staining showed the reduced vacuolar Na+ sequestration in RNAi cells. Taken together, our results evidenced that SeVHA-A participates in vacuolar Na+ sequestration regulating V-ATPase and V-PPase activities and thereby vacuolar pH in S. europaea. The possible mechanisms underlying the reduction of vacuolar V-PPase activity in SeVHA-A-RNAi cells were also discussed.

  18. Arctigenin antagonizes mineralocorticoid receptor to inhibit the transcription of Na/K-ATPase.

    Science.gov (United States)

    Cheng, Ye; Zhou, Meili; Wang, Yan

    2016-01-01

    Hypertension is one of the most important risk factors in cardiovascular disease and is the most common chronic disease. Mineralocorticoid receptor (MR) antagonists have been successfully used in clinic for the treatment of hypertension. Our study aims to investigate whether Arctigenin can antagonize MR and inhibit the transcription of Na/K-ATPase. The yeast two-hybrid assay was used to screen natural products and Arctigenin was identified as an MR antagonist. The direct binding of Arctigenin to MR was determined using assays based on surface plasmon resonance, differential scanning calorimetry and fluorescence quenching. Furthermore, results from mammalian one-hybrid and transcriptional activation experiments also confirmed that Arctigenin can potently antagonize MR in cells. We demonstrated that Arctigenin can decrease the level of Na/K-ATPase mRNA by antagonizing MR in HK-2 cells. Our findings show that Arctigenin can effectively decrease Na/K-ATPase transcription; thus highlight its potential as an anti-hypertensive drug lead compound. Our current findings demonstrate that Arctigenin is an antagonist of MR and effectively decreases the Na/K-ATPase 1 gene expression. Our work provides a hint for the drug discovery against cardiovascular disease.

  19. Common evolutionary origin for the rotor domain of rotary ATPases and flagellar protein export apparatus.

    Directory of Open Access Journals (Sweden)

    Jun-ichi Kishikawa

    Full Text Available The V1- and F1- rotary ATPases contain a rotor that rotates against a catalytic A3B3 or α3β3 stator. The rotor F(1-γ or V1-DF is composed of both anti-parallel coiled coil and globular-loop parts. The bacterial flagellar type III export apparatus contains a V1/F1-like ATPase ring structure composed of FliI6 homo-hexamer and FliJ which adopts an anti-parallel coiled coil structure without the globular-loop part. Here we report that FliJ of Salmonella enterica serovar Typhimurium shows a rotor like function in Thermus thermophilus A3B3 based on both biochemical and structural analysis. Single molecular analysis indicates that an anti-parallel coiled-coil structure protein (FliJ structure protein functions as a rotor in A3B3. A rotary ATPase possessing an F1-γ-like protein generated by fusion of the D and F subunits of V1 rotates, suggesting F(1-γ could be the result of a fusion of the genes encoding two separate rotor subunits. Together with sequence comparison among the globular part proteins, the data strongly suggest that the rotor domains of the rotary ATPases and the flagellar export apparatus share a common evolutionary origin.

  20. Partial Purification of a Tonoplast ATPase from Corn Coleoptiles 1

    Science.gov (United States)

    Mandala, Suzanne; Taiz, Lincoln

    1985-01-01

    The tonoplast ATPase from corn coleoptile membranes was solubilized using a two-step procedure consisting of a pretreatment with 0.15% (w/v) deoxycholate to remove 60% of the protein, and 40 millimolar octyl-glucoside to solubilize the ATPase. During ultracentrifugation, the solublized ATPase entered a linear sucrose gradient faster than the majority of the protein, resulting in an 11-fold purification over the initial specific activity. The partially purified ATPase was almost completely inhibited by KNO3 with an estimated Ki of 10 millimolar. The specific activity of the KNO3-sensitive ATPase was increased 29-fold during purification. N,N′-Dicyclohexylcarbodiimide also completely inhibited the ATPase with half-maximal effects at a concentration of 4 micromolar. Neither vanadate nor azide inhibited enzyme activity. The purified ATPase was stimulated by Cl− and preferred Mg-ATP as substrate. Analysis of frations from the sucrose gradient by sodium dodecyl sulfate-polyacrylamide gel electrophoresis led to the identification of two major polypeptides at 72,000 and 62,000 daltons which were best correlated with ATPase activity. Several minor bands also appeared to copurify with enzyme activity, but were less consistent. Radiation inactivation experiments with intact membranes indicated that the functional molecular size of the tonoplast ATPase was nearly 400,000 daltons. This suggests that the ATPase is composed of several polypeptides, possibly including the 72,000- and 62,000-dalton proteins. Images Fig. 4 PMID:16664239

  1. Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

    Science.gov (United States)

    Stransky, Laura A.; Forgac, Michael

    2015-01-01

    The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

  2. Overproduction of PIB-Type ATPases

    DEFF Research Database (Denmark)

    Liu, Xiangyu; Sitsel, Oleg; Wang, Kaituo

    2016-01-01

    Understanding of the functions and mechanisms of fundamental processes in the cell requires structural information. Structural studies of membrane proteins typically necessitate large amounts of purified and preferably homogenous target protein. Here, we describe a rapid overproduction and purifi...... and purification strategy of a bacterial PIB-type ATPase for isolation of milligrams of target protein per liter Escherichia coli cell culture, with a final quality of the sample which is sufficient for generating high-resolution crystals....

  3. Type IV traffic ATPase TrwD as molecular target to inhibit bacterial conjugation.

    Science.gov (United States)

    Ripoll-Rozada, Jorge; García-Cazorla, Yolanda; Getino, María; Machón, Cristina; Sanabria-Ríos, David; de la Cruz, Fernando; Cabezón, Elena; Arechaga, Ignacio

    2016-06-01

    Bacterial conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit bacterial conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic ATPase, as the molecular target for fatty acid-mediated inhibition of conjugation. Moreover, 2-alkynoic fatty acids, which are also potent inhibitors of bacterial conjugation, are also powerful inhibitors of the ATPase activity of TrwD. Characterization of the kinetic parameters of ATPase inhibition has led us to identify the catalytic mechanism by which fatty acids exert their activity. These results open a new avenue for the rational design of inhibitors of bacterial conjugation in the fight against the dissemination of antibiotic resistance genes. © 2016 John Wiley & Sons Ltd.

  4. V-ATPase-dependent luminal acidification is required for endocytic recycling of a yeast cell wall stress sensor, Wsc1p

    Energy Technology Data Exchange (ETDEWEB)

    Ueno, Kazuma; Saito, Mayu; Nagashima, Makiko; Kojima, Ai; Nishinoaki, Show [Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Toshima, Junko Y., E-mail: yama_jun@aoni.waseda.jp [Faculty of Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480 (Japan); Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Toshima, Jiro, E-mail: jtosiscb@rs.noda.tus.ac.jp [Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan)

    2014-01-10

    Highlights: •A targeted genome screen identified 5 gene groups affecting Wsc1p recycling. •V-ATPase-dependent luminal acidification is required for Wsc1p recycling. •Activity of V-ATPase might be required for cargo recognition by the retromer complex. -- Abstract: Wsc1p is a major cell wall sensor protein localized at the polarized cell surface. The localization of Wsc1p is maintained by endocytosis and recycling from endosomes back to the cell surface, but changes to the vacuole when cells are subjected to heat stress. Exploiting this unique property of Wsc1p, we screened for yeast single-gene deletion mutants exhibiting defects in Wsc1p trafficking. By expressing 3GFP-tagged Wsc1p in mutants with deleted genes whose function is related to intracellular trafficking, we identified 5 gene groups affecting Wsc1p trafficking, impaired respectively in endocytic internalization, multivesicular body sorting, the GARP complex, endosomal maturation/vacuolar fusion, and V-ATPase. Interestingly, deletion of the VPH1 gene, encoding the V{sub o} subunit of vacuolar-type H{sup +}-ATPase (V-ATPase), led to mis-localization of Wsc1p from the plasma membrane to the vacuole. In addition, disruption of other V-ATPase subunits (vma mutants) also caused defects of Wsc1p trafficking and vacuolar acidification similar to those seen in the vph1Δ mutant. Moreover, we found that deletion of the VPS26 gene, encoding a subunit of the retromer complex, also caused a defect in Wsc1p recycling and mis-localization of Wsc1p to the vacuole. These findings clarified the previously unidentified Wsc1p recycling pathway and requirement of V-ATPase-dependent luminal acidification for Wsc1p recycling.

  5. Alternating Hemiplegia of Childhood mutations have a differential effect on Na(+),K(+)-ATPase activity and ouabain binding

    NARCIS (Netherlands)

    Weigand, K.M.; Messchaert, M.; Swarts, H.G.P.; Russel, F.G.M.; Koenderink, J.B.

    2014-01-01

    De novo mutations in ATP1A3, the gene encoding the alpha3-subunit of Na(+),K(+)-ATPase, are associated with the neurodevelopmental disorder Alternating Hemiplegia of Childhood (AHC). The aim of this study was to determine the functional consequences of six ATP1A3 mutations (S137Y, D220N, I274N,

  6. V-ATPase Is Involved in Silkworm Defense Response against Bombyx mori Nucleopolyhedrovirus.

    Directory of Open Access Journals (Sweden)

    Peng Lü

    Full Text Available Silkworms are usually susceptible to the infection of Bombyx mori (B. mori nucleopolyhedrovirus (BmNPV, which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells of BmNPV-resistant strains (NB and BC8, the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi, the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses.

  7. A Global Survey of ATPase Activity in Plasmodium falciparum Asexual Blood Stages and Gametocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, Corrie; Frando, Andrew; Webb-Robertson, Bobbie-Jo; Anderson, Lindsey N.; Fleck, Neil; Flannery, Erika L.; Fishbaugher, Matthew; Murphree, Taylor A.; Hansen, Joshua R.; Smith, Richard D.; Kappe, Stefan H. I.; Wright, Aaron T.; Grundner, Christoph

    2017-10-27

    Effective malaria control and elimination in hyperendemic areas of the world will require treatment of disease-causing Plasmodium falciparum (Pf) blood stage infection but also blocking parasite transmission from humans to mosquito to prevent disease spread. Numerous antimalarial drugs have become ineffective due to parasite drug resistance and many currently used therapies do not kill gametocytes, highly specialized sexual parasite stages with distinct physiology that are necessary for transmission from the human host to the mosquito vector. Further confounding next generation drug development against Pf is the lack of known biochemical activity for most parasite gene products as well as the unknown metabolic needs of non-replicating gametocyte. Here, we take a systematic activity-based proteomics approach to survey the large and druggable ATPase family that is associated with replicating blood stage asexual parasites and transmissible gametocytes. We experimentally confirm existing annotation and predict ATPase function for 38 uncharacterized proteins. ATPase activity broadly changes during the transition from asexual schizonts to gametocytes, indicating altered metabolism and regulatory roles of ATPases specific for each lifecycle stage. By mapping the activity of ATPases associated with gametocytogenesis, we assign biochemical activity to a large number of uncharacterized proteins and identify new candidate transmission blocking targets.

  8. Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

    Energy Technology Data Exchange (ETDEWEB)

    Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.

    1986-01-01

    The H translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ approx. = 70,000 and approx. = 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-( UC)ethylmaleimide and 7-chloro-4-nitro( UC)benzo-2-oxa-1,3-diazole, labeled the M/sub r/ approx. = 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-( UC)dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ approx. = 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10V, 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ approx. = 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F0F1 ATPases.

  9. Phosphorylation of the Na+,K+-ATPase and the H+,K+-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Morth, Jens Preben; Jensen, Jan Egebjerg

    2010-01-01

    Phosphorylation is a widely used, reversible means of regulating enzymatic activity. Among the important phosphorylation targets are the Na(+),K(+)- and H(+),K(+)-ATPases that pump ions against their chemical gradients to uphold ionic concentration differences over the plasma membrane. The two...

  10. Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed

    2008-01-01

    Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence, but not in the absence of K+. Studies with purified membranes revealed that CPZ reduced Na+-dependent phosphorylation by interference with Na+ binding from the intracellular side of the memb...

  11. [Dynamic changes of aciduric virulence factor membrane-bound proton-translocating ATPase of Streptococcus mutans in the development of dental caries].

    Science.gov (United States)

    Gao, Jing; Huang, Wenming

    2016-04-01

    To observe the dynamic changes of membrane-bound proton-translocating ATPase (F-ATPase) in the development of dental caries, the expression of Streptococcus mutans F-ATPase under different pH concentrations and during the development of dental caries is analyzed. Streptococcus mutans cultured under different pH (pH4.0-7.0) concentrations and containing 5% glucose and no glucose containing BHI were collected. RNA was extracted. Subsequently, F-ATPase gene was detected using real-time polymerase chain reaction. Male Wistar rats were divided randomly into caries group and control group. The rats in the caries group were fed caries feed and 5% glucose water, whereas those of control group were fed normal feed. Total RNA was extracted from plaque samples, which were collected from rats' oral cavity every two weeks. F-ATPase gene was detected by real-time PCR. In the 11th week, the upper and lower jaw bone specimens of rats were taken, and molar caries damage assessed. The expression of F-ATPase in the caries group was higher than that in the control group (Pcaries development in both groups; expression in the caries group was higher than that in control group (Pdental caries.

  12. Review: P4-ATPases as Phospholipid Flippases-Structure, Function, and Enigmas

    DEFF Research Database (Denmark)

    Andersen, Jens P; Vestergaard, Anna L; Mikkelsen, Stine A

    2016-01-01

    coagulation, apoptosis, bile and cholesterol homeostasis, and neuronal cell survival. Some P4-ATPases transport phosphatidylserine and phosphatidylethanolamine across the plasma membrane or intracellular membranes whereas other P4-ATPases are specific for phosphatidylcholine. The importance of P4-ATPases...... on properties of mammalian and yeast P4-ATPases for which most mechanistic insight is available. However, the structure, function and enigmas associated with mammalian and yeast P4-ATPases most likely extend to P4-ATPases of plants and other organisms....

  13. TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds.

    Science.gov (United States)

    Appelhagen, Ingo; Nordholt, Niclas; Seidel, Thorsten; Spelt, Kees; Koes, Ronald; Quattrochio, Francesca; Sagasser, Martin; Weisshaar, Bernd

    2015-06-01

    Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H(+) -ATPases in the plasma membrane, and multimeric vacuolar-type H(+) -ATPases (V-ATPases) and vacuolar H(+) -pyrophosphatases (H(+) -PPases) in endomembranes. Here, we show that reduced accumulation of proanthocyanidins (PAs) and hence the diminished brown seed coloration found in the Arabidopsis thaliana mutant transparent testa 13 (tt13) is caused by disruption of the gene encoding the P3A -ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants. Cells of the tt13 seed coat endothelium do not contain PA-filled central vacuoles as observed in the wild-type. tt13 phenocopies tt12, a mutant that is defective in vacuolar import of the PA precursor epicatechin. Our data show that vacuolar loading with PA precursors depends on TT13. Consistent with the tt13 phenotype, but in contrast to other isoforms of P-type H(+) -ATPases, TT13 localizes to the tonoplast. PA accumulation in tt13 is partially restored by expression of the tonoplast localized H(+) -PPase VHP1. Our findings indicate that the P3A -ATPase TT13 functions as a proton pump in the tonoplast of seed coat endothelium cells, and generates the driving force for TT12-mediated transport of PA precursors to the vacuole. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  14. Regulation of the Plasma Membrane H+-ATPase

    DEFF Research Database (Denmark)

    Falhof, Janus

    The plasma membrane (PM) H+-ATPase is responsible for generating the electrochemical gradientthat drives the secondary transport of nutrients across the cellular membrane. It belongs to a familyof cation and lipid transporters that are vital to many organisms. PM H+-ATPases are Type P3AATPases...

  15. Changes in erythrocyte ATPase activity under different pathological ...

    African Journals Online (AJOL)

    Conclusion: Na+- K+ATPase specific activity is a biomarker for the diagnosis of individuals with different physiological diseases. Keywords: Na+-K+ATPase ... aging and that changes in young females were more re- markable than that in young .... activity which might be due to increased / decreased con- centration of ions ...

  16. The loss-of-function disease-mutation G301R in the Na+/K+-ATPase α2 isoform decreases lesion volume and improves functional outcome after acute spinal cord injury in mice

    DEFF Research Database (Denmark)

    Ellman, Ditte; Isaksen, Toke Jost; Lund, Minna

    2017-01-01

    BACKGROUND: The Na(+)/K(+)-ATPases are transmembrane ion pumps important for maintenance of ion gradients across the plasma membrane that serve to support multiple cellular functions, such as membrane potentials, regulation of cellular volume and pH, and co-transport of signaling transmitters...... to anaerobic metabolism and lactate accumulation. During ischemia, Na(+)/K(+)-ATPase-related functions will naturally increase the energy demand of the Na(+)/K(+)-ATPase ion pump. However, the role of the α2Na(+)/K(+)-ATPase in contusion injury to the spinal cord remains unknown. We used mice heterozygous mice...... for the loss-of-function disease-mutation G301R in the Atp1a2 gene (α 2(+/G301R) ) to study the effect of reduced α2Na(+)/K(+)-ATPase expression in a moderate contusion spinal cord injury (SCI) model. RESULTS: We found that α 2(+/G301R) mice display significantly improved functional recovery and decreased...

  17. RNAi-directed downregulation of vacuolar H(+ -ATPase subunit a results in enhanced stomatal aperture and density in rice.

    Directory of Open Access Journals (Sweden)

    Huiying Zhang

    Full Text Available Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L. vacuolar H(+-ATPase subunit A (OsVHA-A gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H(+-ATPase activity and an enhancement of plasma membrane H(+-ATPase activity, thereby increasing the concentrations of extracellular H(+ and intracellular K(+ and Na(+ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H(+-ATPase 3 and downregulation of CAM1 (calmodulin 1, CAM3 (calmodulin 3 and YDA1 (YODA, a MAPKK gene. Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.

  18. Autolysosome biogenesis and developmental senescence are regulated by both Spns1 and v-ATPase.

    Science.gov (United States)

    Sasaki, Tomoyuki; Lian, Shanshan; Khan, Alam; Llop, Jesse R; Samuelson, Andrew V; Chen, Wenbiao; Klionsky, Daniel J; Kishi, Shuji

    2017-02-01

    Spns1 (Spinster homolog 1 [Drosophila]) in vertebrates, as well as Spin (Spinster) in Drosophila, is a hypothetical lysosomal H+-carbohydrate transporter, which functions at a late stage of macroautophagy (hereafter autophagy). The Spin/Spns1 defect induces aberrant autolysosome formation that leads to developmental senescence in the embryonic stage and premature aging symptoms in adulthood. However, the molecular mechanism by which loss of Spin/Spns1 leads to the specific pathogenesis remains to be elucidated. Using chemical, genetic and CRISPR/Cas9-mediated genome-editing approaches in zebrafish, we investigated and determined a mechanism that suppresses embryonic senescence as well as autolysosomal impairment mediated by Spns1 deficiency. Unexpectedly, we found that a concurrent disruption of the vacuolar-type H+-ATPase (v-ATPase) subunit gene, atp6v0ca (ATPase, H+ transporting, lysosomal, V0 subunit ca) led to suppression of the senescence induced by the Spns1 defect, whereas the sole loss of Atp6v0ca led to senescent embryos similar to the single spns1 mutation. Moreover, we discovered that the combined stable defect seen in the presence of both the spns1 and atp6v0ca mutant genes still subsequently induced premature autophagosome-lysosome fusion marked by insufficient acidity, while extending developmental life span, compared with the solely mutated spns1 defect. Our data suggest that Spns1 and the v-ATPase orchestrate proper autolysosomal biogenesis with optimal acidification that is critically linked to developmental senescence and survival.

  19. Analysis of Conditional Paralytic Mutants in Drosophila Sarco-Endoplasmic Reticulum Calcium ATPase Reveals Novel Mechanisms for Regulating Membrane Excitability

    OpenAIRE

    Sanyal, S.; Consoulas, C.; Kuromi, H.; Basole, A.; Mukai, L.; Kidokoro, Y.; Krishnan, K. S.; Ramaswami, M.

    2005-01-01

    Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a gene...

  20. Downregulation of TGF-β Receptor-2 Expression and Signaling through Inhibition of Na/K-ATPase.

    Directory of Open Access Journals (Sweden)

    Jennifer La

    Full Text Available Transforming growth factor-beta (TGF-β is a multi-functional cytokine implicated in the control of cell growth and differentiation. TGF-β signals through a complex of TGF-β receptors 1 and 2 (TGFβR1 and TGFβR2 that phosphorylate and activate Smad2/3 transcription factors driving transcription of the Smad-target genes. The Na+/K+-ATPase is an integral plasma membrane protein critical for maintaining the electro-chemical gradient of Na+ and K+ in the cell. We found that inhibition of the Na+/K+ ATPase by ouabain results in a dramatic decrease in the expression of TGFβR2 in human lung fibrobalsts (HLF at the mRNA and protein levels. This was accompanied by inhibition of TGF-β-induced Smad phosphorylation and the expression of TGF-β target genes, such as fibronectin and smooth muscle alpha-actin. Inhibition of Na+/K+ ATPase by an alternative approach (removal of extracellular potassium had a similar effect in HLF. Finally, treatment of lung alveolar epithelial cells (A549 with ouabain also resulted in the downregulation of TGFβR2, the inhibition of TGF-β-induced Smad phosphorylation and of the expression of mesenchymal markers, vimentin and fibronectin. Together, these data demonstrate a critical role of Na+/K+-ATPase in the control of TGFβR2 expression, TGF-β signaling and cell responses to TGF-β.

  1. Salt regulation of transcript levels for the c subunit of a leaf vacuolar H(+)-ATPase in the halophyte Mesembryanthemum crystallinum.

    Science.gov (United States)

    Tsiantis, M S; Bartholomew, D M; Smith, J A

    1996-05-01

    The halophyte Mesembryanthemum crystallinum is an inducible crassulacean acid metabolism (CAM) plant native to seasonally arid coastal environments that has been widely used to study plant responses to environmental stress. On exposure of plants to salt, the activities of both the tonoplast (vacuolar) H(+)-ATPase (V-ATPase) and Na+/H+ antiporter increase in leaf cells, thereby energizing vacuolar salt accumulation. To investigate the molecular basis of this response, a cDNA (Vmac1) encoding the H(+)-conducting c subunit (16.6 kDa) of an M. crystallinum V-ATPase has been cloned. Northern analysis of RNA from leaves of plants treated with NaCl or with isoosmotic mannitol solutions demonstrated (i) that NaCl increased steady-state transcript levels for the V-ATPase c subunit, and (ii) that this effect was caused by the ionic rather than the osmotic component of salt stress. Southern analysis of genomic DNA suggested the probable existence of more than one gene for this subunit of the V-ATPase in M. crystallinum. Expression studies using the 3'-untranslated region of the Vmac1 cDNa as a probe showed that the corresponding salt-inducible transcript was preferentially expressed in leaves. Induction by salt was also observed in juvenile plants in addition to adult ones. These findings, as well as the inability of mannitol to upregulate mRNA levels for this gene, clearly differentiate between the induction of transcript for the V-ATPase c subunit and for genes involved in the CAM pathway in M. crystallinum. Further, the plant growth regulator abscisic acid (ABA) was able to mimic the effect of salt on transcript levels for the V-ATPase c subunit, suggesting the possible involvement of ABA in a distinct signal-transduction pathway linked to vacuolar salt accumulation in this highly salt-tolerant species.

  2. Novel mutations affecting the Na, K ATPase alpha model complex neurological diseases and implicate the sodium pump in increased longevity.

    Science.gov (United States)

    Ashmore, Lesley J; Hrizo, Stacy L; Paul, Sarah M; Van Voorhies, Wayne A; Beitel, Greg J; Palladino, Michael J

    2009-09-01

    Mutations affecting the Na(+), K(+) ATPase alpha subunit have been implicated in at least two distinct human diseases, rapid-onset dystonia Parkinsonism (RDP), and familial hemiplegic migraine (FHM). Over 40 mutations have been mapped to the human ATP1A2 and ATP1A3 genes and are known to result in RDP, FHM or a variant of FHM with neurological complications. To develop a genetically tractable model system for investigating the role of the Na(+), K(+) ATPase in neural pathologies we performed genetic screens in Drosophila melanogaster to isolate loss-of-function alleles affecting the Na(+), K(+) ATPase alpha subunit. Flies heterozygous for these mutations all exhibit reduced respiration, consistent with a loss-of-function in the major ATPase. However, these mutations do not affect all functions of the Na(+), K(+) ATPase alpha protein since embryos homozygous for these mutations have normal septate junction paracellular barrier function and tracheal morphology. Importantly, all of these mutations cause neurological phenotypes and, akin to the mutations that cause RDP and FHM, these new alleles are missense mutations. All of these alleles exhibit progressive stress-induced locomotor impairment suggesting neuromuscular dysfunction, yet neurodegeneration is observed in an allele-specific manner. Surprisingly, studies of longevity demonstrate that mild hypomorphic mutations in the sodium pump significantly improve longevity, which was verified using the Na(+), K(+) ATPase antagonist ouabain. The isolation and characterization of a series of new missense alleles of ATPalpha in Drosophila provides the foundation for further studies of these neurological diseases and the role of sodium pump impairment in animal longevity.

  3. Metal Fluoride Complexes of Na,K-ATPase

    Science.gov (United States)

    Cornelius, Flemming; Mahmmoud, Yasser A.; Toyoshima, Chikashi

    2011-01-01

    The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K+·Pi with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na+, after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na+ demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway. PMID:21708939

  4. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    Science.gov (United States)

    Caldwell, C.

    1983-01-01

    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  5. Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase for anti-fungal therapy

    Directory of Open Access Journals (Sweden)

    Summer R. Hayek

    2014-01-01

    Full Text Available Vacuolar proton-translocating ATPase (V-ATPase is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae (S. cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans (C. albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens.

  6. Coumarin enhances nitrate uptake in maize roots throughout a modulation of plasma membrane H+ -ATPase activity.

    Science.gov (United States)

    Lupini, Antonio; Araniti, Fabrizio; Mauceri, Antonio; Princi, Maria Polsia; Sorgonà, Agostino; Sunseri, Francesco; Varanini, Zeno; Abenavoli, Maria Rosa

    2017-11-27

    1.Coumarin is one of the simplest plant secondary metabolite widely distributed in plant kingdom affecting root form and function, including anatomy, morphology and nutrient uptake. Although, some plant responses to coumarin have been described, comprehensive knowledge of the physiological and molecular mechanisms is lacking. 2.Maize seedlings exposed to different coumarin concentrations, alone or in combination with 200 μM nitrate (NO3- ), were analyzed, through a physiological and molecular approach, to elucidate action of coumarin on net NO3- uptake rate (NNUR). In detail, the time course of NNUR, plasma membrane (PM) H+ -ATPase activity, the proton pumping, and related gene expressions (ZmNPF6.3, ZmNRT2.1, ZmNAR2.1, ZmHA3 and ZmHA4) were evaluated. 3.Coumarin alone did not affect nitrate uptake, the PM H+ -ATPase activity as well as the transcript levels of ZmNRT2.1 and ZmHA3. By contrast, coumarin alone increased ZmNPF6.3, ZmNAR2.1 and ZmHA4 expression, as observed in response to abiotic stress. When coumarin and NO3- were concurrently added to the nutrient solution, a significant increase in the NNUR, PM H+ -ATPase activity together with ZmNAR2.1:ZmNRT2.1 and ZmHA4 expressions were observed, suggesting that coumarin affected the inducible component of high affinity transport system (iHATS), and this effect appeared to be mediated by nitrate. Moreover, the results with vanadate, an inhibitor of the PM H+ -ATPase, suggested that this enzyme could be a main target of coumarin. 4.Surprisingly, coumarin did not affect the PM H+ -ATPase activity by direct contact with plasma membrane vesicle isolated from maize roots, indicating its possible elicitor role in the gene transcription. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. Elucidating Functional Aspects of P-type ATPases

    DEFF Research Database (Denmark)

    Autzen, Henriette Elisabeth

    2015-01-01

    and helped enlighten how thapsigargin, a potent inhibitor of SERCA1a, depends on a water mediated hydrogen bond network when bound to SERCA1a. Furthermore, molecular dynamics (MD) simulations of the same P-type ATPase were used to assess a long-standing question whether cholesterol affects SERCA1a through......P-type ATPases are proteins that act to maintain ion homeostasis and electrochemical gradients through the translocation of cations across cell membranes. Underscoring their significance in humans, dysfunction of the ATPases can lead to crucial diseases. Dysfunction of the sarco......; however, encouraging hits were obtained with AgNO3 and E. coli lipids, potentially underscoring the importance of utilizing native lipids. Finally, atomistic MD simulations of a Zn2+-ATPase (ZntA) were set up and computed in order to elucidate the dynamics of its ion exit pathway, and helped reveal how...

  8. OSMOTIC FRAGILITY AND Na + -K + + ATPase ACTIVITY OF ...

    African Journals Online (AJOL)

    + -K+ ATPase activity of the erythrocytes of HIV/AIDS patients. Whole blood was taken from subjects at the Human Virology Laboratory of the Nigerian Institute of Medical Research. Subjects were judged suitable for the various investigations by ...

  9. Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening.

    Science.gov (United States)

    Elbatsh, Ahmed M O; Haarhuis, Judith H I; Petela, Naomi; Chapard, Christophe; Fish, Alexander; Celie, Patrick H; Stadnik, Magda; Ristic, Dejan; Wyman, Claire; Medema, René H; Nasmyth, Kim; Rowland, Benjamin D

    2016-02-18

    Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin's highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin's two ATPase sites. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Proton Pumping of the Yeast Plasma Membrane H+-ATPase

    Science.gov (United States)

    1993-08-16

    eucaryotic cells would be more resistant to damage by expression of the ATPase. We started with a plasmid from Dr. Fink’s laboratory tnat uontains the...have begun construction of a Baculovirus expression vector that we will ti-ansfect into insect cells . We hope that this 6 system will not suffer from...causes the bacteria to stop growing. We decided to try to produce the ATPase in insect cells using the baculovirus expression system hoping that the

  11. Structural divergence between the two subgroups of P5 ATPases

    DEFF Research Database (Denmark)

    Sørensen, Danny Mollerup; Buch-Pedersen, Morten Jeppe; Palmgren, Michael Broberg

    2010-01-01

    been identified in the endoplasmic reticulum and seem to have basic functions in protein maturation and secretion. P5B ATPases localize to vacuolar/lysosomal or apical membranes and in animals play a role in hereditary neuronal diseases. Here we have used a bioinformatical approach to identify....... Together these findings indicate that P5A and P5B ATPases are structurally and functionally different....

  12. A plasma membrane H ATPase gene is germination- induced in ...

    African Journals Online (AJOL)

    ONOS

    2010-01-18

    Jan 18, 2010 ... Biol. 36(6): 580-585. Cox KH, Deleon DV, Angerer LM, Angerer RC (1984). Detection of. mRNA in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101: 485-502. Cuming AC (1993). Seed Development. In: Dey PM and Harborne JB. (eds) Methods in Plant Biochemistry.

  13. The V-ATPase a3 subunit mutation R740S is dominant negative and results in osteopetrosis in mice.

    Science.gov (United States)

    Ochotny, Noelle; Flenniken, Ann M; Owen, Celeste; Voronov, Irina; Zirngibl, Ralph A; Osborne, Lucy R; Henderson, Janet E; Adamson, S Lee; Rossant, Janet; Manolson, Morris F; Aubin, Jane E

    2011-07-01

    A mouse founder with high bone mineral density and an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) screen. It was found to carry a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit of the vacuolar type H(+)-ATPase (V-ATPase), resulting in replacement of a highly conserved amino acid (R740S). The +/R740S mice have normal appearance, size, and weight but exhibit high bone density. Osteoblast parameters are unaffected in bones of +/R740S mice, whereas osteoclast number and marker expression are increased, concomitant with a decrease in the number of apoptotic osteoclasts. Consistent with reduced osteoclast apoptosis, expression of Rankl and Bcl2 is elevated, whereas Casp3 is reduced. Transmission electron microscopy revealed that unlike other known mutations in the a3 subunit of V-ATPase, polarization and ruffled border formation appear normal in +/R740S osteoclasts. However, V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport, whereas ATP hydrolysis is not significantly affected. We show for the first time that a point mutation within the a3 subunit, R740S, which is dominant negative for proton pumping and bone resorption, also uncouples proton pumping from ATP hydrolysis but has no effect on ruffled border formation or polarization of osteoclasts. These results suggest that the V(0) complex has proton-pumping-independent functions in mammalian cells. Copyright © 2011 American Society for Bone and Mineral Research.

  14. The vacuolar H+ ATPase V0 subunit d2 is associated with chondrocyte hypertrophy and supports chondrocyte differentiation

    Directory of Open Access Journals (Sweden)

    Babatunde A. Ayodele

    2017-12-01

    Full Text Available Chondrocyte hypertrophy makes important contributions to bone development and growth. We have investigated a number of novel cartilage genes identified in a recent transcriptomic study to determine whether they are differentially expressed between different zones of equine foetal growth cartilage. Twelve genes (ATP6V0D2, BAK1, DDX5, GNB1, PIP4K2A, RAP1B, RPS7, SRSF3, SUB1, TMSB4, TPI1 and WSB2 were found to be more highly expressed in the zone of hypertrophic chondrocytes than in the reserve or proliferative zones, whereas FOXA3 and SERPINA1 were expressed at lower levels in the hypertrophic zone than in the reserve zone. ATP6V0D2, which encodes vacuolar H+ ATPase (V-ATPase V0 subunit d2 (ATP6V0D2, was selected for further study. Immunohistochemical analysis of ATP6V0D2 in growth cartilage showed stronger staining in hypertrophic than in reserve zone or proliferative chondrocytes. Expression of ATP6V0D2 mRNA and protein was up-regulated in the mouse chondrocytic ATDC5 cell line by conditions inducing expression of hypertrophy-associated genes including Col10a1 and Mmp13 (differentiation medium. In ATDC5 cells cultured in control medium, knockdown of Atp6v0d2 or inhibition of V-ATPase activity using bafilomycin A1 caused a decrease in Col2a1 expression, and in cells cultured in differentiation medium the two treatments caused a decrease in nuclear area. Inhibition of V-ATPase, but not Atp6v0d2 knockdown, prevented the upregulation of Col10a1, Mmp13 and Vegf by differentiation medium, while Atp6v0d2 knockdown, but not inhibition of V-ATPase, caused an increase in the number of ATDC5 cells cultured in differentiation medium. These observations identify ATP6V0D2 as a novel chondrocyte hypertrophy-associated gene. The results are consistent with roles for V-ATPase, both ATP6V0D2-dependent and -independent, in supporting chondrocyte differentiation and hypertrophy.

  15. Monoclonal antibody modification of the ATPase activity of Escherichia coli F1 ATPase.

    Science.gov (United States)

    Aggeler, R; Mendel-Hartvig, J; Capaldi, R A

    1990-11-13

    Monoclonal antibodies (mAbs) have been made against each of the five subunits of ECF1 (alpha, beta, gamma, delta, and epsilon), and these have been used in topology studies and for examination of the role of individual subunits in the functioning of the enzyme. All of the mAbs obtained reacted with ECF1, while several failed to react with ECF1F0, including three mAbs against the gamma subunit (gamma II, gamma III, and gamma IV), one mAb against delta, and two mAbs against epsilon (epsilon I and epsilon II). These topology data are consistent with the gamma, delta, and epsilon subunits being located at the interface between the F1 and F0 parts of the complex. Two forms of ECF1 were used to study the effects of mAbs on the ATPase activity of the enzyme: ECF1 with the epsilon subunit tightly bound and acting to inhibit activity and ECF1* in which the delta and epsilon subunits had been removed by organic solvent treatment. ECF1* had an ATPase activity under standard conditions of 93 mumol of ATP hydrolyzed min-1 mg-1, cf. an activity of 7.5 units mg-1 for our standard ECF1 preparation and 64 units mg-1 for enzyme in which the epsilon subunit had been removed by trypsin treatment. The protease digestion of ECF1* reduced activity to 64 units mg-1 in a complicated process involving an inhibition of activity by cleavage of the alpha subunit, activation by cleavage of gamma, and inhibition with cleavage of the beta subunit. mAbs to the gamma subunit, gamma II and gamma III, activated ECF1 by 4.4- and 2.4-fold, respectively, by changing the affinity of the enzyme for the epsilon subunit, as evidenced by density gradient centrifugation experiments. The gamma-subunit mAbs did not alter the ATPase activity of ECF1*- or trypsin-treated enzyme. The alpha-subunit mAb (alpha I) activated ECF1 by a factor of 2.5-fold and ECF1F0 by 1.3-fold, but inhibited the ATPase activity of ECF1* by 30%.

  16. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe

    2011-01-01

    Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary tran......(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps....

  17. Identification of small-molecule inhibitors of Yersinia pestis Type III secretion system YscN ATPase.

    Science.gov (United States)

    Swietnicki, Wieslaw; Carmany, Daniel; Retford, Michael; Guelta, Mark; Dorsey, Russell; Bozue, Joel; Lee, Michael S; Olson, Mark A

    2011-01-01

    Yersinia pestis is a gram negative zoonotic pathogen responsible for causing bubonic and pneumonic plague in humans. The pathogen uses a type III secretion system (T3SS) to deliver virulence factors directly from bacterium into host mammalian cells. The system contains a single ATPase, YscN, necessary for delivery of virulence factors. In this work, we show that deletion of the catalytic domain of the yscN gene in Y. pestis CO92 attenuated the strain over three million-fold in the Swiss-Webster mouse model of bubonic plague. The result validates the YscN protein as a therapeutic target for plague. The catalytic domain of the YscN protein was made using recombinant methods and its ATPase activity was characterized in vitro. To identify candidate therapeutics, we tested computationally selected small molecules for inhibition of YscN ATPase activity. The best inhibitors had measured IC(50) values below 20 µM in an in vitro ATPase assay and were also found to inhibit the homologous BsaS protein from Burkholderia mallei animal-like T3SS at similar concentrations. Moreover, the compounds fully inhibited YopE secretion by attenuated Y. pestis in a bacterial cell culture and mammalian cells at µM concentrations. The data demonstrate the feasibility of targeting and inhibiting a critical protein transport ATPase of a bacterial virulence system. It is likely the same strategy could be applied to many other common human pathogens using type III secretion system, including enteropathogenic E. coli, Shigella flexneri, Salmonella typhimurium, and Burkholderia mallei/pseudomallei species.

  18. Identification of small-molecule inhibitors of Yersinia pestis Type III secretion system YscN ATPase.

    Directory of Open Access Journals (Sweden)

    Wieslaw Swietnicki

    Full Text Available Yersinia pestis is a gram negative zoonotic pathogen responsible for causing bubonic and pneumonic plague in humans. The pathogen uses a type III secretion system (T3SS to deliver virulence factors directly from bacterium into host mammalian cells. The system contains a single ATPase, YscN, necessary for delivery of virulence factors. In this work, we show that deletion of the catalytic domain of the yscN gene in Y. pestis CO92 attenuated the strain over three million-fold in the Swiss-Webster mouse model of bubonic plague. The result validates the YscN protein as a therapeutic target for plague. The catalytic domain of the YscN protein was made using recombinant methods and its ATPase activity was characterized in vitro. To identify candidate therapeutics, we tested computationally selected small molecules for inhibition of YscN ATPase activity. The best inhibitors had measured IC(50 values below 20 µM in an in vitro ATPase assay and were also found to inhibit the homologous BsaS protein from Burkholderia mallei animal-like T3SS at similar concentrations. Moreover, the compounds fully inhibited YopE secretion by attenuated Y. pestis in a bacterial cell culture and mammalian cells at µM concentrations. The data demonstrate the feasibility of targeting and inhibiting a critical protein transport ATPase of a bacterial virulence system. It is likely the same strategy could be applied to many other common human pathogens using type III secretion system, including enteropathogenic E. coli, Shigella flexneri, Salmonella typhimurium, and Burkholderia mallei/pseudomallei species.

  19. The evolutionary history of sarco(endo)plasmic calcium ATPase (SERCA).

    Science.gov (United States)

    Altshuler, Ianina; Vaillant, James J; Xu, Sen; Cristescu, Melania E

    2012-01-01

    Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na(+)/K(+) transporters, H(+)/K(+) transporters, and plasma membrane Ca(2+) pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endo)plasmic reticulum calcium ATPase (SERCA), which maintains calcium homeostasis in the cell by actively pumping Ca(2+) into the sarco(endo)plasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech Helobdella robusta. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain.

  20. A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases.

    Directory of Open Access Journals (Sweden)

    Gianluca Bartolommei

    Full Text Available The detection of small amounts (nanomoles of inorganic phosphate has a great interest in biochemistry. In particular, phosphate detection is useful to evaluate the rate of hydrolysis of phosphatases, that are enzymes able to remove phosphate from their substrate by hydrolytic cleavage. The hydrolysis rate is correlated to enzyme activity, an extremely important functional parameter. Among phosphatases there are the cation transporting adenosinetriphosphatases (ATPases, that produce inorganic phosphate by cleavage of the γ-phosphate of ATP. These membrane transporters have many fundamental physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. We have optimized a molybdenum-based protocol that makes use of potassium antimony (III oxide tartrate (originally employed for phosphate detection in environmental analysis to allow its use with phosphatase enzymes. In particular, the method was successfully applied to native and recombinant ATPases to demonstrate its reliability, validity, sensitivity and versatility. Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements. Therefore, it may be valuable in biochemical and biomedical investigations of ATPase enzymes, in combination with more specific tests, as well as in high throughput drug screening.

  1. Two isoforms of sarco/endoplasmic reticulum calcium ATPase (SERCA) are essential in Caenorhabditis elegans.

    Science.gov (United States)

    Cho, J H; Bandyopadhyay, J; Lee, J; Park, C S; Ahnn, J

    2000-12-31

    SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase), a membrane bound Ca(2+)- /Mg(2+)- dependent ATPase that sequesters Ca(2+) into the SR/ER lumen, is one of the essential components for the maintenance of intracellular Ca(2+) homeostasis. Here we describe the identification and functional characterization of a C. elegans SERCA gene (ser-1). ser-1 is a single gene alternatively spliced at its carboxyl terminus to form two isoforms (SER-1A and SER-1B) and displays a high homology (70% identity, 80% similarity) with mammalian SERCAs. Green fluorescent protein (GFP) and whole-mount immunostaining analyses reveal that SER-1 expresses in neuronal cells, body-wall muscles, pharyngeal and vulval muscles, excretory cells, and vulva epithelial cells. Furthermore, SER-1::GFP expresses during embryonic stages and the expression is maintained through the adult stages. Double-stranded RNA injection (also known as RNAi) targeted to each SER-1 isoform results in severe phenotypic defects: ser-1A(RNAi) animals show embryonic lethality, whereas ser-1B(RNAi) results in L1 larval arrest phenotype. These findings suggest that both isoforms of C. elegans SERCA, like in mammals, are essential for embryonic development and post-embryonic growth and survival.

  2. Autoinhibitory Regulation of Plasma Membrane H+-ATPases

    DEFF Research Database (Denmark)

    Pedersen, Jesper Torbøl

    Electrochemical gradients across cell membranes are essential for nutrient uptake. In plant and fungal cells the electrochemical gradient across the plasma membrane (PM) can build much higher than in mammalian cells. The protein responsible for this gradient is the essential PM H+-ATPase that uses...... a huge amount of energy in form of ATP, to pump out protons. To avoid complete energy depletion in the cells, tight regulation of the PM H+-ATPase is a necessity. The proteins two terminal domains have been identified as autoinhibitory domains that regulate the pumping activity, but due to lack of a high....... In contrast to fungal PM H+-ATPases the terminal phosphorylation sites in the plant counterpart was found highly conserved even in the earliest land plants. The phosphorylation sites were, however, not found in algae, the sister group of land plants. We therefore hypotheses that the delicate regulation...

  3. The coupled chemomechanics of the F(1)-ATPase molecular motor.

    Science.gov (United States)

    Xu, Lizhong

    2008-11-01

    The enzyme F(1)-ATPase is a rotary nanomotor in which the central gamma subunit rotates inside the cavity made of alpha(3)beta(3) subunits. The experiments showed that the rotation proceeds in steps of 120 degrees and each 120 degrees step consists of 80 degrees and 40 degrees substeps. Here the Author proposes a stochastic wave mechanics of the F(1)-ATPase motor and combines it with the structure-based kinetics of the F(1)-ATPase to form a chemomechanic coupled model. The model can reproduce quantitatively and explain the experimental observations about the F(1) motor. Using the model, several rate-limited situations about gamma subunit rotation are proposed, the effects of the friction and the load on the substeps are investigated and the chemomechanic coupled time during ATP hydrolysis cycle is determined.

  4. Structural and functional studies of heavy metal ATPases

    DEFF Research Database (Denmark)

    Sitsel, Oleg

    2015-01-01

    of SsZntA using a broad range of techniques reveals an array of unique Zn2+-ATPase features relating to ion uptake, binding, discharge and countertransport. These results are then used to comprehensively compare Zn2+-ATPases with their Cu+-transporting counterparts, showing how the two have managed...... to handle heavy metal ions. LpCopA is then compared to its two human homologues ATP7A and ATP7B, which cause the severe Menkes and Wilson diseases when malfunctioning. The differences between the three proteins are described and disease-causing mutations in the human proteins are analyzed. The crystal...... to adapt to transport of distinct ion types while still maintaining all the major features of the P1B-ATPase subclass....

  5. Transport ATPases: structure, motors, mechanism and medicine: a brief overview.

    Science.gov (United States)

    Pedersen, Peter L

    2005-12-01

    Today we know there are four different types of ATPases that operate within biological membranes with the purpose of moving many different types of ions or molecules across these membranes. Some of these ions or molecules are transported into cells, some out of cells, and some in or out of organelles within cells. These ATPases span the biological world from bacteria to eukaryotic cells and have become most simply and commonly known as "transport ATPases." The price that each cell type pays for transport work is counted in molecules of hydrolyzed ATP, a metabolic currency that is itself regenerated by a transport ATPase working in reverse, i.e., the ATP synthase. Four major classes of transport ATPases, the P, V, F, and ABC types are now known. In addition to being involved in many different types of biological/physiological processes, mutations in these proteins also account for a large number of diseases. The purpose of this introductory article to a mini-review series on transport ATPases is to provide the reader with a very brief and focused look at this important area of research that has an interesting history and bears significance to cell physiology, biochemistry, immunology, nanotechnology, and medicine, including drug discovery. The latter involves potential applications to a whole host of diseases ranging from cancer to those that affect bones (osteoporosis), ears (hearing), eyes (macromolecular degeneration), the heart (hypercholesterolemia/cardiac arrest,), immune system (immune deficiency disease), kidney (nephrotoxicity), lungs (cystic fibrosis), pancreas (diabetes and cystic fibrosis), skin (Darier disease), and stomach (ulcers).

  6. Mercuric chloride-induced inhibition of different ATPases in the intestine of mudskipper, Boleophthalmus dentatus.

    Science.gov (United States)

    Lakshmi, R; Kundu, R; Thomas, E; Mansuri, A P

    1991-02-01

    This paper deals with the toxicity of mercuric chloride to different ATPases in the intestine of mudskipper (Boleophthalmus dentatus). Mudskippers were exposed to four sublethal concentrations of mercuric chloride for three durations. The specific activities of Na+, K(+)-ATPase, Ca(2+)-ATPase, Mg(2+)-ATPase, Ca2+, HCO3(-)-ATPase, and Mg2+, HCO3(-)-ATPase were estimated. There was linear inhibition of all the enzymes with increasing mercuric chloride concentration as well as exposure duration. The Na+,K(+)-ATPase was found to be the enzyme most affected, followed by other ion-dependent ATPases. Inhibition of all the enzymes indicates severe damage to the intestinal cells, resulting in a blockage of the transport of substances across the membrane.

  7. Purification and characterization of ATPase from Nitrobacter winogradskyi.

    Science.gov (United States)

    Hara, T; Villalobos, A P; Fukumori, Y; Yamanaka, T

    1991-07-15

    An ATPase was purified from Nitrobacter winogradskyi, and some of its molecular and enzymatic properties were determined. The enzyme was composed of two subunits of 64 and 59 kDa, respectively. The enzyme had its pH optimum at 9.5 and showed a specific activity of 7 units per mg protein. This activity was about 14% and 18% of that of F1-ATPases obtained from Escherichia coli and Sulfolobus acidocaldarius, respectively. The enzyme was 29% and 6% inhibited by 100 microM dicyclohexylcarbodiimide (DCCD) and 100 microM NaN3, respectively. It was not inhibited by 20 mM NaNO3.

  8. Cloning of sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) from Caribbean spiny lobster Panulirus argus.

    Science.gov (United States)

    Mandal, A; Arunachalam, S C; Meleshkevitch, E A; Mandal, P K; Boudko, D Y; Ahearn, G A

    2009-02-01

    We have previously reported on calcium transport mechanisms in American lobster, Homarus americanus, using (45)Ca(2+) coupled with vesicle preparations of hepatopancreatic endoplasmic reticulum. The active transport of calcium across membranes bordering calcium-sequestering stores such as sarcoplasmic or endoplasmic reticulum is catalyzed by membrane-spanning proteins, the sarco-endoplasmic Ca(2+)-ATPases (SERCAs). In the study described here we used advanced bioinformatics and molecular techniques to clone SERCA from the economically important Caribbean spiny lobster, Panulirus argus. We report the complete cloning of a full-length SERCA from P. argus antenna cDNA (GenBank accession number AY702617). This cDNA has a 1020-amino acid residue open reading frame which is 90% identical to published sequences of other crustacean SERCA proteins. Our data support the hypothesis that one crustacean and three vertebrate genes controlling calcium transport were derived from a common ancestral gene.

  9. Evolution of tonoplast P-ATPase transporters involved in vacuolar acidification

    NARCIS (Netherlands)

    Li, Y.; Provenzano, S.; Bliek, M.; Spelt, C.; Appelhagen, I.; Machado de Faria, L.; Verweij, W.; Schubert, A.; Sagasser, M.; Seidel, T.; Weisshaar, B.; Koes, R.; Quattrocchio, F.

    2016-01-01

    Petunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H+ P3A -ATPase, whereas PH1 is the only known eukaryoticP3B -ATPase. As

  10. Structural and functional studies of a Cu+-ATPase from Legionella pneumophila

    DEFF Research Database (Denmark)

    Mattle, Daniel

    During his studies, Daniel Mattle explored the copper(I) export mechanism of a P-type Cu+ ATPase from Legionella pneumophila – a homologue to the human Cu+ ATPases. Cu+ ATPases are responsible for the homeostatic control of the physiological relevant – but toxic – copper(I) cations. To assess...

  11. Na+,K+-ATPase Na+ affinity in rat skeletal muscle fiber types

    DEFF Research Database (Denmark)

    Kristensen, Michael; Juel, Carsten

    2010-01-01

    Previous studies in expression systems have found different ion activation of the Na(+)/K(+)-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na(+),K(+)-ATPase activity, and the Na(+) affinity of Na(+),K(+)-ATPase...

  12. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe

    2011-01-01

    Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary...... transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na...

  13. Effects of aqueous extract of Hibiscus sabdariffa on renal Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in Wistar rats.

    Science.gov (United States)

    Olatunji, Lawrence A; Usman, Taofeek O; Adebayo, Joseph O; Olatunji, Victoria A

    2012-09-01

    To investigate the effects of oral administration of aqueous extract of Hibiscus sabdariffa on renal Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in rats. The 25 and 50 mg/(kg·d) of aqueous extracts of H. sabdariffa were respectively given to rats in the experimental groups for 28 d, and rats in the control group received an appropriate volume of distilled water as vehicle. Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase activities in the kidney were assayed by spectrophotometric method. Administrations of 25 and 50 mg/(kg·d) of aqueous extract of H. sabdariffa significantly decreased the Ca(2+)-Mg(2+)-ATPase activity in the kidney of rats (Psabdariffa may preserve the renal function despite a decreased renal Ca(2+)-Mg(2+)-ATPase activity.

  14. Regulatory Mechanisms in the P4-ATPase Complex

    DEFF Research Database (Denmark)

    Costa, Sara

    Eukaryotic cell membranes are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Several relevant physiological processes depend on trans-bilayer phospholipid asymmetry, including vesiculation in the se......Eukaryotic cell membranes are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Several relevant physiological processes depend on trans-bilayer phospholipid asymmetry, including vesiculation...... in the secretory and endocytic pathways. The inward-oriented translocation of phospholipids is catalyzed by proteins called P4-ATPases or flippases, which belong to a subfamily of P-type ATPases. P4-ATPases, together with their CDC50 β-subunits, are essential for eukaryotic life but the key features...... of their activity and regulation remain to be elucidated. Therefore, these studies focus on the role of the catalytic and CDC50 β-subunit in the phospholipid translocation and the regulation processes behind it. Recent studies suggested that P4-ATPase complex functionality is highly dependent on the conformation...

  15. On Allosteric Modulation of P-Type Cu+-ATPases

    DEFF Research Database (Denmark)

    Mattle, Daniel; Sitsel, Oleg; Autzen, Henriette E.

    2013-01-01

    of intramembranous Cu+ binding, and we suggest an alternative role for the proposed second site in copper translocation and proton exchange. The class-specific features demonstrate that topological diversity in P-type ATPases may tune a general energy coupling scheme to the translocation of compounds with remarkably...

  16. Changes in erythrocyte ATPase activity under different pathological ...

    African Journals Online (AJOL)

    Background: Studies have shown that Na+-K+ ATPase activity was altered in disrupted red blood cell membranes and this enzyme is believed to be the site of active transport of Na+ and K+ in intact red blood cells. The enzyme is often referred to as Na+- K+ pump because it pumps Na+ out and K+ into the cell against ...

  17. Towards the structure of yeast and mammalian P4-ATPases

    DEFF Research Database (Denmark)

    Lyons, Joseph; Laban, Milena; Mikkelsen, Stine

    2017-01-01

    a basis for the analysis of reported mutagenesis data, we aim to solve the first molecular structures of the PS transporting P4-ATPases using electron microscopy. To date, negative stain EM analysis, on detergent, amphipol and saposin-lipoprotein nanoparticle (Salipro) reconstituted of both Drs2p/CDC50p...

  18. The Kdp-ATPase system and its regulation

    Indian Academy of Sciences (India)

    2007-03-15

    Mar 15, 2007 ... K+, the dominant intracellular cation, is required for various physiological processes like turgor homeostasis, pH regulation etc. Bacterial cells have evolved many diverse K+ transporters to maintain the desired concentration of internal K+. In E. coli, the KdpATPase (comprising of the KdpFABC complex), ...

  19. Effects of intermittent fasting on age-related changes on Na,K-ATPase activity and oxidative status induced by lipopolysaccharide in rat hippocampus.

    Science.gov (United States)

    Vasconcelos, Andrea Rodrigues; Kinoshita, Paula Fernanda; Yshii, Lidia Mitiko; Marques Orellana, Ana Maria; Böhmer, Ana Elisa; de Sá Lima, Larissa; Alves, Rosana; Andreotti, Diana Zukas; Marcourakis, Tania; Scavone, Cristoforo; Kawamoto, Elisa Mitiko

    2015-05-01

    Chronic neuroinflammation is a common characteristic of neurodegenerative diseases, and lipopolysaccharide (LPS) signaling is linked to glutamate-nitric oxide-Na,K-ATPase isoforms pathway in central nervous system (CNS) and also causes neuroinflammation. Intermittent fasting (IF) induces adaptive responses in the brain that can suppress inflammation, but the age-related effect of IF on LPS modulatory influence on nitric oxide-Na,K-ATPase isoforms is unknown. This work compared the effects of LPS on the activity of α1,α2,3 Na,K-ATPase, nitric oxide synthase gene expression and/or activity, cyclic guanosine monophosphate, 3-nitrotyrosine-containing proteins, and levels of thiobarbituric acid-reactive substances in CNS of young and older rats submitted to the IF protocol for 30 days. LPS induced an age-related effect in neuronal nitric oxide synthase activity, cyclic guanosine monophosphate, and levels of thiobarbituric acid-reactive substances in rat hippocampus that was linked to changes in α2,3-Na,K-ATPase activity, 3-nitrotyrosine proteins, and inducible nitric oxide synthase gene expression. IF induced adaptative cellular stress-response signaling pathways reverting LPS effects in rat hippocampus of young and older rats. The results suggest that IF in both ages would reduce the risk for deficits on brain function and neurodegenerative disorders linked to inflammatory response in the CNS. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. AAV-2 Rep78 and HPV-16 E1 interact in vitro, modulating their ATPase activity.

    Science.gov (United States)

    Bandyopadhyay, Sarmistha; Raney, Kevin D; Liu, Yong; Hermonat, Paul L

    2008-01-15

    Adeno-associated virus (AAV) is a nonpathogenic single-stranded human parvovirus which usually requires the presence of a "helper" virus for strong DNA replication. In addition to adeno- and herpes viruses, human papillomavirus (HPV) can serve as an AAV helper. We recently published that HPV type 16 (HPV-16) E1 protein contributes significantly as an individual helper gene for AAV-2 DNA replication and transcription. As Rep78 and E1 are the corresponding DNA helicase/replication proteins of AAV and HPV, respectively, and Rep78 and E1 have a degree of homology, we assayed whether these two proteins interact physically. The full length proteins were purified from bacteria as GST-E1 and MBP-Rep78 and used in five assays to observe Rep78-E1 interactions. All five assays (pull-down, coimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), chemical cross-linking, and ATPase activity) provided evidence consistent with Rep78-E1 interaction. Most intriguing, an overall decrease in ATPase activity was observed when both proteins were present together. These data strongly suggest that E1 and Rep78 interact and that this interaction modulates at least some of their individual biochemical functions. This study adds to our understanding of AAV-HPV interaction biology, E1's modulation of Rep78 biochemistry, Rep78's modulation of E1 biochemistry and provides initial clues which may lead to the underlying mechanism of HPV E1 helper function for AAV DNA replication.

  1. Loop 2 in Saccharomyces cerevisiae Rad51 protein regulates filament formation and ATPase activity.

    Science.gov (United States)

    Zhang, Xiao-Ping; Galkin, Vitold E; Yu, Xiong; Egelman, Edward H; Heyer, Wolf-Dietrich

    2009-01-01

    Previous studies showed that the K342E substitution in the Saccharomyces cerevisiae Rad51 protein increases the interaction with Rad54 protein in the two-hybrid system, leads to increased sensitivity to the alkylating agent MMS and hyper-recombination in an oligonucleotide-mediated gene targeting assay. K342 localizes in loop 2, a region of Rad51 whose function is not well understood. Here, we show that Rad51-K342E displays DNA-independent and DNA-dependent ATPase activities, owing to its ability to form filaments in the absence of a DNA lattice. These filaments exhibit a compressed pitch of 81 A, whereas filaments of wild-type Rad51 and Rad51-K342E on DNA form extended filaments with a 97 A pitch. Rad51-K342E shows near normal binding to ssDNA, but displays a defect in dsDNA binding, resulting in less stable protein-dsDNA complexes. The mutant protein is capable of catalyzing the DNA strand exchange reaction and is insensitive to inhibition by the early addition of dsDNA. Wild-type Rad51 protein is inhibited under such conditions, because of its ability to bind dsDNA. No significant changes in the interaction between Rad51-K342E and Rad54 could be identified. These findings suggest that loop 2 contributes to the primary DNA-binding site in Rad51, controlling filament formation and ATPase activity.

  2. Transporters, chaperones, and P-type ATPases controlling grapevine copper homeostasis.

    Science.gov (United States)

    Leng, Xiangpeng; Mu, Qian; Wang, Xiaomin; Li, Xiaopeng; Zhu, Xudong; Shangguan, Lingfei; Fang, Jinggui

    2015-11-01

    With more copper and copper-containing compounds used as bactericides and fungicides in viticulture, copper homeostasis in grapevine (Vitis) has become one of the serious environmental crises with great risk. To better understand the regulation of Cu homeostasis in grapevine, grapevine seedlings cultured in vitro with different levels of Cu were utilized to investigate the tolerance mechanisms of grapevine responding to copper availability at physiological and molecular levels. The results indicated that Cu contents in roots and leaves arose with increasing levels of Cu application. With copper concentration increasing, malondialdehyde (MDA) content increased in roots and leaves and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased to protect the plant itself from damage. The expression patterns of 19 genes, encoding transporters, chaperones, and P-type ATPases involved in copper homeostasis in root and leaf of grapevine seedling under various levels of Cu(2+) were further analyzed. The expression patterns indicated that CTr1, CTr2, and CTr8 transporters were significantly upregulated in response both to Cu excess and deficiency. ZIP2 was downregulated in response to Cu excess and upregulated under Cu-deficient conditions, while ZIP4 had an opposite expression pattern under similar conditions. The expression of chaperones and P-type ATPases in response to Cu availability in grapevine were also briefly studied.

  3. A cytotoxic type III secretion effector of Vibrio parahaemolyticus targets vacuolar H+-ATPase subunit c and ruptures host cell lysosomes.

    Directory of Open Access Journals (Sweden)

    Shigeaki Matsuda

    Full Text Available Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1. VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H(+-ATPase (V-ATPase, the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC, which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death.

  4. ATPaseTb2, a unique membrane-bound FoF1-ATPase component, is essential in bloodstream and dyskinetoplastic trypanosomes.

    Directory of Open Access Journals (Sweden)

    Karolína Šubrtová

    2015-02-01

    Full Text Available In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt membrane potential (Δψm is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential FoF1-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψm that manifests as a decreased growth phenotype, indicating that the FoF1-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk and thus subunit a, an essential component of the proton pore in the membrane Fo-moiety. These Dk cells generate the Δψm by combining the hydrolytic activity of the matrix-facing F1-ATPase and the electrogenic exchange of ATP4- for ADP3- by the ATP/ADP carrier (AAC. Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric FoF1-ATPase complexes were identified. In fact, the immunoprecipitation of a F1-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψm of Dk cells. Thus, even in the absence of subunit a, a portion of the FoF1-ATPase is assembled in Dk cells.

  5. Involvement of MoVMA11, a Putative Vacuolar ATPase c' Subunit, in Vacuolar Acidification and Infection-Related Morphogenesis of Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Guoqing Chen

    Full Text Available Many functions of vacuole depend on the activity of vacuolar ATPase which is essential to maintain an acidic lumen and create the driving forces for massive fluxes of ions and metabolites through vacuolar membrane. In filamentous fungus Magnaportheoryzae, subcellular colocalization and quinacrine staining suggested that the V1V0 domains of V-ATPase were fully assembled and the vacuoles were kept acidic during infection-related developments. Targeted gene disruption of MoVMA11 gene, encoding the putative c' subunit of V-ATPase, impaired vacuolar acidification and mimicked the phenotypes of yeast V-ATPase mutants in the poor colony morphology, abolished asexual and sexual reproductions, selective carbon source utilization, and increased calcium and heavy metals sensitivities, however, not in the typical pH conditional lethality. Strikingly, aerial hyphae of the MoVMA11 null mutant intertwined with each other to form extremely thick filamentous structures. The results also implicated that MoVMA11 was involved in cell wall integrity and appressorium formation. Abundant non-melanized swollen structures and rare, small appressoria without penetration ability were produced at the hyphal tips of the ΔMovma11 mutant on onion epidermal cells. Finally, the MoVMA11 null mutant lost pathogenicity on both intact and wounded host leaves. Overall, our data indicated that MoVMA11, like other fungal VMA genes, is associated with numerous cellular functions and highlighted that V-ATPase is essential for infection-related morphogenesis and pathogenesis in M. oryzae.

  6. Regulation of Na+/K+-ATPase by Estradiol and IGF-1 in Cardio-Metabolic Diseases.

    Science.gov (United States)

    Obradovic, Milan; Stanimirovic, Julijana; Panic, Anastasija; Bogdanovic, Nikola; Sudar-Milovanovic, Emina; Cenic-Milosevic, Desanka; Isenovic, Esma R

    2017-01-01

    The sodium/potassium- adenosine- triphosphatase (Na+/K+-ATPase) is an important mediator in vasculature tone and contractility, and its abnormal regulation has been implicated in many diseases such as obesity, insulin resistance, diabetes, and hypertension. Decreased Na+/K+-ATPase abundance and its altered isoform expression induce cardiomyocytes death and cardiac dysfunction, possibly leading to the development of myocardial dilation and heart failure. Therefore, the regulation of Na+/K+-ATPase activity/expression could be important in treatment and possible prevention of cardio-metabolic diseases. A number of hormones and environmental factors regulate the function of Na+/K+-ATPase in response to changing cellular requirements. Estradiol and insulin like growth factor-1 (IGF-1) are among potent hormones that positively regulate Na+/K+- ATPase activity or de novo synthesis of α - and β - subunits. Both estradiol and IGF-1 have a huge therapeutic potential in treatment of vasculopathy in cardio-metabolic diseases. We searched the MEDLINE and PUBMED databases for all English and non-English articles with an English abstract from April 1978 to May 2016. The main data search terms were: Na+/K+-ATPase; estradiol and Na+/K+-ATPase; estradiol, Na+/K+-ATPase and CVS; estradiol, Na+/K+-ATPase and CVD; estradiol, Na+/K+- ATPase and obesity; estradiol, Na+/K+-ATPase and diabetes; estradiol, Na+/K+-ATPase and hypertension; IGF-1; IGF-1 and Na+/K+-ATPase; IGF-1, Na+/K+-ATPase and CVS; IGF-1, Na+/K+-ATPase and CVD; IGF-1, Na+/K+- ATPase and obesity; IGF-1, Na+/K+-ATPase and diabetes; IGF-1, Na+/K+-ATPase and hypertension. The present review discusses the latest data from animal and human studies which focus on the effects of estradiol and IGF-1 on Na+/K+-ATPase regulation in physiological and pathophysiological conditions in cardiovascular system. Understanding the molecular mechanisms of estradiol and IGF-1 action on Na+/K+-ATPase in humans, may help resolving outstanding

  7. Decreased ATPase activity in adriamycin nephrosis is independent of proteinuria

    Energy Technology Data Exchange (ETDEWEB)

    Bakker, W.W.; Kalicharan, D.; Donga, J.; Hulstaert, C.E.; Hardonk, M.J.

    1987-03-01

    In previous studies from this laboratory it has been shown that ATP-ase activity in situ in the glomerular basement membrane (GBM) is clearly reduced in rats rendered nephrotic after treatment with adriamycin (ADR). The question was raised whether this reduction of ATP-ase activity in the GBM is due to toxic activity of ADR or rather a result of the nephrotic condition per se. Therefore, we studied ATP-ase activity using the cerium-based method in kidneys from ADR-treated rats without proteinuria (48 hr after ADR injection), or with proteinuria (approximately 150 mg/24 hr) several weeks after ADR injection. Also kidneys from rats rendered nephrotic by surgical ablation and from non-nephrotic rats treated with local X-irradiation (2000 rads) as well as from normal control rats were studied. The results show that in the GBM of ADR-treated or irradiated rats, clear reduction of ATP-ase activity is observed irrespective of their proteinuria, whereas in the GBM of rats rendered nephrotic by renal ablation (approximately 156 mg/24 hr mean protein excretion) no reduction of enzyme activity is found. It is concluded that decreased ATP-ase activity of the glomerular filtration barrier in ADR-treated rats is due to an early toxic activity of this drug and not a result of the nephrotic state per se. In view of the identical results in X-irradiated rats, it is likely that ADR may act through production of toxic radicals leading to damage of this membrane-associated enzyme system.

  8. Expression of gill vacuolar-type H+-ATPase B subunit, and Na+, K+-ATPase alpha- and beta- subunit messenger RNAs in smolting Salmo salar

    DEFF Research Database (Denmark)

    Seidelin, Michel; Madsen, Steffen; Cutler, Christopher P

    2001-01-01

    Changes in gill vacuolar-type H+-ATPase B subunit, and Na+,K+-ATPase alpha and beta subunit mRNA expression were examined during the course of smoltification in Salmo salar. We cloned and sequenced cDNA fragments of S. salar gill i) vacuolar-type H+-ATPase (V-H+-ATPase) B subunit, ii) Na......+,K+-ATPase alpha (1) subunit, and iii) Na+,K+-ATPase beta (1) subunit, and used these as Northern blotting probes. During smoltification, the salmon showed a typical increase in gill Na+,K+-ATPase activity and improved hypo-osmoregulatory ability as judged by their ability to regulate plasma [Cl-] in a 24-hr...... seawater challenge test (35 ppt). Gill Na+,K+-ATPase alpha (1) and beta (1) subunit mRNA levels were regulated at a constant ratio during smoltification. Both transcripts were elevated during the build-up of gill Na+,K+-ATPase activity, underlining the importance of increased mRNA levels for increased...

  9. Epigallocatechin-3-Gallate Protects Erythrocyte Ca2+-ATPase and Na+/K+-ATPase Against Oxidative Induced Damage During Aging in Humans

    Directory of Open Access Journals (Sweden)

    Prabhanshu Kumar

    2014-10-01

    Full Text Available Purpose: The main purpose of this study was to investigate the protective role of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced oxidative damage in erythrocyte during aging in humans. Methods: Human erythrocyte membrane bound Ca2+-ATPase and Na+/K+-ATPase activities were determined as a function of human age. Protective role of epigallocatechin-3-gallate was evaluated by in vitro experiments by adding epigallocatechin-3-gallate in concentration dependent manner (final concentration range 10-7M to 10-4M to the enzyme assay medium. Oxidative stress was induced in vitro by incubating washed erythrocyte ghosts with tertiary butyl hydroperoxide (10-5 M final concentration. Results: We have reported concentration dependent effect of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced damage on activities of Ca2+-ATPase and Na+/K+-ATPase during aging in humans. We have detected a significant (p < 0.001 decreased activity of Ca2+-ATPase and Na+/K+ -ATPase as a function of human age. Epigallocatechin-3-gallate protected ATPases against tertiary butyl hydroperoxide induced damage in concentration dependent manner during aging in humans. Conclusion: Epigallocatechin-3-gallate is a powerful antioxidant that is capable of protecting erythrocyte Ca2+-ATPase and Na+/K+ -ATPase against oxidative stress during aging in humans. We may propose hypothesis that a high intake of catechin rich diet may provide some protection against development of aging and age related diseases.

  10. Clp ATPases and ClpP proteolytic complexes regulate vital biological processes in low GC, Gram-positive bacteria.

    Science.gov (United States)

    Frees, Dorte; Savijoki, Kirsi; Varmanen, Pekka; Ingmer, Hanne

    2007-03-01

    Clp proteolytic complexes consisting of a proteolytic core flanked by Clp ATPases are widely conserved in bacteria, and their biological roles have received considerable interest. In particular, mutants in the clp genes in the low-GC-content Gram-positive phyla Bacillales and Lactobacillales display a diverse range of phenotypic changes including general stress sensitivity, aberrant cell morphology, failure to initiate developmental programs, and for pathogens, severely attenuated virulence. Extensive research dedicated to unravelling the molecular mechanisms underlying these complex phenotypes has led to fascinating new insights that will be covered by this review. First, Clp ATPases and ClpP-containing proteolytic complexes play indispensable roles in cellular protein quality control systems by refolding or degrading damaged proteins in both stressed and non-stressed cells. Secondly, ClpP proteases and the chaperone activity of Clp ATPases are important for controlling stability and activity of central transcriptional regulators, thereby exerting tremendous impact on cell physiology. Targets include major stress regulators like Spx (oxidative stress), the antisigma factor RsiW (alkaline stress) and HdiR (DNA damage) in addition to regulators of developmental programs like ComK (competence development), sigmaH and Sda (sporulation). Thus, Clp proteins are central in co-ordinating developmental decisions and stress response in low GC Gram-positive bacteria.

  11. Cell-specific expression of SERCA, the exogenous Ca2+ transport ATPase, in cardiac myocytes.

    Science.gov (United States)

    Ma, Hailun; Sumbilla, Carlota M; Farrance, Iain K G; Klein, Michael G; Inesi, Giuseppe

    2004-03-01

    We evaluated various constructs to obtain cell-specific expression of the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) gene in cardiac myocytes after cDNA transfer by means of transfections or infections with adenovirus vectors. Expression of exogenous enhanced green fluorescent protein (EGFP) and SERCA genes was studied in cultured chicken embryo and neonatal rat cardiac myocytes, skeletal and smooth muscle cells, fibroblasts, and hepatocytes. Whereas the cytomegalovirus (CMV) promoter yielded high levels of protein expression in all cells studied, cardiac troponin T (cTnT) promoter segments demonstrated high specificity for cardiac myocytes. Their efficiency for protein expression was lower than that of the CMV promoter, but higher than that of cardiac myosin light chain or beta-myosin heavy chain promoter segments. A double virus system for Cre-dependent expression under control of the CMV promoter and Cre expression under control of a cardiac-specific promoter yielded high protein levels in cardiac myocytes, but only partial cell specificity due to significant Cre expression in hepatocytes. Specific intracellular targeting of gene products was demonstrated in situ by specific immunostaining of exogenous SERCA1 and endogenous SERCA2 and comparative fluorescence microscopy. The -374 cTnT promoter segment was the most advantageous of the promoters studied, producing cell-specific SERCA expression and a definite increase over endogenous Ca2+ -ATPase activity as well as faster removal of cytosolic calcium after membrane excitation. We conclude that analysis of promoter efficiency and cell specificity is of definite advantage when cell-specific expression of exogenous SERCA is wanted in cardiac myocytes after cDNA delivery to mixed cell populations.

  12. Vacuolar H+-ATPase: An Essential Multitasking Enzyme in Physiology and Pathophysiology

    Directory of Open Access Journals (Sweden)

    L. Shannon Holliday

    2014-01-01

    Full Text Available Vacuolar H+-ATPases (V-ATPases are large multisubunit proton pumps that are required for housekeeping acidification of membrane-bound compartments in eukaryotic cells. Mammalian V-ATPases are composed of 13 different subunits. Their housekeeping functions include acidifying endosomes, lysosomes, phagosomes, compartments for uncoupling receptors and ligands, autophagosomes, and elements of the Golgi apparatus. Specialized cells, including osteoclasts, intercalated cells in the kidney and pancreatic beta cells, contain both the housekeeping V-ATPases and an additional subset of V-ATPases, which plays a cell type specific role. The specialized V-ATPases are typically marked by the inclusion of cell type specific isoforms of one or more of the subunits. Three human diseases caused by mutations of isoforms of subunits have been identified. Cancer cells utilize V-ATPases in unusual ways; characterization of V-ATPases may lead to new therapeutic modalities for the treatment of cancer. Two accessory proteins to the V-ATPase have been identified that regulate the proton pump. One is the (prorenin receptor and data is emerging that indicates that V-ATPase may be intimately linked to renin/angiotensin signaling both systemically and locally. In summary, V-ATPases play vital housekeeping roles in eukaryotic cells. Specialized versions of the pump are required by specific organ systems and are involved in diseases.

  13. Beneficial renal and pancreatic phenotypes in a mouse deficient in FXYD2 regulatory subunit of Na,K-ATPase

    Directory of Open Access Journals (Sweden)

    Elena eArystarkhova

    2016-03-01

    Full Text Available The fundamental role of Na,K-ATPase in eukaryotic cells calls for complex and efficient regulation of its activity. Besides alterations in gene expression and trafficking, kinetic properties of the pump are modulated by reversible association with single span membrane proteins, the FXYDs. Seven members of the family are expressed in a tissue-specific manner, affecting pump kinetics in all possible permutations. This mini-review focuses on functional properties of FXYD2 studied in transfected cells, and on noteworthy and unexpected phenotypes discovered in a Fxyd2-/- mouse. FXYD2, the gamma subunit, reduces activity of Na,K-ATPase either by decreasing affinity for Na+, or reducing Vmax. FXYD2 mRNA splicing and editing provide another layer for regulation of Na,K-ATPase. In kidney of knockouts, there was elevated activity for Na,K-ATPase and for NCC and NKCC2 apical sodium transporters. That should lead to sodium retention and hypertension, however, the mice were in sodium balance and normotensive. Adult Fxyd2-/- mice also exhibited a mild pancreatic phenotype with enhanced glucose tolerance, elevation of circulating insulin, but no insulin resistance. There was an increase in beta cell proliferation and beta cell mass that correlated with activation of the PI3K-Akt pathway. The Fxyd2-/- mice are thus in a highly desirable state: the animals are resistant to Na+ retention, and showed improved glucose control, i.e. they display favorable metabolic adaptations to protect against development of salt-sensitive hypertension and diabetes. Investigation of the mechanisms of these adaptations in the mouse has the potential to unveil a novel therapeutic FXYD2-dependent strategy.

  14. Reproductive organ and vascular specific promoter of the rice plasma membrane Ca2+ATPase mediates environmental stress responses in plants.

    Directory of Open Access Journals (Sweden)

    Kazi Md Kamrul Huda

    Full Text Available Plasma membrane Ca(2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca(2+ from the cell, hence regulating Ca(2+ level within cells. Though plant Ca(2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied.The 1478 bp promoter sequence of rice plasma membrane Ca(2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca(2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The -1478 to -886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for -1210 and -886 bp flanking region. The -1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The -1210 and -886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the -886 bp and -519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs.The rice plasma membrane Ca(2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible

  15. Effects of different kinds of fluorides on enolase and ATPase activity of a fluoride-sensitive and fluoride-resistant Streptococcus mutans strain.

    Science.gov (United States)

    van Loveren, C; Hoogenkamp, M A; Deng, D M; ten Cate, J M

    2008-01-01

    Enolase and ATPase are sensitive to fluoride. It is unclear whether this sensitivity differs for F-sensitive and F-resistant cells or for different types of fluoride. Permeabilized cells of the fluoride-sensitive strain Streptococcus mutans C180-2 and its fluoride-resistant mutant strain C180-2 FR were preincubated at pH 7 or 4 with NaF, the amine fluorides Olaflur and Dectaflur and amine chloride controls. After preincubations, enolase and ATPase activities of the cells were assessed. Enolase activity was more inhibited after preincubation at pH 7 with NaF than with Olaflur. Amine chloride stimulated, although not with statistical significance, the enolase activity of both strains. After preincubation at pH 4 the enolases were strongly inactivated, but the fluoride-resistant strain's enolase to a lesser extent. The results suggested that amine acts to protect enolase activity against the detrimental low pH effect. Gene sequencing showed that the enolase genes of the fluoride-resistant and fluoride-sensitive strain were identical. ATPase activity was not reduced after NaF preincubation at either pH 7 or pH 4. The amine fluorides and their chloride controls in the preincubation mixture reduced the ATPase activity significantly at both pH values. In conclusion, our results showed that preincubation with amine fluoride did not inhibit enolase activity more effectively than NaF. The amine part of the molecule may protect enolase activity against preincubations at low pH. ATPase activity was not inhibited by NaF preincubation but was significantly inhibited after preincubation with amine fluorides and amine chlorides. (c) 2008 S. Karger AG, Basel.

  16. Regulation of Na+/K+ ATPase transport velocity by RNA editing.

    Directory of Open Access Journals (Sweden)

    Claudia Colina

    2010-11-01

    Full Text Available Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+/K(+ pumps in order to maintain ion homeostasis. In this study we show that Na(+/K(+ pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na(+/K(+ ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na(+ release to the extracellular solution, causing a higher turnover rate at the resting membrane potential.

  17. V-ATPase-Mediated Granular Acidification Is Regulated by the V-ATPase Accessory Subunit Ac45 in POMC-Producing Cells

    Science.gov (United States)

    Jansen, Eric J. R.; Hafmans, Theo G. M.

    2010-01-01

    The vacuolar (H+)-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit of the pump. Here we studied as a candidate V-ATPase regulator the neuroendocrine V-ATPase accessory subunit Ac45. We transgenically manipulated the expression levels of the Ac45 protein specifically in Xenopus intermediate pituitary melanotrope cells and analyzed in detail the functioning of the transgenic cells. We found in the transgenic melanotrope cells the following: i) significantly increased granular acidification; ii) reduced sensitivity for a V-ATPase-specific inhibitor; iii) enhanced early processing of proopiomelanocortin (POMC) by prohormone convertase PC1; iv) reduced, neutral pH–dependent cleavage of the PC2 chaperone 7B2; v) reduced 7B2-proPC2 dissociation and consequently reduced proPC2 maturation; vi) decreased levels of mature PC2 and consequently reduced late POMC processing. Together, our results show that the V-ATPase accessory subunit Ac45 represents the first regulator of the proton pump and controls V-ATPase-mediated granular acidification that is necessary for efficient prohormone processing. PMID:20702583

  18. Vacuolar H(+)-ATPase and plasma membrane H(+)-ATPase contribute to the tolerance against high-pressure carbon dioxide treatment in Saccharomyces cerevisiae.

    Science.gov (United States)

    Watanabe, Taisuke; Furukawa, Soichi; Kitamoto, Katsuhiko; Takatsuki, Akira; Hirata, Ryogo; Ogihara, Hirokazu; Yamasaki, Makari

    2005-11-25

    As a non-thermal sterilization process, high-pressure carbon dioxide treatment (HPCT) is considered to be promising. The main sterilizing effect of HPCT is thought to be acidification in cytoplasm of microorganisms. We investigated the tolerance mechanism of Saccharomyces cerevisiae to HPCT with special reference to vacuolar and plasma membrane H(+)-ATPases. HPCT was imposed at 35 degrees C, 4 to 10 MPa, for 10 min. slp1 mutant defective in vacuole morphogenesis was more sensitive to HPCT than its isogenic parent. Concanamycin A, a specific inhibitor of vacuolar H(+)-ATPase (V-ATPase), at 10 microM rendered the parent more HPCT-sensitive to the level of slp1. To confirm further the contribution of V-ATPase to the tolerance against HPCT in S. cerevisiae, we compared vma1 mutant of V-ATPase with its isogenic parent for their HPCT sensitivity. vma1 mutant was more sensitive to HPCT than its parent. Addition of 10 microM vanadate, an inhibitor of plasma membrane H(+)-ATPase (P-ATPase), to the wild type strains also increased the inactivation ratio. These results clearly show that V- and P-ATPases contribute to the tolerance against HPCT in S. cerevisiae by accumulating excess H(+) from cytoplasm to vacuole and excluding H(+) outside of the cell, respectively.

  19. Activation of Na+-K+-ATPase with DRm217 attenuates oxidative stress-induced myocardial cell injury via closing Na+-K+-ATPase/Src/Ros amplifier.

    Science.gov (United States)

    Yan, Xiaofei; Xun, Meng; Dou, Xiaojuan; Wu, Litao; Zhang, Fujun; Zheng, Jin

    2017-04-01

    Reduced Na+-K+-ATPase activity has close relationship with cardiomyocyte death. Reactive oxygen species (ROS) also plays an important role in cardiac cell damage. It has been proved that Na+-K+-ATPase and ROS form a feed-forward amplifier. The aim of this study was to explore whether DRm217, a proved Na+/K+-ATPase's DR-region specific monoclonal antibody and direct activator, could disrupt Na+-K+-ATPase/ROS amplifier and protect cardiac cells from ROS-induced injury. We found that DRm217 protected myocardial cells against hydrogen peroxide (H2O2)-induced cardiac cell injury and mitochondrial dysfunction. DRm217 also alleviated the effect of H2O2 on inhibition of Na+-K+-ATPase activity, Na+-K+-ATPase cell surface expression, and Src phosphorylation. H2O2-treatment increased intracellular ROS, mitochondrial ROS and induced intracellular Ca2+, mitochondrial Ca2+ overload. DRm217 closed Na+-K+-ATPase/ROS amplifier, alleviated Ca2+ accumulation and finally inhibited ROS and mitochondrial ROS generation. These novel results may help us to understand the important role of the Na+-K+-ATPase in oxidative stress and oxidative stress-related disease.

  20. WRNIP1 accumulates at laser light irradiated sites rapidly via its ubiquitin-binding zinc finger domain and independently from its ATPase domain

    Energy Technology Data Exchange (ETDEWEB)

    Nomura, Hironoshin [Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki Aoba 6-3, Aobaku, Sendai 980-8578 (Japan); Yoshimura, Akari, E-mail: akari_yo@musashino-u.ac.jp [Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan); Edo, Takato [Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki Aoba 6-3, Aobaku, Sendai 980-8578 (Japan); Kanno, Shin-ichiro [Division of Dynamic Proteome, Institute of Development, Aging and Cancer, Tohoku University, Seiryomachi 4-1, Aobaku, Sendai 980-8575 (Japan); Tada, Syusuke [Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Ichihara, Chiba 290-0193 (Japan); Seki, Masayuki [Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki Aoba 6-3, Aobaku, Sendai 980-8578 (Japan); Yasui, Akira [Division of Dynamic Proteome, Institute of Development, Aging and Cancer, Tohoku University, Seiryomachi 4-1, Aobaku, Sendai 980-8575 (Japan); Enomoto, Takemi [Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer WRNIP1 accumulates in laser light irradiated sites very rapidly via UBZ domain. Black-Right-Pointing-Pointer The ATPase domain of WRNIP1 is dispensable for its accumulation. Black-Right-Pointing-Pointer The accumulation of WRNIP1 seems not to be dependent on the interaction with WRN. -- Abstract: WRNIP1 (Werner helicase-interacting protein 1) was originally identified as a protein that interacts with the Werner syndrome responsible gene product. WRNIP1 contains a ubiquitin-binding zinc-finger (UBZ) domain in the N-terminal region and two leucine zipper motifs in the C-terminal region. In addition, it possesses an ATPase domain in the middle of the molecule and the lysine residues serving as ubiquitin acceptors in the entire of the molecule. Here, we report that WRNIP1 accumulates in laser light irradiated sites very rapidly via its ubiquitin-binding zinc finger domain, which is known to bind polyubiquitin and to be involved in ubiquitination of WRNIP1 itself. The accumulation of WRNIP1 in laser light irradiated sites also required the C-terminal region containing two leucine zippers, which is reportedly involved in the oligomerization of WRNIP1. Mutated WRNIP1 with a deleted ATPase domain or with mutations in lysine residues, which serve as ubiquitin acceptors, accumulated in laser light irradiated sites, suggesting that the ATPase domain of WRNIP1 and ubiquitination of WRNIP1 are dispensable for the accumulation.

  1. Oscillations in glycolysis in Saccharomyces cerevisiae: the role of autocatalysis and intracellular ATPase activity.

    Science.gov (United States)

    Kloster, Antonina; Olsen, Lars Folke

    2012-05-01

    We have investigated the glycolytic oscillations, measured as NADH autofluorescence, in the yeast Saccharomyces cerevisiae in a batch reactor. Specifically, we have tested the effect of cell density and a number of inhibitors or activators of ATPase activity on the amplitude of the oscillations. The amplitude dependence on cell density shows the same behavior as that observed in cells in a CSTR. Furthermore, the amplitude decreases with increasing inhibition of the three ATPases (i) F(0)F(1) ATPase, (ii) plasma membrane ATPase (Pma1p) and (iii) vacuolar ATPase (V-ATPase). The amplitude of the oscillations also decreases by stimulating the ATPase activity, e.g. by FCCP or Amphotericin B. Thus, ATPase activity strongly affects the glycolytic oscillations. We discuss these data in relation to a simple autocatalytic model of glycolysis which can reproduce the experimental data and explain the role of membrane-bound ATPases . In addition we also studied a recent detailed model of glycolysis and found that, although this model faithfully reproduces the oscillations of glycolytic intermediates observed experimentally, it is not able to explain the role of ATPase activity on the oscillations. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Saccharomyces cerevisiae vacuolar H+-ATPase regulation by disassembly and reassembly: one structure and multiple signals.

    Science.gov (United States)

    Parra, Karlett J; Chan, Chun-Yuan; Chen, Jun

    2014-06-01

    Vacuolar H(+)-ATPases (V-ATPases) are highly conserved ATP-driven proton pumps responsible for acidification of intracellular compartments. V-ATPase proton transport energizes secondary transport systems and is essential for lysosomal/vacuolar and endosomal functions. These dynamic molecular motors are composed of multiple subunits regulated in part by reversible disassembly, which reversibly inactivates them. Reversible disassembly is intertwined with glycolysis, the RAS/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, and phosphoinositides, but the mechanisms involved are elusive. The atomic- and pseudo-atomic-resolution structures of the V-ATPases are shedding light on the molecular dynamics that regulate V-ATPase assembly. Although all eukaryotic V-ATPases may be built with an inherent capacity to reversibly disassemble, not all do so. V-ATPase subunit isoforms and their interactions with membrane lipids and a V-ATPase-exclusive chaperone influence V-ATPase assembly. This minireview reports on the mechanisms governing reversible disassembly in the yeast Saccharomyces cerevisiae, keeping in perspective our present understanding of the V-ATPase architecture and its alignment with the cellular processes and signals involved. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. Nonequilibrium Energetics of a Single F1-ATPase Molecule

    OpenAIRE

    Toyabe, Shoichi; Watanabe-Nakayama, Takahiro; Okamoto, Tetsuaki; Kudo, Seishi; Muneyuki, Eiro

    2010-01-01

    Molecular motors drive mechanical motions utilizing the free energy liberated from chemical reactions such as ATP hydrolysis. Although it is essential to know the efficiency of this free energy transduction, it has been a challenge due to the system's microscopic scale. Here, we evaluate the single-molecule energetics of a rotary molecular motor, F1-ATPase, by applying a recently derived nonequilibrium equality together with an electrorotation method. We show that the sum of the heat flow thr...

  4. Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening

    OpenAIRE

    Elbatsh, Ahmed M.O.; Haarhuis, Judith H.I.; Petela, Naomi; Chapard, Christophe; Fish, Alexander; Celie, Patrick H.; Stadnik, Magda; Ristic, Dejan; Wyman, Claire; Medema, Rene; Nasmyth, Kim; Rowland, Benjamin D.

    2016-01-01

    textabstractCohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sor...

  5. Identification of Small-Molecule Inhibitors of Yersinia pestis Type III Secretion System YscN ATPase

    OpenAIRE

    Swietnicki, Wieslaw; Carmany, Daniel; Retford, Michael; Guelta, Mark; Dorsey, Russell; Bozue, Joel; Lee, Michael S.; Olson, Mark A.

    2011-01-01

    Yersinia pestis is a gram negative zoonotic pathogen responsible for causing bubonic and pneumonic plague in humans. The pathogen uses a type III secretion system (T3SS) to deliver virulence factors directly from bacterium into host mammalian cells. The system contains a single ATPase, YscN, necessary for delivery of virulence factors. In this work, we show that deletion of the catalytic domain of the yscN gene in Y. pestis CO92 attenuated the strain over three million-fold in the Swiss-Webst...

  6. Saccharomyces cerevisiae mutants affected in vacuole assembly or vacuolar H+-ATPase are hypersensitive to lead (Pb) toxicity

    OpenAIRE

    Sousa, Cátia A.; Perez, Rita R.; Soares, Eduardo V.

    2014-01-01

    Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16Δ), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16Δ mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H+-ATPase (V-ATPas...

  7. V-ATPase as an effective therapeutic target for sarcomas

    Energy Technology Data Exchange (ETDEWEB)

    Perut, Francesca, E-mail: francesca.perut@ior.it [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Avnet, Sofia; Fotia, Caterina; Baglìo, Serena Rubina; Salerno, Manuela [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Hosogi, Shigekuni [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kusuzaki, Katsuyuki [Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Baldini, Nicola [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna (Italy)

    2014-01-01

    Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment. - Highlights: • Osteosarcoma, rhabdomyosarcoma, and chondrosarcoma survive in acidic microenvironment. • At acidic extracellular pH, sarcoma survival is dependent on V-ATPase expression. • Esomeprazole administration induce a significant dose-dependent toxicity.

  8. Rotary ATPases: models, machine elements and technical specifications.

    Science.gov (United States)

    Stewart, Alastair G; Sobti, Meghna; Harvey, Richard P; Stock, Daniela

    2013-01-01

    Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual "machine elements" to the requirement of the right "fuel" and "oil" for different types of motors.

  9. Spectrophotometric assay of renal ouabain-resistant Na(+)-ATPase and its regulation by leptin and dietary-induced obesity.

    Science.gov (United States)

    Bełtowski, Jerzy; Jamroz-Wiśniewska, Anna; Nazar, Jarosław; Wójcicka, Grazyna

    2004-01-01

    Apart from Na(+),K(+)-ATPase, a second sodium pump, Na(+)-stimulated, K(+)-independent ATPase (Na(+)-ATPase) is expressed in proximal convoluted tubule of the mammalian kidney. The aim of this study was to develop a method of Na(+)-ATPase assay based on the method previously used by us to measure Na(+),K(+)-ATPase activity. The ATPase activity was assayed as the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Na(+)-ATPase activity was calculated as the difference between the activities measured in the presence and in the absence of 50 mM NaCl. Na(+)-ATPase activity was detected in the renal cortex (3.5 +/- 0.2 mumol phosphate/h per mg protein), but not in the renal medulla. Na(+)-ATPase was not inhibited by ouabain or an H(+),K(+)-ATPase inhibitor, Sch 28080, but was almost completely blocked by 2 mM furosemide. Leptin administered intraperitoneally (1 mg/kg) decreased the Na(+),K(+)-ATPase activity in the renal medulla at 0.5 and 1 h by 22.1% and 27.1%, respectively, but had no effect on Na(+)-ATPase in the renal cortex. Chronic hyperleptinemia induced by repeated subcutaneous leptin injections (0.25 mg/kg twice daily for 7 days) increased cortical Na(+),K(+)-ATPase, medullary Na(+),K(+)-ATPase and cortical Na(+)-ATPase by 32.4%, 84.2% and 62.9%, respectively. In rats with dietary-induced obesity, the Na(+),K(+)- ATPase activity was higher in the renal cortex and medulla by 19.7% and 34.3%, respectively, but Na(+)-ATPase was not different from control. These data indicate that both renal Na(+)-dependent ATPases are separately regulated and that up-regulation of Na(+)-ATPase may contribute to Na(+) retention and arterial hypertension induced by chronic hyperleptinemia.

  10. A Yersinia pestis YscN ATPase mutant functions as a live attenuated vaccine against bubonic plague in mice.

    Science.gov (United States)

    Bozue, Joel; Cote, Christopher K; Webster, Wendy; Bassett, Anthony; Tobery, Steven; Little, Stephen; Swietnicki, Wieslaw

    2012-07-01

    Yersinia pestis is the causative agent responsible for bubonic and pneumonic plague. The bacterium uses the pLcr plasmid-encoded type III secretion system to deliver virulence factors into host cells. Delivery requires ATP hydrolysis by the YscN ATPase encoded by the yscN gene also on pLcr. A yscN mutant was constructed in the fully virulent CO92 strain containing a nonpolar, in-frame internal deletion within the gene. We demonstrate that CO92 with a yscN mutation was not able to secrete the LcrV protein (V-Antigen) and attenuated in a subcutaneous model of plague demonstrating that the YscN ATPase was essential for virulence. However, if the yscN mutant was complemented with a functional yscN gene in trans, virulence was restored. To evaluate the mutant as a live vaccine, Swiss-Webster mice were vaccinated twice with the ΔyscN mutant at varying doses and were protected against bubonic plague in a dose-dependent manner. Antibodies to F1 capsule but not to LcrV were detected in sera from the vaccinated mice. These preliminary results suggest a proof-of-concept for an attenuated, genetically engineered, live vaccine effective against bubonic plague. Published 2012. This article is a US Government work and is in the public domain in the USA.

  11. The role of the plasma membrane H+-ATPase in plant-microbe interactions.

    Science.gov (United States)

    Elmore, James Mitch; Coaker, Gitta

    2011-05-01

    Plasma membrane (PM) H+-ATPases are the primary pumps responsible for the establishment of cellular membrane potential in plants. In addition to regulating basic aspects of plant cell function, these enzymes contribute to signaling events in response to diverse environmental stimuli. Here, we focus on the roles of the PM H+-ATPase during plant-pathogen interactions. PM H+-ATPases are dynamically regulated during plant immune responses and recent quantitative proteomics studies suggest complex spatial and temporal modulation of PM H+-ATPase activity during early pathogen recognition events. Additional data indicate that PM H+-ATPases cooperate with the plant immune signaling protein RIN4 to regulate stomatal apertures during bacterial invasion of leaf tissue. Furthermore, pathogens have evolved mechanisms to manipulate PM H+-ATPase activity during infection. Thus, these ubiquitous plant enzymes contribute to plant immune responses and are targeted by pathogens to increase plant susceptibility.

  12. Biochemical and biological characterization of wild-type and ATPase-deficient Cockayne syndrome B repair protein.

    Science.gov (United States)

    Citterio, E; Rademakers, S; van der Horst, G T; van Gool, A J; Hoeijmakers, J H; Vermeulen, W

    1998-05-08

    Cockayne syndrome (CS) is a nucleotide excision repair disorder characterized by sun (UV) sensitivity and severe developmental problems. Two genes have been shown to be involved: CSA and CSB. Both proteins play an essential role in preferential repair of transcription-blocking lesions from active genes. In this study we report the purification and characterization of baculovirus-produced HA-His6-tagged CSB protein (dtCSB), using a highly efficient three-step purification protocol. Microinjection of dtCSB protein in CS-B fibroblasts shows that it is biologically functional in vivo. dtCSB exhibits DNA-dependent ATPase activity, stimulated by naked as well as nucleosomal DNA. Using structurally defined DNA oligonucleotides, we show that double-stranded DNA and double-stranded DNA with partial single-stranded character but not true single-stranded DNA act as efficient cofactors for CSB ATPase activity. Using a variety of substrates, no overt DNA unwinding by dtCSB could be detected, as found with other SNF2/SWI2 family proteins. By site-directed mutagenesis the invariant lysine residue in the NTP-binding motif of CSB was substituted with a physicochemically related arginine. As expected, this mutation abolished ATPase activity. Surprisingly, the mutant protein was nevertheless able to partially rescue the defect in recovery of RNA synthesis after UV upon microinjection in CS-B fibroblasts. These results indicate that integrity of the conserved nucleotide-binding domain is important for the in vivo function of CSB but that also other properties independent from ATP hydrolysis may contribute to CSB biological functions.

  13. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  14. F-actin reorganization by V-ATPase inhibition in prostate cancer.

    Science.gov (United States)

    Licon-Munoz, Yamhilette; Michel, Vera; Fordyce, Colleen A; Parra, Karlett J

    2017-11-15

    The vacuolar ATPase (V-ATPase) proton pump sustains cellular pH homeostasis, and its inhibition triggers numerous stress responses. However, the cellular mechanisms involved remain largely elusive in cancer cells. We studied V-ATPase in the prostate cancer (PCa) cell line PC-3, which has characteristics of highly metastatic PCa. V-ATPase inhibitors impaired endo-lysosomal pH, vesicle trafficking, migration, and invasion. V-ATPase accrual in the Golgi and recycling endosomes suggests that traffic of internalized membrane vesicles back to the plasma membrane was particularly impaired. Directed movement provoked co-localization of V-ATPase containing vesicles with F-actin near the leading edge of migrating cells. V-ATPase inhibition prompted prominent F-actin cytoskeleton reorganization. Filopodial projections were reduced, which related to reduced migration velocity. F-actin formed novel cytoplasmic rings. F-actin rings increased with extended exposure to sublethal concentrations of V-ATPase inhibitors, from 24 to 48 h, as the amount of alkalinized endo-lysosomal vesicles increased. Studies with chloroquine indicated that F-actin rings formation was pH-dependent. We hypothesize that these novel F-actin rings assemble to overcome widespread traffic defects caused by V-ATPase inhibition, similar to F-actin rings on the surface of exocytic organelles. © 2017. Published by The Company of Biologists Ltd.

  15. [Effects of cocaine on activities of ATPase, LDH and SDH in mouse splenocytes].

    Science.gov (United States)

    Sun, Wen-ping; Lu, Yan-xu; Zhang, Xiao-yu; Tang, Wei-wei; Huang, Qing-yu

    2010-04-01

    To examine the effects of cocaine on the activities of ATPase, LDH and SDH in cultured mouse splenocytes in vitro. The ATPase, LDH and SDH activities in mouse splenocytes were detected at day 7 after continuous culturing the mouse cells exposed to cocaine hydrochloride in final concentration of 10, 20 and 100 microg/mL in vitro. The activities of ATPase, LDH and SDH in mouse splenocytes exposed to cocaine hydrochloride in final concentration of 10, 20 and 100 microg/mL were significantly decreased after continuous culturing for 7 days. The present study demonstrated that cocaine could inhibit the activities of ATPase, LDH and SDH in cultured splenocytes in vitro.

  16. Abscisic acid induction of vacuolar H+-ATPase activity in mesembryanthemum crystallinum is developmentally regulated

    Science.gov (United States)

    Barkla; Vera-Estrella; Maldonado-Gama; Pantoja

    1999-07-01

    Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways.

  17. Size of the plasma membrane H+-ATPase from Neurospora crassa determined by radiation inactivation and comparison with the sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle.

    Science.gov (United States)

    Bowman, B J; Berenski, C J; Jung, C Y

    1985-07-25

    Using radiation inactivation, we have measured the size of the H+-ATPase in Neurospora crassa plasma membranes. Membranes were exposed to either high energy electrons from a Van de Graaff generator or to gamma irradiation from 60Co. Both forms of radiation caused an exponential loss of ATPase activity in parallel with the physical destruction of the Mr = 104,000 polypeptide of which this enzyme is composed. By applying target theory, the size of the H+-ATPase in situ was found to be approximately 2.3 X 10(5) daltons. We also used radiation inactivation to measure the size of the Ca2+-ATPase of sarcoplasmic reticulum and got a value of approximately 2.4 X 10(5) daltons, in agreement with previous reports. By irradiating a mixture of Neurospora plasma membranes and rabbit sarcoplasmic reticulum, we directly compared the sizes of these two ATPases and found them to be essentially the same. We conclude that both H+-ATPase and Ca2+-ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides.

  18. The Plasma Membrane Calcium ATPases and Their Role as Major New Players in Human Disease.

    Science.gov (United States)

    Stafford, Nicholas; Wilson, Claire; Oceandy, Delvac; Neyses, Ludwig; Cartwright, Elizabeth J

    2017-07-01

    The Ca2+ extrusion function of the four mammalian isoforms of the plasma membrane calcium ATPases (PMCAs) is well established. There is also ever-increasing detail known of their roles in global and local Ca2+ homeostasis and intracellular Ca2+ signaling in a wide variety of cell types and tissues. It is becoming clear that the spatiotemporal patterns of expression of the PMCAs and the fact that their abundances and relative expression levels vary from cell type to cell type both reflect and impact on their specific functions in these cells. Over recent years it has become increasingly apparent that these genes have potentially significant roles in human health and disease, with PMCAs1-4 being associated with cardiovascular diseases, deafness, autism, ataxia, adenoma, and malarial resistance. This review will bring together evidence of the variety of tissue-specific functions of PMCAs and will highlight the roles these genes play in regulating normal physiological functions and the considerable impact the genes have on human disease. Copyright © 2017 the American Physiological Society.

  19. Regulation of Vacuolar H+-ATPase (V-ATPase) Reassembly by Glycolysis Flow in 6-Phosphofructo-1-kinase (PFK-1)-deficient Yeast Cells.

    Science.gov (United States)

    Chan, Chun-Yuan; Dominguez, Dennis; Parra, Karlett J

    2016-07-22

    Yeast 6-phosphofructo-1-kinase (PFK-1) has two subunits, Pfk1p and Pfk2p. Deletion of Pfk2p alters glucose-dependent V-ATPase reassembly and vacuolar acidification (Chan, C. Y., and Parra, K. J. (2014) Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. J. Biol. Chem. 289, 19448-19457). This study capitalized on the mechanisms suppressing vacuolar H(+)-ATPase (V-ATPase) in pfk2Δ to gain new knowledge of the mechanisms underlying glucose-dependent V-ATPase regulation. Because V-ATPase is fully assembled in pfk2Δ, and glycolysis partially suppressed at steady state, we manipulated glycolysis and assessed its direct involvement on V-ATPase function. At steady state, the ratio of proton transport to ATP hydrolysis increased 24% after increasing the glucose concentration from 2% to 4% to enhance the glycolysis flow in pfk2Δ. Tighter coupling restored vacuolar pH when glucose was abundant and glycolysis operated below capacity. After readdition of glucose to glucose-deprived cells, glucose-dependent V1Vo reassembly was proportional to the glycolysis flow. Readdition of 2% glucose to pfk2Δ cells, which restored 62% of ethanol concentration, led to equivalent 60% V1Vo reassembly levels. Steady-state level of assembly (100% reassembly) was reached at 4% glucose when glycolysis reached a threshold in pfk2Δ (≥40% the wild-type flow). At 4% glucose, the level of Pfk1p co-immunoprecipitated with V-ATPase decreased 58% in pfk2Δ, suggesting that Pfk1p binding to V-ATPase may be inhibitory in the mutant. We concluded that V-ATPase activity at steady state and V-ATPase reassembly after readdition of glucose to glucose-deprived cells are controlled by the glycolysis flow. We propose a new mechanism by which glucose regulates V-ATPase catalytic activity that occurs at steady state without changing V1Vo assembly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity.

    Science.gov (United States)

    Pantazatos, S P; Huang, Y-Y; Rosoklija, G B; Dwork, A J; Arango, V; Mann, J J

    2017-05-01

    Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in the dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden death medication-free individuals post mortem. Using small RNA-seq, we also examined miRNA expression (nine samples per group). DeSeq2 identified 35 genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted Pdepression, altered genes include humanin-like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted Psuicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted Pdepression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders.

  1. Structure and Function of a Novel ATPase that Interacts with Holliday Junction Resolvase Hjc and Promotes Branch Migration

    Science.gov (United States)

    Zhai, Binyuan; DuPrez, Kevin; Doukov, Tzanko I.; Li, Huan; Huang, Mengting; Shang, Guijun; Ni, Jinfeng; Gu, Lichuan; Shen, Yulong; Fan, Li

    2017-01-01

    Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicus PilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage. PMID:28238763

  2. Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

    Directory of Open Access Journals (Sweden)

    Narendranath Reddy Chintagari

    2010-02-01

    Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

  3. Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase.

    Science.gov (United States)

    Zhao, Jianhua; Benlekbir, Samir; Rubinstein, John L

    2015-05-14

    Eukaryotic vacuolar H(+)-ATPases (V-ATPases) are rotary enzymes that use energy from hydrolysis of ATP to ADP to pump protons across membranes and control the pH of many intracellular compartments. ATP hydrolysis in the soluble catalytic region of the enzyme is coupled to proton translocation through the membrane-bound region by rotation of a central rotor subcomplex, with peripheral stalks preventing the entire membrane-bound region from turning with the rotor. The eukaryotic V-ATPase is the most complex rotary ATPase: it has three peripheral stalks, a hetero-oligomeric proton-conducting proteolipid ring, several subunits not found in other rotary ATPases, and is regulated by reversible dissociation of its catalytic and proton-conducting regions. Studies of ATP synthases, V-ATPases, and bacterial/archaeal V/A-ATPases have suggested that flexibility is necessary for the catalytic mechanism of rotary ATPases, but the structures of different rotational states have never been observed experimentally. Here we use electron cryomicroscopy to obtain structures for three rotational states of the V-ATPase from the yeast Saccharomyces cerevisiae. The resulting series of structures shows ten proteolipid subunits in the c-ring, setting the ATP:H(+) ratio for proton pumping by the V-ATPase at 3:10, and reveals long and highly tilted transmembrane α-helices in the a-subunit that interact with the c-ring. The three different maps reveal the conformational changes that occur to couple rotation in the symmetry-mismatched soluble catalytic region to the membrane-bound proton-translocating region. Almost all of the subunits of the enzyme undergo conformational changes during the transitions between these three rotational states. The structures of these states provide direct evidence that deformation during rotation enables the smooth transmission of power through rotary ATPases.

  4. Effects of phenol on ATPase activities in crude gill homogenates of rainbow trout (Salmo gairdneri Richardson)

    Energy Technology Data Exchange (ETDEWEB)

    Poston, T.M.

    1979-01-01

    The ATPase specific activities from crude gill homogenates of rainbow trout were lower than those from microsomal preparations reported in the literature. Sodium pump activity (ouabain sensitive NaK-ATPase) was demonstrable at 37/sup 0/C. An ouabain insensitive NaK-ATPase was demonstrable at temperatures below 30/sup 0/C and may represent a Na-ATPase activity reported by others. Energy of activation at 25/sup 0/C for total NaK-ATPase ws 10,500 cal.mole/sup -1/. Mg-baseline activity had an energy of activation at 25/sup 0/C of 15,600 cal.mole/sup -1/. Mg-baseline activity was thermally labile at temperatures in excess of 30/sup 0/C. Concentrations of Mg/sup +2/ in excess of 5 mM appeared to inhibit total NaK-ATPase activity. At 37/sup 0/C, Na/sup +/ and K/sup +/ exerted little, if any, stimulatory effect on ATPase activities, in spite of the fact that 37/sup 0/C was the only temperature at which sodium pump activity was demonstrable. MS-222 failed to produce any discernible changes in any of the demonstrable ATPase activities in crude gill homogenates. Total NaK-ATPase activities were more sensitive than Mg-baseline activities to in vitro inhibition by phenol. Concentrations of phenol which produce 50% inhibition in total NaK-ATPase produced only 35% inhibition in Mg-baseline activity. The nature of in vitro inhibition was uncompetitive. Sodium pump activity was unaffected by phenol at concentrations as high as 25 mM. An effort was made to demonstrate an in vivo effects of phenol on rainbow trout gill ATPase activites. An infestation of a parasite (Gyrodactilus) on the experimental fish precludes any definative assessment of in vivo effects.

  5. Peran Enzim Ca2+ - ATPase pada Membran Sel Darah Merah

    Directory of Open Access Journals (Sweden)

    Ferry P. Gultom

    2015-10-01

    Full Text Available Ca2+ - ATP ase enzyme is transmembrane protein which is found in membrane cell. This protein works as a pump in many cases such as thalassemia which causes leakage of the cell as there is oxidation of sulphidril chain from amino acid in membrane. The calcium-ion intake must be pumped out to get red blood cell homeostatic condition. The aim of this paper is to determine the activity of Ca2+ - ATPase enzyme as a pump of Ca ion in membrane cell.

  6. New ATPase regulators-p97 goes to the PUB

    DEFF Research Database (Denmark)

    Madsen, Louise; Seeger, Michael; Semple, Colin A

    2009-01-01

    The conserved eukaryotic AAA-type ATPase complex, known as p97 or VCP in mammals and Cdc48 in yeast, is involved in a number of cellular pathways, including fusion of homotypic membranes, protein degradation, and activation of membrane-bound transcription factors. Most likely, p97 is directed....... Recently, a small, conserved family of proteins, containing PUB domains, was found to function as p97 adaptors. Intriguingly, their association with p97 is regulated by tyrosine phosphorylation, suggesting that they act as a relay between signalling pathways and p97 functions. Here we give an overview...

  7. Cloning and characterization of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish axial muscle. Sarco/Endoplasmic Reticulum Ca(2+)-ATPase.

    Science.gov (United States)

    Zhang, Z; Chen, D; Wheatly, M G

    2000-11-01

    The discontinuous pattern of muscle growth during the moulting cycle of a freshwater crustacean (the crayfish Procambarus clarkii) was used as a model system to examine the regulation of the expression of Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA). We describe the cloning, sequencing and characterization of a novel SERCA cDNA (3856 bp) obtained from crayfish axial abdominal muscle by reverse transcription/polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). This complete sequence contains a 145 base pair (bp) noncoding region at the 5' end, a 3006 bp open reading frame coding for 1002 amino acid residues with a molecular mass of 110 kDa and 705 bp of untranslated region at the 3' end. This enzyme contains all the conserved domains found in 'P'-type ATPases, and the hydropathy profile suggests a transmembrane organization typical of other SERCAs. It exhibits 80% amino acid identity with Drosophila melanogaster SERCA, 79% identity with Artemia franciscana SERCA, 72% identity with rabbit fast-twitch muscle neonatal isoform SERCA1b, 71% identity with slow-twitch muscle isoform SERCA2 and 67% identity with SERCA3. Sequence alignment revealed that regions anchoring the cytoplasmic domain in the membrane were highly conserved and that most differences were in the NH(2) terminus, the central loop region and the COOH terminus. Northern analysis of total RNA from crayfish tissues probed with the 460 bp fragment initially isolated showed four bands (7.6, 7.0, 5.8 and 4.5 kilobases) displaying tissue-specific expression. SERCA was most abundant in muscle (axial abdominal, cardiac and stomach), where it is involved in Ca(2+) resequestration during relaxation, and in eggs, where it may be implicated in early embryogenesis. The level of SERCA mRNA expression in axial abdominal muscle varied during the moulting cycle as determined by slot-blot analysis. SERCA expression was greatest during intermoult and decreased to approximately 50% of

  8. Auxin induces exocytosis and the rapid synthesis of a high-turnover pool of plasma-membrane H(+)-ATPase.

    Science.gov (United States)

    Hager, A; Debus, G; Edel, H G; Stransky, H; Serrano, R

    1991-11-01

    Auxin causes elongation growth of plant cells by increasing the plastic extensibility of the cell wall. Putative cellular events involved in this hormone action were studied using maize (Zea mays L.) coleoptiles with the following results: (i) Auxin enhances membrane flow from the endoplasmic reticulum to the plasma membrane (PM). This effect was demonstrated by pulse-labeling of the endoplasmic reticulum with myo-[(3)H]inositol in coleoptile segments and by measuring the distribution of the label within isolated and separated microsomal membrane fractions, (ii) Auxin rapidly increases the amount of antibody-detectable H(+)-ATPase in the PM. This augmentation is already significant 10 min after the addition of indole-3-acetic acid (IAA) and reaches a new higher steady-state level after about 30 min. (iii) Cycloheximide, a potent inhibitor of both protein synthesis and extension growth, quickly diminishes the auxin-enhanced level of the PM H(+)-ATPase, indicating an apparent half-life of the enzyme of around 12 min. (iv) Cordycepin, which blocks the synthesis of mRNAs, reduces the auxin-elevated level of the H(+)-ATPase similar to cycloheximide. (v) Changes in the growth rate of coleoptile segments in response to IAA, cycloheximide, and cordycepin exactly reflect the changes of the H(+)-ATPase level in the PM. (vi) The elongation growth induced by fusicoccin, or ester compounds, or by an elevated CO2 concentration in the incubation medium, is not related to an increased number of H(+)-ATPase molecules within the PM. (vii) The necessity of H(+) for cell-wall-loosening processes is again demonstrated by growth experiments with abraded coleoptile segments. The adjustment of the cell wall to a pH of ≥6.5 completely abolishes the auxin-induced elongation growth; no inhibition occurs with non-abraded segments. Buffer solutions of pH ≤6.0 induce "acid growth" of abraded segments for several hours. It is suggested that auxin activates a cluster of genes responsible (i

  9. Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging.

    Science.gov (United States)

    Iyer, Lakshminarayan M; Makarova, Kira S; Koonin, Eugene V; Aravind, L

    2004-01-01

    Recently, it has been shown that a predicted P-loop ATPase (the HerA or MlaA protein), which is highly conserved in archaea and also present in many bacteria but absent in eukaryotes, has a bidirectional helicase activity and forms hexameric rings similar to those described for the TrwB ATPase. In this study, the FtsK-HerA superfamily of P-loop ATPases, in which the HerA clade comprises one of the major branches, is analyzed in detail. We show that, in addition to the FtsK and HerA clades, this superfamily includes several families of characterized or predicted ATPases which are predominantly involved in extrusion of DNA and peptides through membrane pores. The DNA-packaging ATPases of various bacteriophages and eukaryotic double-stranded DNA viruses also belong to the FtsK-HerA superfamily. The FtsK protein is the essential bacterial ATPase that is responsible for the correct segregation of daughter chromosomes during cell division. The structural and evolutionary relationship between HerA and FtsK and the nearly perfect complementarity of their phyletic distributions suggest that HerA similarly mediates DNA pumping into the progeny cells during archaeal cell division. It appears likely that the HerA and FtsK families diverged concomitantly with the archaeal-bacterial division and that the last universal common ancestor of modern life forms had an ancestral DNA-pumping ATPase that gave rise to these families. Furthermore, the relationship of these cellular proteins with the packaging ATPases of diverse DNA viruses suggests that a common DNA pumping mechanism might be operational in both cellular and viral genome segregation. The herA gene forms a highly conserved operon with the gene for the NurA nuclease and, in many archaea, also with the orthologs of eukaryotic double-strand break repair proteins MRE11 and Rad50. HerA is predicted to function in a complex with these proteins in DNA pumping and repair of double-stranded breaks introduced during this process and

  10. Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography

    Directory of Open Access Journals (Sweden)

    Geisler Markus

    1998-01-01

    Full Text Available In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993 et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 &mgr;M and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

  11. Role of matrix metalloprotease-2 in oxidant activation of Ca ATPase ...

    Indian Academy of Sciences (India)

    Unknown

    Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H2O2. (1 mM) stimulated Ca2+ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca2+ATPase activity by H2O2 in the smooth muscle plasma membrane. The smooth ...

  12. Interaction of the indole class of vacuolar H+-ATPase inhibitors with lipid bilayers

    NARCIS (Netherlands)

    Fernandes, F.; Loura, L.; Koehorst, R.B.M.; Dixon, N.; Kee, T.P.; Hemminga, M.A.; Prieto, M.

    2006-01-01

    The selective inhibitor of osteoclastic V-ATPase (2Z,4E)-5-(5,6-dichloro-2-indolyi)-2-methoxy-N-(1,2,2,6,6-pentamethylpip eridin-4-yl)-2,4-pentadienamide (SB 242784), member of the indole class of V-ATPase inhibitors, is expected to target the membrane-bound domain of the enzyme. A structural study

  13. Excess capacity of H+ ATPase and inverse respiratory control in Escherichia coli

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Westerhoff, Hans V.; Michelsen, Ole

    1993-01-01

    With succinate as free-energy source, Escherichia coli generating virtually all ATP by oxidative phosphorylation might be expected heavily to tax its ATP generating capacity. To examine this the H+-ATPase (ATP synthase) was modulated over a 30-fold range. Decreasing the amount of H+-ATPase reduced...

  14. Na+,K+-ATPase concentration in rodent and human heart and skeletal muscle

    DEFF Research Database (Denmark)

    Kjeldsen, K; Bjerregaard, P; Richter, Erik

    1988-01-01

    0.02) and the heart to body weight ratio by 14% (p less than 0.005). The increase in Na+,K+-ATPase concentration was only slowly reversible. After three weeks of deconditioning an increase of 12% (p less than 0.05) was still observed. In comparison skeletal muscle Na+,K+-ATPase concentration...

  15. CA2+-ATPASE INHIBITORY ACTIVITY OF A LOCKED ANALOG OF THAPSIGARGIN

    DEFF Research Database (Denmark)

    Andersen, A.; Treiman, M.; Poulsen, J. C. J.

    1994-01-01

    The preparation of a nonionic desoxy-analogue of thapsigargin possessing a Ca2+-ATPase inhibitory potency similar to that of thapsioargin is described.......The preparation of a nonionic desoxy-analogue of thapsigargin possessing a Ca2+-ATPase inhibitory potency similar to that of thapsioargin is described....

  16. Crystal structure of a copper-transporting PIB-type ATPase

    DEFF Research Database (Denmark)

    Gourdon, Pontus; Liu, Xiang-Yu; Skjørringe, Tina

    2011-01-01

    copper entry point at the intracellular interface. Comparisons to Ca(2+)-ATPase suggest an ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site. The structure also provides a framework to analyse missense mutations in the human ATP7A and ATP7B...... proteins associated with Menkes' and Wilson's diseases....

  17. Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model

    Science.gov (United States)

    Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

    2010-01-01

    An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

  18. An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour

    NARCIS (Netherlands)

    Verweij, W.; Spelt, C.; di Sansebastiano, G.-P.; Vermeer, J.; Reale, L.; Ferranti, F.; Koes, R.; Quattrocchio, F.

    2008-01-01

    The regulation of pH in cellular compartments is crucial for intracellular trafficking of vesicles and proteins and the transport of small molecules, including hormones. In endomembrane compartments, pH is regulated by vacuolar H+-ATPase1 (V-ATPase), which, in plants, act together with

  19. A possible mechanism for low affinity of silkworm Na+/K+-ATPase for K.

    Science.gov (United States)

    Homareda, Haruo; Otsu, Masahiro; Yamamoto, Sachiko; Ushimaru, Makoto; Ito, Sayaka; Fukutomi, Toshiyuki; Jo, Taeho; Eishi, Yoshinobu; Hara, Yukichi

    2017-12-01

    The affinity for K+ of silkworm nerve Na+/K+-ATPase is markedly lower than that of mammalian Na+/K+-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K+ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na+/K+-ATPase α and β subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and β subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na+/K+-ATPase α1 subunit. On the other hand, the amino acid identity of the β subunit with mammalian counterparts was as low as 30%. Cloned α and β cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na+/K+-ATPase. Na+/K+-ATPase expressed in the cultured cells showed a low affinity for K+ and a high affinity for Na+, characteristic of the silkworm nerve Na+/K+-ATPase. These results suggest that the β subunit is responsible for the affinity for K+ of Na+/K+-ATPase.

  20. Leptin decreases renal medullary Na(+), K(+)-ATPase activity through phosphatidylinositol 3-kinase dependent mechanism.

    Science.gov (United States)

    Bełtowski, J; Marciniak, A; Wójcicka, G

    2004-06-01

    We examined the effect of leptin on renal function and renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase activities in the rat. Leptin was infused under general anaesthesia into the abdominal aorta proximally to the renal arteries. Leptin infused at doses of 1 and 10 microg/kg/min increased urine output by 40% and 140%, respectively. Urinary Na(+) excretion increased in rats receiving leptin at doses of 0.1, 1, and 10 microg/kg/min by 57.6%, 124.2% and 163.6%, respectively. Leptin had no effect on creatinine clearance, potassium excretion and phosphate excretion. Na(+),K(+)-ATPase activity in the renal medulla of rats treated with 1 and 10 microg/kg/min leptin was lower than in control animals by 25.5% and 33.2%, respectively. In contrast, cortical Na(+),K(+)-ATPase as well as either cortical or medullary ouabain-sensitive H(+),K(+)-ATPase activities did not differ between leptin-treated and control animals. The effect of leptin on Na(+),K(+)-ATPase activity was abolished by actin depolymerizing agents, cytochalazin D and latrunculin B, and by phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002. These results indicate that: 1). natriuretic effect of leptin is mediated, at least in part, by decrease in renal medullary Na(+),K(+)-ATPase activity, 2). inhibition of medullary Na(+),K(+)-ATPase by leptin is mediated by PI3K and requires integrity of actin cytoskeleton.

  1. Hemin reconstitutes proton extrusion in an H+-ATPase-negative mutant of Lactococcus lactis

    DEFF Research Database (Denmark)

    Blank, L.M.; Købmann, Brian Jensen; Michelsen, Ole

    2001-01-01

    H+-ATPase is considered essential for growth of Lactococcus lactis. However, media containing hemin restored the aerobic growth of an H+-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown L. lactis cells...

  2. Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast.

    Science.gov (United States)

    Banerjee, Subhrajit; Kane, Patricia M

    2017-09-15

    Luminal pH and phosphoinositide content are fundamental features of organelle identity. Vacuolar H+-ATPases (V-ATPases) drive organelle acidification in all eukaryotes, and membrane-bound a-subunit isoforms of the V-ATPase are implicated in organelle-specific targeting and regulation. Earlier work demonstrated that the endolysosomal lipid PI(3,5)P2 activates V-ATPases containing the vacuolar a-subunit isoform in Saccharomyces cerevisiae Here we demonstrate that PI(4)P, the predominant Golgi phosphatidylinositol (PI) species, directly interacts with the cytosolic amino terminal (NT) domain of the yeast Golgi V-ATPase a-isoform Stv1. Lysine-84 of Stv1NT is essential for interaction with PI(4)P in vitro and in vivo, and interaction with PI(4)P is required for efficient localization of Stv1-containing V-ATPases. The cytosolic NT domain of the human V-ATPase a2 isoform specifically interacts with PI(4)P in vitro, consistent with its Golgi localization and function. We propose that NT domains of Vo a-subunit isoforms interact specifically with PI lipids in their organelles of residence. These interactions can transmit organelle-specific targeting or regulation information to V-ATPases. © 2017 Banerjee and Kane. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  3. Relationship between Na(+),K(+)-ATPase and cell attachment.

    Science.gov (United States)

    Contreras, R G; Shoshani, L; Flores-Maldonado, C; Lázaro, A; Cereijido, M

    1999-12-01

    A prolonged ouabain blockade of the Na(+),K(+)-ATPase detaches cells from each other and from the substrate. This suggests the existence of a link between pump (P) and attachment (A). In the present work, we report that MDCK-W cells treated with ouabain increase tyrosine phosphorylation and content of active MAP kinase, redistribute molecules involved in cell attachment (occludin, ZO-1, desmoplakin, cytokeratin, alpha-actinin, vinculin and actin), and detach. Genistein and UO126, inhibitors of protein tyrosine kinase and of MAP kinase kinase, respectively, block this detachment. The content of P190(Rho-GAP), a GTPase activating protein of the Rho small G-protein subfamily, is increased by ouabain, suggesting that both the Rho/Rac and MAPK pathways are involved. Another clone of MDCK cells whose Na(+),K(+)-ATPase has a negligible affinity for the drug, show none of the effects described for MDCK-W and remain attached. Ma104 cells, a line that has a high affinity for ouabain and stops pumping, fail to modify phosphorylation, as well as the pattern of distribution of attaching molecules, and remain in the monolayer. Taken together, these results suggest that there is a mechanism (P-->A) that transduces a blockade of the pump in a detachment of the cell from neighbors and substrate, in which Ma104 cells are faulty.

  4. Examining zinc transporting P-type ATPases by genome editing

    DEFF Research Database (Denmark)

    Østerberg, Jeppe Thulin

    Zinc is an essential micronutrient in plants. It is taken up from the soil by the plant roots and transported from there throughout the plant tissues, as a final destination ending up in the plant progeny, the seed. In this route from soil to seed, several bottlenecks occur. The export of zinc...... into apoplastic gaps is required both for xylem loading in the roots and for loading into the apoplastic space in the seeds, the tissue surrounding the next plant generation. The movement of the positively charged zinc ions out of the symplast is against the electrochemical gradient and thus requires active...... transport. Both in the model plant Arabidopsis thaliana and in the cereals, the P1B2-ATPases have been demonstrated to be involved in the loading of zinc into the xylem, and here we demonstrate that the Arabidopsis P1B2-ATPase AtHMA2 and AtHMA4 are involved in loading of zinc into the seed apoplast. The P1B...

  5. Stimulation of Na{sup +}/K{sup +} ATPase activity and Na{sup +} coupled glucose transport by {beta}-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Sopjani, Mentor [Department of Physiology, University of Tuebingen (Germany); Department of Chemistry, University of Prishtina, Kosovo (Country Unknown); Alesutan, Ioana; Wilmes, Jan [Department of Physiology, University of Tuebingen (Germany); Dermaku-Sopjani, Miribane [Department of Physiology, University of Tuebingen (Germany); Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Lam, Rebecca S. [Department of Physiology, University of Tuebingen (Germany); Department of Molecular Neurogenetics, Max Planck Institute of Biophysics, Frankfurt/Main (Germany); Koutsouki, Evgenia [Department of Physiology, University of Tuebingen (Germany); Jakupi, Muharrem [Faculty of Medicine, University of Prishtina, Kosovo (Country Unknown); Foeller, Michael [Department of Physiology, University of Tuebingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen (Germany)

    2010-11-19

    Research highlights: {yields} The oncogenic transcription factor {beta}-catenin stimulates the Na{sup +}/K{sup +}-ATPase. {yields} {beta}-Catenin stimulates SGLT1 dependent Na{sup +}, glucose cotransport. {yields} The effects are independent of transcription. {yields} {beta}-Catenin sensitive transport may contribute to properties of proliferating cells. -- Abstract: {beta}-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. {beta}-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that {beta}-catenin influences membrane transport. To this end, {beta}-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of {beta}-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na{sup +}/K{sup +}-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of {beta}-catenin on the endogenous Na{sup +}/K{sup +}-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of {beta}-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of {beta}-catenin expression. The stimulating effect of {beta}-catenin on both Na{sup +}/K{sup +} ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of {beta}-catenin, i.e. the regulation of transport.

  6. Nitric oxide and Na,K-ATPase activity in rat skeletal muscle

    DEFF Research Database (Denmark)

    Juel, Carsten

    2016-01-01

    activity was depressed by oxidized glutathione. Conclusion: NO and cGMP stimulate the Na,K-ATPase in glycolytic skeletal muscle. Direct S-nitrosylation and interference with S-glutathionylation seem to be excluded. In addition, phosphorylation of phospholemman at serine 68 is not involved. Most likely......Aim: It has been suggested that nitric oxide (NO) stimulates the Na,K-ATPase in cardiac myocytes. Therefore, the aims of this study were to investigate whether NO increases Na,K-ATPase activity in skeletal muscle and, if that is the case, to identify the underlying mechanism. Method: The study used...... isolated rat muscle, muscle homogenates and purified membranes as model systems. Na,K-ATPase activity was quantified from phosphate release due to ATP hydrolysis. Results: Exposure to the NO donor spermine NONOate (10 μm) increased the maximal Na,K-ATPase activity by 27% in isolated glycolytic muscles...

  7. Tetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungal activity

    DEFF Research Database (Denmark)

    Bublitz, Maike; Kjellerup, Lasse; Cohrt, Karen O.Hanlon

    2018-01-01

    We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles...... inhibit adenosine triphosphate (ATP) hydrolysis of the fungal H+-ATPase, depolarize the fungal plasma membrane and exhibit broad-spectrum antifungal activity. Comparative inhibition studies indicate that many tetrahydrocarbazoles also inhibit the mammalian Ca2+-ATPase (SERCA) and Na......+,K+-ATPase with an even higher potency than Pma1. We have located the binding site for this compound class by crystallographic structure determination of a SERCA-tetrahydrocarbazole complex to 3.0 Å resolution, finding that the compound binds to a region above the ion inlet channel of the ATPase. A homology model...

  8. The GHKL ATPase MORC1 Modulates Species-Specific Plant Immunity in Solanaceae.

    Science.gov (United States)

    Manosalva, Patricia; Manohar, Murli; Kogel, Karl-Heinz; Kang, Hong-Gu; Klessig, Daniel F

    2015-08-01

    The microrchidia (MORC) proteins, a subset of the GHKL ATPase superfamily, were recently described as components involved in transcriptional gene silencing and plant immunity in Arabidopsis. To assess the role of MORC1 during resistance to Phytophthora infestans in solanaceous species, we altered the expression of the corresponding MORC1 homologs in potato, tomato, and Nicotiana benthamiana. Basal resistance to P. infestans was compromised in StMORC1-silenced potato and enhanced in overexpressing lines, indicating that StMORC1 positively affects immunity. By contrast, silencing SlMORC1 expression in tomato or NbMORC1 expression in N. benthamiana enhanced basal resistance to this oomycete pathogen. In addition, silencing SlMORC1 further enhanced resistance conferred by two resistance genes in tomato. Transient expression of StMORC1 in N. benthamiana accelerated cell death induced by infestin1 (INF1), whereas SlMORC1 or NbMORC1 suppressed it. Domain-swapping and mutational analyses indicated that the C-terminal region dictates the species-specific effects of the solanaceous MORC1 proteins on INF1-induced cell death. This C-terminal region also was required for homodimerization and phosphorylation of recombinant StMORC1 and SlMORC1, and its transient expression induced spontaneous cell death in N. benthamiana. Thus, this C-terminal region likely plays important roles in both determining and modulating the biological activity of MORC1 proteins.

  9. Secretory pathway Ca2+-ATPases promote in vitro microcalcifications in breast cancer cells.

    Science.gov (United States)

    Dang, Donna; Prasad, Hari; Rao, Rajini

    2017-11-01

    Calcification of the breast is often an outward manifestation of underlying molecular changes that drive carcinogenesis. Up to 50% of all non-palpable breast tumors and 90% of ductal carcinoma in situ present with radiographically dense mineralization in mammographic scans. However, surprisingly little is known about the molecular pathways that lead to microcalcifications in the breast. Here, we report on a rapid and quantitative in vitro assay to monitor microcalcifications in breast cancer cell lines, including MCF7, MDA-MB-231, and Hs578T. We show that the Secretory Pathway Ca 2+ -ATPases SPCA1 and SPCA2 are strongly induced under osteogenic conditions that elicit microcalcifications. SPCA gene expression is significantly elevated in breast cancer subtypes that are associated with microcalcifications. Ectopic expression of SPCA genes drives microcalcifications and is dependent on pumping activity. Conversely, knockdown of SPCA expression significantly attenuates formation of microcalcifications. We propose that high levels of SPCA pumps may initiate mineralization in the secretory pathway by elevating luminal Ca 2+ . Our new findings offer mechanistic insight and functional implications on a widely observed, yet poorly understood radiographic signature of breast cancer. © 2017 Wiley Periodicals, Inc.

  10. [Nucleotide sequence and organization of tRNA-Gly (UCC), tRNA-Arg (UCU), and alpha-subunits of CF1ATPase in chloroplast DNA of Allium porrum].

    Science.gov (United States)

    Tabidze, V D; Kartvelishvili, M G; Dzhincharadze, A G; Beridze, T G

    1994-03-01

    The nucleotide sequence of 1029 bp BamHI-fragment of leek chloroplast DNA (Allium porrum, fam. Liliaceae) has been determined. The fragment contains the 3'-terminal part of the tRNA-Gly (UCC) gene, the tRNA-Arg (UCU) gene and the 3'-terminal domain of the CF1ATPase alpha-subunit gene. The gene arrangement and the nucleotide sequence of this fragment are similar to those of the tobacco chloroplast DNA but differ significantly from that of other monocots in which the region containing these genes underwent extensive recombination.

  11. Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hyeon-Ok [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Hong, Sung-Eun [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Kim, Chang Soon [Department of Microbiological Engineering, Kon-Kuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 143–701 (Korea, Republic of); Park, Jin-Ah; Kim, Jin-Hee; Kim, Ji-Young; Kim, Bora [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Chang, Yoon Hwan; Hong, Seok-Il; Hong, Young Jun [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Park, In-Chul, E-mail: parkic@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Lee, Jin Kyung, E-mail: jklee@kirams.re.kr [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of)

    2015-08-15

    Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H + ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. - Highlights: • Co-treatment with EGFR TKIs and vATPase inhibitors induces synergistic cell death • EGFR TKIs enhance cell sensitivity to vATPase inhibitors via Hif-1α downregulation • Co-treatment of these inhibitors is potentially effective for the treatment of NSCLC.

  12. Retrieval of the vacuolar H-ATPase from phagosomes revealed by live cell imaging.

    Directory of Open Access Journals (Sweden)

    Margaret Clarke

    2010-01-01

    Full Text Available The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.

  13. Na,K-ATPase activity in mouse muscle is regulated by AMPK and PGC-1α.

    Science.gov (United States)

    Ingwersen, Maria S; Kristensen, Michael; Pilegaard, Henriette; Wojtaszewski, Jørgen F P; Richter, Erik A; Juel, Carsten

    2011-07-01

    Na,K-ATPase activity, which is crucial for skeletal muscle function, undergoes acute and long-term regulation in response to muscle activity. The aim of the present study was to test the hypothesis that AMP kinase (AMPK) and the transcriptional coactivator PGC-1α are underlying factors in long-term regulation of Na,K-ATPase isoform (α,β and PLM) abundance and Na(+) affinity. Repeated treatment of mice with the AMPK activator AICAR decreased total PLM protein content but increased PLM phosphorylation, whereas the number of α- and β-subunits remained unchanged. The K(m) for Na(+) stimulation of Na,K-ATPase was reduced (higher affinity) after AICAR treatment. PLM abundance was increased in AMPK kinase-dead mice compared with control mice, but PLM phosphorylation and Na,K-ATPase Na(+) affinity remained unchanged. Na,K-ATPase activity and subunit distribution were also measured in mice with different degrees of PGC-1α expression. Protein abundances of α1 and α2 were reduced in PGC-1α +/- and -/- mice, and the β(1)/β(2) ratio was increased with PGC-1α overexpression (TG mice). PLM protein abundance was decreased in TG mice, but phosphorylation status was unchanged. Na,K-ATPase V (max) was decreased in PCG-1α TG and KO mice. Experimentally in vitro induced phosphorylation of PLM increased Na,K-ATPase Na(+) affinity, confirming that PLM phosphorylation is important for Na,K-ATPase function. In conclusion, both AMPK and PGC-1α regulate PLM abundance, AMPK regulates PLM phosphorylation and PGC-1α expression influences Na,K-ATPase α(1) and α(2) content and β(1)/β(2) isoform ratio. Phosphorylation of the Na,K-ATPase subunit PLM is an important regulatory mechanism.

  14. Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis.

    Science.gov (United States)

    Takahashi, Koji; Hayashi, Ken-ichiro; Kinoshita, Toshinori

    2012-06-01

    The phytohormone auxin is a major regulator of diverse aspects of plant growth and development. The ubiquitin-ligase complex SCF(TIR1/AFB) (for Skp1-Cul1-F-box protein), which includes the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) auxin receptor family, has recently been demonstrated to be critical for auxin-mediated transcriptional regulation. Early-phase auxin-induced hypocotyl elongation, on the other hand, has long been explained by the acid-growth theory, for which proton extrusion by the plasma membrane H(+)-ATPase is a functional prerequisite. However, the mechanism by which auxin mediates H(+)-ATPase activation has yet to be elucidated. Here, we present direct evidence for H(+)-ATPase activation in etiolated hypocotyls of Arabidopsis (Arabidopsis thaliana) by auxin through phosphorylation of the penultimate threonine during early-phase hypocotyl elongation. Application of the natural auxin indole-3-acetic acid (IAA) to endogenous auxin-depleted hypocotyl sections induced phosphorylation of the penultimate threonine of the H(+)-ATPase and increased H(+)-ATPase activity without altering the amount of the enzyme. Changes in both the phosphorylation level of H(+)-ATPase and IAA-induced elongation were similarly concentration dependent. Furthermore, IAA-induced H(+)-ATPase phosphorylation occurred in a tir1-1 afb2-3 double mutant, which is severely defective in auxin-mediated transcriptional regulation. In addition, α-(phenylethyl-2-one)-IAA, the auxin antagonist specific for the nuclear auxin receptor TIR1/AFBs, had no effect on IAA-induced H(+)-ATPase phosphorylation. These results suggest that the TIR1/AFB auxin receptor family is not involved in auxin-induced H(+)-ATPase phosphorylation. Our results define the activation mechanism of H(+)-ATPase by auxin during early-phase hypocotyl elongation; this is the long-sought-after mechanism that is central to the acid-growth theory.

  15. Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein.

    Directory of Open Access Journals (Sweden)

    Jianhua Zhao

    2017-06-01

    Full Text Available Intracellular pathogenic bacteria evade the immune response by replicating within host cells. Legionella pneumophila, the causative agent of Legionnaires' Disease, makes use of numerous effector proteins to construct a niche supportive of its replication within phagocytic cells. The L. pneumophila effector SidK was identified in a screen for proteins that reduce the activity of the proton pumping vacuolar-type ATPases (V-ATPases when expressed in the yeast Saccharomyces cerevisae. SidK is secreted by L. pneumophila in the early stages of infection and by binding to and inhibiting the V-ATPase, SidK reduces phagosomal acidification and promotes survival of the bacterium inside macrophages. We determined crystal structures of the N-terminal region of SidK at 2.3 Å resolution and used single particle electron cryomicroscopy (cryo-EM to determine structures of V-ATPase:SidK complexes at ~6.8 Å resolution. SidK is a flexible and elongated protein composed of an α-helical region that interacts with subunit A of the V-ATPase and a second region of unknown function that is flexibly-tethered to the first. SidK binds V-ATPase strongly by interacting via two α-helical bundles at its N terminus with subunit A. In vitro activity assays show that SidK does not inhibit the V-ATPase completely, but reduces its activity by ~40%, consistent with the partial V-ATPase deficiency phenotype its expression causes in yeast. The cryo-EM analysis shows that SidK reduces the flexibility of the A-subunit that is in the 'open' conformation. Fluorescence experiments indicate that SidK binding decreases the affinity of V-ATPase for a fluorescent analogue of ATP. Together, these results reveal the structural basis for the fine-tuning of V-ATPase activity by SidK.

  16. TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

    NARCIS (Netherlands)

    Appelhagen, I.; Nordholt, N.; Seidel, T.; Spelt, K.; Koes, R.; Quattrochio, F.; Sagasser, M.; Weisshaar, B.

    2015-01-01

    Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H(+) -ATPases in the plasma membrane, and multimeric vacuolar-type H(+) -ATPases (V-ATPases) and vacuolar H(+)

  17. 75 FR 1798 - Prospective Grant of Exclusive License: Development of V-ATPase Inhibitor Compounds for the...

    Science.gov (United States)

    2010-01-13

    ... use will specifically exclude rheumatoid arthritis and other osteo-specific autoimmune diseases. DATES... Alzheimer's diseases. Briefly, vacuolar type (H+) ATPase (V-ATPase) has been described as ``a universal...-ATPase Inhibitor Compounds for the Treatment of Human Cancers and Osteoclastic Bone Diseases Excluding...

  18. The influence of Na+,K+-ATPase on glutamate signaling in neurodegenerative diseases and senescence

    Directory of Open Access Journals (Sweden)

    Paula Fernanda Kinoshita

    2016-06-01

    Full Text Available Decreased Na+,K+-ATPase (NKA activity causes energy deficiency, which is commonly observed in neurodegenerative diseases. The NKA is constituted of three subunits: α, β and γ, with four distinct isoforms of the catalytic α subunit (α1-4. Genetic mutations in the ATP1A2 gene and ATP1A3 gene, encoding the α2 and α3 subunit isoforms, respectively can cause distinct neurological disorders, concurrent to impaired NKA activity. Within the central nervous system (CNS, the α2 isoform is expressed mostly in glial cells and the α3 isoform is neuron-specific. Mutations in ATP1A2 gene can result in familial hemiplegic migraine (FHM2, while mutations in the ATP1A3 gene can cause Rapid-onset dystonia-Parkinsonism (RDP and alternating hemiplegia of childhood (AHC, as well as the cerebellar ataxia, areflexia, pescavus, optic atrophy and sensorineural hearing loss (CAPOS syndrome. Data indicates that the central glutamatergic system is affected by mutations in the α2 isoform, however further investigations are required to establish a connection to mutations in the α3 isoform, especially given the diagnostic confusion and overlap with glutamate transporter disease. The age-related decline in brain α2/3 activity may arise from changes in the cyclic guanosine monophosphate (cGMP and cGMP‐dependent protein kinase (PKG pathway. Glutamate, through nitric oxide synthase (NOS, cGMP and PKG, stimulates brain α2/3 activity, with the glutamatergic N-methyl-D-aspartate (NMDA receptor cascade able to drive an adaptive, neuroprotective response to inflammatory and challenging stimuli, including amyloid‐β. Here we review the NKA, both as an ion pump as well as a receptor that interacts with NMDA, including the role of NKA subunits mutations. Failure of the NKA-associated adaptive response mechanisms may render neurons more susceptible to degeneration over the course of aging.

  19. Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex

    Directory of Open Access Journals (Sweden)

    Kluge Christoph

    2004-08-01

    Full Text Available Abstract Background Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET. Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i a less conserved cytoplasmically orientated N-terminus and (ii a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. Results To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP in transfected cells. Conclusions All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA

  20. Quantitative Gait Analysis Using a Motorized Treadmill System Sensitively Detects Motor Abnormalities in Mice Expressing ATPase Defective Spastin.

    Directory of Open Access Journals (Sweden)

    James W Connell

    Full Text Available The hereditary spastic paraplegias (HSPs are genetic conditions in which there is progressive axonal degeneration in the corticospinal tract. Autosomal dominant mutations, including nonsense, frameshift and missense changes, in the gene encoding the microtubule severing ATPase spastin are the most common cause of HSP in North America and northern Europe. In this study we report quantitative gait analysis using a motorized treadmill system, carried out on mice knocked-in for a disease-associated mutation affecting a critical residue in the Walker A motif of the spastin ATPase domain. At 4 months and at one year of age homozygous mutant mice had a number of abnormal gait parameters, including in stride length and stride duration, compared to heterozygous and wild-type littermates. Gait parameters in heterozygous animals did not differ from wild-type littermates. We conclude that quantitative gait analysis using the DigiGait system sensitively detects motor abnormalities in a hereditary spastic paraplegia model, and would be a useful method for analyzing the effects of pharmacological treatments for HSP.

  1. Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase.

    Science.gov (United States)

    Capell, Anja; Liebscher, Sabine; Fellerer, Katrin; Brouwers, Nathalie; Willem, Michael; Lammich, Sven; Gijselinck, Ilse; Bittner, Tobias; Carlson, Aaron M; Sasse, Florenz; Kunze, Brigitte; Steinmetz, Heinrich; Jansen, Rolf; Dormann, Dorothee; Sleegers, Kristel; Cruts, Marc; Herms, Jochen; Van Broeckhoven, Christine; Haass, Christian

    2011-02-02

    Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRN haploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.

  2. Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca{sup 2+}-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Pestov, Nikolay B., E-mail: korn@mail.ibch.ru [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Dmitriev, Ruslan I.; Kostina, Maria B. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Korneenko, Tatyana V. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shakhparonov, Mikhail I. [Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871 (Russian Federation); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Full-length secretory pathway Ca-ATPase (SPCA2) cloned from rat duodenum. Black-Right-Pointing-Pointer ATP2C2 gene (encoding SPCA2) exists only in genomes of Tetrapoda. Black-Right-Pointing-Pointer Rat and pig SPCA2 are expressed in intestines, lung and some secretory glands. Black-Right-Pointing-Pointer Subcellular localization of SPCA2 may depend on tissue type. Black-Right-Pointing-Pointer In rat duodenum, SPCA2 is localized in plasma membrane-associated compartments. -- Abstract: Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2

  3. PI3Kβ Plays a Key Role in Apolipoprotein A-I-Induced Endothelial Cell Proliferation Through Activation of the Ecto-F1-ATPase/P2Y1 Receptors

    Directory of Open Access Journals (Sweden)

    Audrey Castaing-Berthou

    2017-06-01

    Full Text Available Background/Aims: High-density lipoproteins (HDL exert multiple cardioprotective functions on the arterial wall, including the promotion of endothelial cell survival and proliferation. Among mechanism contributing to endothelial protection, it has been reported that apolipoprotein A-I (apoA-I, the major protein in HDL, binds and activates the endothelial ecto-F1-ATPase receptor. This generates extracellular ADP, which in turn promotes endothelial cell survival. In this study we aimed to further investigate the signaling pathway involved downstream of apoA-I-induced ecto-F1-ATPase activation. Methods: In human umbilical vein endothelial cells (HUVECs, pharmacological and gene silencing approaches were used to study pathways involved downstream ecto-F1-ATPase activation by apoA-I. Results: ApoA-I and HDL both induced Akt phosphorylation. F1-ATPase inhibitors such as inhibitory factor 1 and oligomycin completely blocked apoA-I-induced Akt phosphorylaton and significantly blocked HDL-induced phosphorylation, indicating that this signaling pathway is dependent on ecto-F1-ATPase activation by apoA-I. Further, we were able to specify roles for the P2Y1-ADPreceptor and the PI3Kβ isoform in this pathway since pharmacological inhibition and silencing of these proteins dramatically inhibited apoA-I-induced Akt phosphorylation and cell proliferation. Conclusion: Altogether, these data highlight a key role of the P2Y1/PI3Kβ axis in endothelial cell proliferation downstream of ecto-F1-ATPase activation by apoA-I. Pharmacological targeting of this pathway could represent a promising approach to enhance vascular endothelial protection.

  4. Use of chromatin remodeling ATPases as RNAi targets for parental control of western corn rootworm (Diabrotica virgifera virgifera) and Neotropical brown stink bug (Euschistus heros).

    Science.gov (United States)

    Fishilevich, Elane; Vélez, Ana M; Khajuria, Chitvan; Frey, Meghan L F; Hamm, Ronda L; Wang, Haichuan; Schulenberg, Greg A; Bowling, Andrew J; Pence, Heather E; Gandra, Premchand; Arora, Kanika; Storer, Nicholas P; Narva, Kenneth E; Siegfried, Blair D

    2016-04-01

    RNA interference (RNAi) is a gene silencing mechanism that is present in animals and plants and is triggered by double stranded RNA (dsRNA) or small interfering RNA (siRNA), depending on the organism. In the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), RNAi can be achieved by feeding rootworms dsRNA added to artificial diet or plant tissues transformed to express dsRNA. The effect of RNAi depends on the targeted gene function and can range from an absence of phenotypic response to readily apparent responses, including lethality. Furthermore, RNAi can directly affect individuals that consume dsRNA or the effect may be transferred to the next generation. Our previous work described the potential use of genes involved in embryonic development as a parental RNAi technology for the control of WCR. In this study, we describe the use of chromatin-remodeling ATPases as target genes to achieve parental gene silencing in two insect pests, a coleopteran, WCR, and a hemipteran, the Neotropical brown stink bug, Euschistus heros Fabricius (Hemiptera: Pentatomidae). Our results show that dsRNA targeting chromatin-remodeling ATPase transcripts, brahma, mi-2, and iswi strongly reduced the fecundity of the exposed females in both insect species. Additionally, knockdown of chd1 reduced the fecundity of E. heros. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. The second sodium pump: from the function to the gene.

    Science.gov (United States)

    Rocafull, Miguel A; Thomas, Luz E; del Castillo, Jesús R

    2012-06-01

    Transepithelial Na(+) transport is mediated by passive Na(+) entry across the luminal membrane and exit through the basolateral membrane by two active mechanisms: the Na(+)/K(+) pump and the second sodium pump. These processes are associated with the ouabain-sensitive Na(+)/K(+)-ATPase and the ouabain-insensitive, furosemide-inhibitable Na(+)-ATPase, respectively. Over the last 40 years, the second sodium pump has not been successfully associated with any particular membrane protein. Recently, however, purification and cloning of intestinal α-subunit of the Na(+)-ATPase from guinea pig allowed us to define it as a unique biochemical and molecular entity. The Na(+)- and Na(+)/K(+)-ATPase genes are at the same locus, atp1a1, but have independent promoters and some different exons. Herein, we spotlight the functional characteristics of the second sodium pump, and the associated Na(+)-ATPase, in the context of its role in transepithelial transport and its response to a variety of physiological and pathophysiological conditions. Identification of the Na(+)-ATPase gene (atna) allowed us, using a bioinformatics approach, to explore the tertiary structure of the protein in relation to other P-type ATPases and to predict regulatory sites in the promoter region. Potential regulatory sites linked to inflammation and cellular stress were identified in the atna gene. In addition, a human atna ortholog was recognized. Finally, experimental data obtained using spontaneously hypertensive rats suggest that the Na(+)-ATPase could play a role in the pathogenesis of essential hypertension. Thus, the participation of the second sodium pump in transepithelial Na(+) transport and cellular Na(+) homeostasis leads us to reconsider its role in health and disease.

  6. Structural studies of Ca2+-ATPase ligand and regulatory complexes

    DEFF Research Database (Denmark)

    Drachmann, Nikolaj Düring

    2015-01-01

    is regulated by the Sarcolipin homologue Phospholamban. They act upon binding by increasing the apparent Ca2+ affinity of the ATPases, thus regulating the activity in the physiologically relevant Ca2+ concentrations. In the first part of the thesis, a purification protocol of native SERCA2a from pig hearts...... is presented. The purified protein was used for X-ray crystallographic studies aiming at determining the three dimensional structure of the SERCA2a isoform in a Ca2+-free conformation. Crystals of the Ca2+ free state of SERCA2a stabilised by the inhibitor cyclopiazonic acid was obtained and a dataset...... was collected scaling to 3.26 Å resolution, allowing a preliminary structural analysis. The overall crystal structure is very similar to SERCA1a. Additionally, co-crystallisation studies have been initiated of SERCA2a and recombinantly expressed Phospholamban. Besides the above mentioned regulatory peptides...

  7. Clamp loader ATPases and the evolution of DNA replication machinery

    Science.gov (United States)

    2012-01-01

    Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life. PMID:22520345

  8. Clamp loader ATPases and the evolution of DNA replication machinery

    Directory of Open Access Journals (Sweden)

    Kelch Brian A

    2012-04-01

    Full Text Available Abstract Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life.

  9. Characterization and effect of light on the plasma membrane H(+) -ATPase of bean leaves

    Science.gov (United States)

    Linnemeyer, P. A.; Van Volkenburgh, E.; Cleland, R. E.

    1990-01-01

    Proton excretion from bean (Phaseolus vulgaris L.) leaf cells is increased by bright white light. To test whether this could be due, at least in part, to an increase in plasma membrane (PM) ATPase activity, PM vesicles were isolated from primary leaves by phase partitioning and used to characterize PM ATPase activity and changes in response to light. ATPase activity was characterized as magnesium ion dependent, vanadate sensitive, and slightly stimulated by potassium chloride. The pH optimum was 6.5, the Km was approximately 0.30 millimolar ATP, and the activity was about 60% latent. PM vesicles were prepared from leaves of plants grown for 11 days in dim red light (growing slowly) or grown for 10 days in dim red light and then transferred to bright white-light for 1 day (growing rapidly). For both light treatments, ATPase specific activity was approximately 600 to 700 nanomoles per milligram protein per minute, and the latency, Km, and sensitivity to potassium chloride were also similar. PM vesicles from plants grown in complete darkness, however, exhibited a twofold greater specific activity. We conclude that the promotion of leaf growth and proton excretion by bright white light is not due to an increase in ATPase specific activity. Light does influence ATPase activity, however; both dim red light and bright white light decreased the ATPase specific activity by nearly 50% as compared with dark-grown leaves.

  10. Specialized Functional Diversity and Interactions of the Na,K-ATPase

    Science.gov (United States)

    Matchkov, Vladimir V.; Krivoi, Igor I.

    2016-01-01

    Na,K-ATPase is a protein ubiquitously expressed in the plasma membrane of all animal cells and vitally essential for their functions. A specialized functional diversity of the Na,K-ATPase isozymes is provided by molecular heterogeneity, distinct subcellular localizations, and functional interactions with molecular environment. Studies over the last decades clearly demonstrated complex and isoform-specific reciprocal functional interactions between the Na,K-ATPase and neighboring proteins and lipids. These interactions are enabled by a spatially restricted ion homeostasis, direct protein-protein/lipid interactions, and protein kinase signaling pathways. In addition to its “classical” function in ion translocation, the Na,K-ATPase is now considered as one of the most important signaling molecules in neuronal, epithelial, skeletal, cardiac and vascular tissues. Accordingly, the Na,K-ATPase forms specialized sub-cellular multimolecular microdomains which act as receptors to circulating endogenous cardiotonic steroids (CTS) triggering a number of signaling pathways. Changes in these endogenous cardiotonic steroid levels and initiated signaling responses have significant adaptive values for tissues and whole organisms under numerous physiological and pathophysiological conditions. This review discusses recent progress in the studies of functional interactions between the Na,K-ATPase and molecular microenvironment, the Na,K-ATPase-dependent signaling pathways and their significance for diversity of cell function. PMID:27252653

  11. Homotypic vacuole fusion in yeast requires organelle acidification and not the V-ATPase membrane domain.

    Science.gov (United States)

    Coonrod, Emily M; Graham, Laurie A; Carpp, Lindsay N; Carr, Tom M; Stirrat, Laura; Bowers, Katherine; Bryant, Nia J; Stevens, Tom H

    2013-11-25

    Studies of homotypic vacuole-vacuole fusion in the yeast Saccharomyces cerevisiae have been instrumental in determining the cellular machinery required for eukaryotic membrane fusion and have implicated the vacuolar H(+)-ATPase (V-ATPase). The V-ATPase is a multisubunit, rotary proton pump whose precise role in homotypic fusion is controversial. Models formulated from in vitro studies suggest that it is the proteolipid proton-translocating pore of the V-ATPase that functions in fusion, with further studies in worms, flies, zebrafish, and mice appearing to support this model. We present two in vivo assays and use a mutant V-ATPase subunit to establish that it is the H(+)-translocation/vacuole acidification function, rather than the physical presence of the V-ATPase, that promotes homotypic vacuole fusion in yeast. Furthermore, we show that acidification of the yeast vacuole in the absence of the V-ATPase rescues vacuole-fusion defects. Our results clarify the in vivo requirements of acidification for membrane fusion. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Inhibitory effect of lidocaine on the sarcoplasmic reticulum Ca2+-dependent atpase from temporalis muscle.

    Science.gov (United States)

    Sánchez, Gabriel A; Casadoumecq, Ana C; Alonso, Guillermo L; Takara, Delia

    2010-01-01

    Myotoxic effects of local anesthetics on skeletal musclefibers involve the inhibition ofsarcoplasmic reticulum Ca2+ -dependent ATPase activity and Ca2 transport. Lidocaine is a local anesthetic frequently used to relieve the symptoms of trigeminal neuralgia. The aim of this work was to test the inhibitory and/or stimulatory effect of lidocaine on sarcoplasmic reticulum Ca2+ -dependent ATPase isolated from rabbit temporalis muscle. Ca2+ -dependent ATPase activity was determined by a colorimetric method Calcium-binding to the Ca dependent ATPase, Ca2+ transport, and phosphorylation of the enzyme by ATP were determined with radioisotopic techniques. Lidocaine inhibited the Ca2+ -dependent ATPase activity in a concentration-dependent manner. The preincubation of the sarcoplasmic reticulum membranes with lidocaine enhanced the Ca2+ dependent ATPase activity in the absence of calcium ionophore. Lidocaine also inhibited both Ca2+ uptake and enzyme phosphorylation by ATP but had no effect on Ca2+ -binding to the enzyme. We conclude that the effect of lidocaine on the sarcoplasmic reticulum Ca2+ -dependent ATPase from temporalis muscle is due to the drug's direct interaction with the enzyme and the increased permeability of the sarcoplasmic reticulum membrane to Ca.

  13. Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

    Energy Technology Data Exchange (ETDEWEB)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Ochotny, Noelle [Department of Pharmacology, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Manolson, Morris F. [Faculty of Dentistry, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Holliday, L. Shannon, E-mail: sholliday@dental.ufl.edu [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610 (United States)

    2009-11-06

    Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

  14. Response of plasma membrane H+-ATPase in rice (Oryza sativa) seedlings to simulated acid rain.

    Science.gov (United States)

    Liang, Chanjuan; Ge, Yuqing; Su, Lei; Bu, Jinjin

    2015-01-01

    Understanding the adaptation of plants to acid rain is important to find feasible approaches to alleviate such damage to plants. We studied effects of acid rain on plasma membrane H(+)-ATPase activity and transcription, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate during stress and recovery periods. Simulated acid rain at pH 5.5 did not affect plasma membrane H(+)-ATPase activity, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate. Plasma membrane H(+)-ATPase activity and transcription in leaves treated with acid rain at pH 3.5 was increased to maintain ion homeostasis by transporting excessive H(+) out of cells. Then intracellular H(+) was close to the control after a 5-day recovery, alleviating damage on membrane and sustaining photosynthetic efficiency and growth. Simulated acid rain at pH 2.5 inhibited plasma membrane H(+)-ATPase activity by decreasing the expression of H(+)-ATPase at transcription level, resulting in membrane damage and abnormal intracellular H(+), and reduction in photosynthetic efficiency and relative growth rate. After a 5-day recovery, all parameters in leaves treated with pH 2.5 acid rain show alleviated damage, implying that the increased plasma membrane H(+)-ATPase activity and its high expression were involved in repairing process in acid rain-stressed plants. Our study suggests that plasma membrane H(+)-ATPase can play a role in adaptation to acid rain for rice seedlings.

  15. Membrane Targeting of P-type ATPases in Plant Cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeffrey F. Harper, Ph.D.

    2004-06-30

    How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems.

  16. Differential roles for the Co2+/Ni2+ transporting ATPases, CtpD and CtpJ, in Mycobacterium tuberculosis virulence

    Science.gov (United States)

    Raimunda, Daniel; Long, Jarukit E.; Padilla-Benavides, Teresita; Sassetti, Christopher M.; Argüello, José M.

    2013-01-01

    SUMMARY The genome of Mycobacterium tuberculosis encodes two paralogous P1B4-ATPases, CtpD (Rv1469) and CtpJ (Rv3743). Both proteins showed ATPase activation by Co2+ and Ni2+, and both appear to be required for metal efflux from the cell. However, using a combination of biochemical and genetic studies we found that these proteins play nonredundant roles in virulence and metal efflux. CtpJ expression is induced by Co2+ and this protein possesses a relatively high turnover rate. A ctpJ deletion mutant accumulated Co2+, indicating that this ATPase controls cytoplasmic metal levels. In contrast, CtpD expression is induced by redox stressors and this protein displays a relatively low turnover rate. A ctpD mutant failed to accumulate metal, suggesting an alternative cellular function. ctpD is co-transcribed with two thioredoxin genes trxA (Rv1470), trxB (Rv1471), and an enoyl-coA hydratase (Rv1472), indicating a possible role for CtpD in the metallation of these redox-active proteins. Supporting this, in vitro metal binding assays showed that TrxA binds Co2+ and Ni2+. Mutation of ctpD, but not ctpJ, reduced bacterial fitness in the mouse lung, suggesting that redox maintenance, but not Co+2 accumulation, is important for growth in vivo. PMID:24255990

  17. Stepwise evolution of resistance to toxic cardenolides via genetic substitutions in the Na+/K+ -ATPase of milkweed butterflies (lepidoptera: Danaini).

    Science.gov (United States)

    Petschenka, Georg; Fandrich, Steffi; Sander, Nils; Wagschal, Vera; Boppré, Michael; Dobler, Susanne

    2013-09-01

    Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to cardenolides, because their target site, the sodium pump (Na(+)/K(+) -ATPase), has evolved amino acid substitutions that reduce cardenolide binding (so-called target site insensitivity, TSI). Because many, but not all, species of milkweed butterflies (Danaini) are associated with cardenolide-containing host plants, we analyzed 16 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na(+)/K(+) -ATPase gene and by enzymatic assays with extracted Na(+)/K(+) -ATPase. Here we report that sensitivity to cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic cardenolides. Thus, the interplay between bottom-up selection by plant compounds and top-down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins. © 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.

  18. Sinorhizobium meliloti Nia is a P(1B-5)-ATPase expressed in the nodule during plant symbiosis and is involved in Ni and Fe transport.

    Science.gov (United States)

    Zielazinski, Eliza L; González-Guerrero, Manuel; Subramanian, Poorna; Stemmler, Timothy L; Argüello, José M; Rosenzweig, Amy C

    2013-12-01

    The P1B-ATPases are a ubiquitous family of metal transporters. These transporters are classified into subfamilies on the basis of substrate specificity, which is conferred by conserved amino acids in the last three transmembrane domains. Five subfamilies have been identified to date, and representative members of four (P1B-1 to P1B-4) have been studied. The fifth family (P1B-5), of which some members contain a C-terminal hemerythrin (Hr) domain, is less well characterized. The S. meliloti Sma1163 gene encodes for a P1B-5-ATPase, denoted Nia (Nickel-iron ATPase), that is induced by exogenous Fe(2+) and Ni(2+). The nia mutant accumulates nickel and iron, suggesting a possible role in detoxification of these two elements under free-living conditions, as well as in symbiosis, when the highest expression levels are measured. This function is supported by an inhibitory effect of Fe(2+) and Ni(2+) on the pNPPase activity, and by the ability of Nia to bind Fe(2+) in the transmembrane domain. Optical and X-ray absorption spectroscopic studies of the isolated Hr domain confirm the presence of a dinuclear iron center and suggest that this domain might function as an iron sensor.

  19. Alteration of aluminium inhibition of synaptosomal (Na(+)/K(+))ATPase by colestipol administration.

    Science.gov (United States)

    Silva, V S; Oliveira, L; Gonçalves, P P

    2013-11-01

    The ability of aluminium to inhibit the (Na(+)/K(+))ATPase activity has been observed by several authors. During chronic dietary exposure to AlCl3, brain (Na(+)/K(+))ATPase activity drops, even if no alterations of catalytic subunit protein expression and of energy charge potential are observed. The aluminium effect on (Na(+)/K(+))ATPase activity seems to implicate the reduction of interacting protomers within the oligomeric ensemble of the membrane-bound (Na(+)/K(+))ATPase. The activity of (Na(+)/K(+))ATPase is altered by the microviscosity of lipid environment. We studied if aluminium inhibitory effect on (Na(+)/K(+))ATPase is modified by alterations in synaptosomal membrane cholesterol content. Adult male Wistar rats were submitted to chronic dietary AlCl3 exposure (0.03 g/day of AlCl3) and/or to colestipol, a hypolidaemic drug (0.31 g/day) during 4 months. The activity of (Na(+)/K(+))ATPase was studied in brain cortex synaptosomes with different cholesterol contents. Additionally, we incubate synaptosomes with methyl-β-cyclodextrin for both enrichment and depletion of membrane cholesterol content, with or without 300 μM AlCl3. This enzyme activity was significantly reduced by micromolar AlCl3 added in vitro and when aluminium was orally administered to rats. The oral administration of colestipol reduced the cholesterol content and concomitantly inhibited the (Na(+)/K(+))ATPase. The aluminium inhibitory effect on synaptosomal (Na(+)/K(+))ATPase was reduced by cholesterol depletion both in vitro and in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Src-independent ERK signaling through the rat α3 isoform of Na/K-ATPase.

    Science.gov (United States)

    Madan, Namrata; Xu, Yunhui; Duan, Qiming; Banerjee, Moumita; Larre, Isabel; Pierre, Sandrine V; Xie, Zijian

    2017-03-01

    The Na/K-ATPase α1 polypeptide supports both ion-pumping and signaling functions. The Na/K-ATPase α3 polypeptide differs from α1 in both its primary structure and its tissue distribution. The expression of α3 seems particularly important in neurons, and recent clinical evidence supports a unique role of this isoform in normal brain function. The nature of this specific role of α3 has remained elusive, because the ubiquitous presence of α1 has hindered efforts to characterize α3-specific functions in mammalian cell systems. Using Na/K-ATPase α1 knockdown pig kidney cells (PY-17), we generated the first stable mammalian cell line expressing a ouabain-resistant form of rat Na/K-ATPase α3 in the absence of endogenous pig α1 detectable by Western blotting. In these cells, Na/K-ATPase α3 formed a functional ion-pumping enzyme and rescued the expression of Na/K-ATPase β1 and caveolin-1 to levels comparable with those observed in PY-17 cells rescued with a rat Na/K-ATPase α1 (AAC-19). The α3-containing enzymes had lower Na+ affinity and lower ouabain-sensitive transport activity than their α1-containing counterparts under basal conditions, but showed a greater capacity to be activated when intracellular Na+ was increased. In contrast to Na/K-ATPase α1, α3 could not regulate Src. Upon exposure to ouabain, Src activation did not occur, yet ERK was activated through Src-independent pathways involving PI3K and PKC. Hence, α3 expression confers signaling and pumping properties that are clearly distinct from that of cells expressing Na/K-ATPase α1. Copyright © 2017 the American Physiological Society.

  1. Identification of Novel Bisbenzimidazole Derivatives as Anticancer Vacuolar (H+-ATPase Inhibitors

    Directory of Open Access Journals (Sweden)

    Renukadevi Patil

    2017-09-01

    Full Text Available The vacuolar (H+-ATPases (V-ATPases are a family of ATP-driven proton pumps and they have been associated with cancer invasion, metastasis, and drug resistance. Despite the clear involvement of V-ATPases in cancer, the therapeutic use of V-ATPase-targeting small molecules has not reached human clinical trials to date. Thus, V-ATPases are emerging as important targets for the identification of potential novel therapeutic agents. We identified a bisbenzimidazole derivative (V as an initial hit from a similarity search using four known V-ATPase inhibitors (I–IV. Based on the initial hit (V, we designed and synthesized a focused set of novel bisbenzimidazole analogs (2a–e. All newly prepared compounds have been screened for selected human breast cancer (MDA-MB-468, MDA-MB-231, and MCF7 and ovarian cancer (A2780, Cis-A2780, and PA-1 cell lines, along with the normal breast epithelial cell line, MCF10A. The bisbenzimidazole derivative (2e is active against all cell lines tested. Remarkably, it demonstrated high cytotoxicity against the triple-negative breast cancer (TNBC cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 μM. Additionally, it has been shown to inhibit the V-ATPase pump that is mainly responsible for acidification. To the best of our knowledge the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. These results strongly suggest that the compound 2e could be further developed as a potential anticancer V-ATPase inhibitor for breast cancer treatment.

  2. Na/K-ATPase/src complex mediates regulation of CD40 in renal parenchyma.

    Science.gov (United States)

    Xie, Jeffrey X; Zhang, Shungang; Cui, Xiaoyu; Zhang, Jue; Yu, Hui; Khalaf, Fatimah K; Malhotra, Deepak; Kennedy, David J; Shapiro, Joseph I; Tian, Jiang; Haller, Steven T

    2017-12-22

    Recent studies have highlighted a critical role for CD40 in the pathogenesis of renal injury and fibrosis. However, little is currently understood about the regulation of CD40 in this setting. We use novel Na/K-ATPase cell lines and inhibitors in order to demonstrate the regulatory function of Na/K-ATPase with regards to CD40 expression and function. We utilize 5/6 partial nephrectomy as well as direct infusion of a Na/K-ATPase ligand to demonstrate this mechanism exists in vivo. We demonstrate that knockdown of the α1 isoform of Na/K-ATPase causes a reduction in CD40 while rescue of the α1 but not the α2 isoform restores CD40 expression in renal epithelial cells. Second, because the major functional difference between α1 and α2 is the ability of α1 to form a functional signaling complex with Src, we examined whether the Na/K-ATPase/Src complex is important for CD40 expression. We show that a gain-of-Src binding α2 mutant restores CD40 expression while loss-of-Src binding α1 reduces CD40 expression. Furthermore, loss of a functional Na/K-ATPase/Src complex also disrupts CD40 signaling. Importantly, we show that use of a specific Na/K-ATPase/Src complex antagonist, pNaKtide, can attenuate cardiotonic steroid (CTS)-induced induction of CD40 expression in vitro. Because the Na/K-ATPase/Src complex is also a key player in the pathogenesis of renal injury and fibrosis, our new findings suggest that Na/K-ATPase and CD40 may comprise a pro-fibrotic feed-forward loop in the kidney and that pharmacological inhibition of this loop may be useful in the treatment of renal fibrosis.

  3. Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated1

    Science.gov (United States)

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Maldonado-Gama, Minerva; Pantoja, Omar

    1999-01-01

    Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways. PMID:10398716

  4. Tissue-specific expression of Sarcoplasmic/Endoplasmic Reticulum Calcium ATPases (ATP2A/SERCA) 1, 2, 3 during Xenopus laevis development.

    Science.gov (United States)

    Pegoraro, Caterina; Pollet, Nicolas; Monsoro-Burq, Anne H

    2011-01-01

    Calcium-ATPase pumps are critical in most cells, to sequester calcium into intracytoplasmic stores and regulate general calcium signalling. In addition, cell-specific needs for calcium signals have been described and employ a diversity of calcium ATPases in adult tissues and oocytes. A major family of such calcium pumps is ATP2A/SERCA family, for Sarcoplasmic/Endoplasmic Reticulum Calcium ATPases. Although largely studied in adults, the developmental expression of the atp2a/serca genes remains unknown. Here, we provide genome organisation in Xenopuslaevis and tropicalis and phylogeny of atp2a/serca genes in craniates. We detail embryonic expression for the three X. laevis atp2a/serca genes. We found that the three atp2a/serca genes are strongly conserved among vertebrates and display complementary and tissue-specific expression in embryos. These expression patterns present variations when compared to the data reported in adults. Atp2a1/serca1 is expressed as soon as the end of gastrulation in a subset of the myod-positive cells, and later labels prospective slow muscle cells in the superficial part of the somite. In contrast atp2a2/serca2 is found in a larger subset of cells, but is not ubiquitous as reported in adults. Notably, atp2a2/serca2 is prominently expressed in the neural-related tissues, i.e. the neural plate, cement gland, but is excluded from premigratory neural crest. Finally, atp2a3/serca3 expression is restricted to the ectoderm throughout development. Copyright © 2010 Elsevier B.V. All rights reserved.

  5. Na,K-ATPase: a molecular target for Leptospira interrogans endotoxin

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    Younes-Ibrahim M.

    1997-01-01

    Full Text Available On the basis of our report that a glycolipoprotein fraction (GLP extracted from Leptospira interrogans contains a potent inhibitor of renal Na,K-ATPase, we proposed that GLP-induced inhibition of Na,K-ATPase might be the primary cellular defect in the physiopathology of leptospirosis. The present study was designed to test this hypothesis by determining whether or not 1 GLP inhibits all the isoforms of Na,K-ATPase which are expressed in the tissues affected by leptospirosis, 2 Na,K-ATPase from leptospirosis-resistant species, such as the rat, is sensitive to GLP, 3 GLP inhibits Na,K-ATPase from intact cells, and 4 GLP inhibits ouabain-sensitive H,K-ATPase. The results indicate that in the rabbit, a leptospirosis-sensitive species, GLP inhibits with similar efficiency (apparent IC50: 120-220 µg protein GLP/ml all isoforms of Na,K-ATPase known to be expressed in target tissues for the disease. Na,K-ATPase from rat kidney displays a sensitivity to GLP similar to that of the rabbit kidney enzyme (apparent IC50: 25-80 and 50-150 µg protein GLP/ml for rat and rabbit, respectively, indicating that resistance to the disease does not result from the resistance of Na,K-ATPase to GLP. GLP also reduces ouabain-sensitive rubidium uptake in rat thick ascending limbs (pmol mm-1 min-1 ± SEM; control: 23.8 ± 1.8; GLP, 88 µg protein/ml: 8.2 ± 0.9, demonstrating that it is active in intact cells. Finally, GLP had no demonstrable effect on renal H,K-ATPase activity, even on the ouabain-sensitive form, indicating that the active principle of GLP is more specific for Na,K-ATPase than ouabain itself. Although the hypothesis remains to be demonstrated in vivo, the present findings are compatible with the putative role of GLP-induced inhibition of Na,K-ATPase as an initial mechanism in the physiopathology of leptospirosis

  6. Crystal Structure of the Vanadate-Inhibited Ca2+-ATPase

    DEFF Research Database (Denmark)

    Clausen, Johannes D.; Bublitz, Maike; Arnou, Bertrand Jean-Paul

    2016-01-01

    Vanadate is the hallmark inhibitor of the P-type ATPase family; however, structural details of its inhibitory mechanism have remained unresolved. We have determined the crystal structure of sarcoplasmic reticulum Ca2+-ATPase with bound vanadate in the absence of Ca2+. Vanadate is bound at the cat...... nucleotide analogs in the E2·VO3− structure with that in E2·BeF3− (E2P ground state analog) reveals multiple binding modes to the Ca2+-ATPase....

  7. Na,K-ATPase activity in mouse muscle is regulated by AMPK and PGC-1a

    DEFF Research Database (Denmark)

    Ingwersen, Maria S; Kristensen, Michael; Pilegaard, Henriette

    2011-01-01

    Na,K-ATPase activity, which is crucial for skeletal muscle function, undergoes acute and long-term regulation in response to muscle activity. The aim of the present study was to test the hypothesis that AMP kinase (AMPK) and the transcriptional coactivator PGC-1a are underlying factors in long-te...... influences Na,K-ATPase a(1) and a(2) content and ß(1)/ß(2) isoform ratio. Phosphorylation of the Na,K-ATPase subunit PLM is an important regulatory mechanism....

  8. P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis.

    Science.gov (United States)

    Hussain, Dawar; Haydon, Michael J; Wang, Yuwen; Wong, Edwin; Sherson, Sarah M; Young, Jeff; Camakaris, James; Harper, Jeffrey F; Cobbett, Christopher S

    2004-05-01

    Arabidopsis thaliana has eight genes encoding members of the type 1(B) heavy metal-transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter- and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.

  9. The Rice AAA-ATPase OsFIGNL1 Is Essential for Male Meiosis

    Directory of Open Access Journals (Sweden)

    Peipei Zhang

    2017-09-01

    Full Text Available Meiosis is crucial in reproduction of plants and ensuring genetic diversity. Although several genes involved in homologous recombination and DNA repair have been reported, their functions in rice (Oryza sativa male meiosis remain poorly understood. Here, we isolated and characterized the rice OsFIGNL1 (OsFidgetin-like 1 gene, encoding a conserved AAA-ATPase, and explored its function and importance in male meiosis and pollen formation. The rice Osfignl1 mutant exhibited normal vegetative growth, but failed to produce seeds and displayed pollen abortion phenotype. Phenotypic comparisons between the wild-type and Osfignl1 mutant demonstrated that OsFIGNL1 is required for anther development, and that the recessive mutation of this gene causes male sterility in rice. Complementation and CRISPR/Cas9 experiments demonstrated that wild-type OsFIGNL1 is responsible for the male sterility phenotype. Subcellular localization showed that OsFIGNL1-green fluorescent protein was exclusively localized in the nucleus of rice protoplasts. Male meiosis in the Osfignl1 mutant exhibited abnormal chromosome behavior, including chromosome bridges and multivalent chromosomes at diakinesis, lagging chromosomes, and chromosome fragments during meiosis. Yeast two-hybrid assays demonstrated OsFIGNL1 could interact with RAD51A1, RAD51A2, DMC1A, DMC1B, and these physical interactions were further confirmed by BiFC assay. Taken together, our results suggest that OsFIGNL1 plays an important role in regulation of male meiosis and anther development.

  10. Sublethal Effects of vATPase-A and Snf7 dsRNAs on Biology of Southern Corn Rootworm, Diabrotica undecimpunctata howardi Barber.

    Science.gov (United States)

    Pereira, Adriano E; Vélez, Ana M; Meinke, Lance J; Siegfried, Blair D

    2017-10-16

    RNA interference is a powerful tool against corn rootworm. Adults and neonates of southern corn rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae), were exposed to the LC50 of vATPase-A and Snf7 double-stranded RNAs (dsRNAs), and the effects on female fecundity, egg viability, male fitness as measured by sperm viability and mating capacity, larval recovery along with dry weight, and instar determination 10 d after exposure to dsRNA, were determined. Significant reductions were observed for a number of parameters in dsRNA-exposed rootworms relative to control treatments. Female fecundity and larval recovery were significantly reduced after exposure to both dsRNAs. In addition, larval dry weight and recovery of 2nd and 3rd instars along with dry weight for 3rd instars were significantly reduced after neonate exposure to vATPase-A dsRNA. Neither dsRNA affected male capacity to mate or sperm viability after exposure to the respective LC50s. After 10 d of feeding on untreated corn roots, neonates that survived exposure for 2 d to the vATPase-A dsRNA LC50 exhibited lower dry weight than the control. There was significant gene knockdown in adult males and females after exposure for 5 d to LC50 of vATPase-A and Snf7 dsRNAs. The parameters are discussed in terms of fitness and possible outcomes after deployment of corn hybrids expressing dsRNAs. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Activity determination of Na+ K+ - ATPase and Mg++ - ATPase enzymes in the gill of Poecilia vivipara (Osteichthyes, Cyprinodontiformes in different salinities

    Directory of Open Access Journals (Sweden)

    Amaral Marcelo da Cunha

    2001-01-01

    Full Text Available This work aimed to know the tolerance mechanisms through the salinity variation by Na+ K+ - ATPase and Mg++ - ATPase and enzymes encountered in the gills of Poecilia vivipara. In field, the presence of this species was observed in salinities of 0 and 28?. In laboratory, these fish were maintained in aquarium with mean salinity of 30? and positive responses were obtained. Some adult specimens, collected in a lagoon of the Coqueiros Beach, were utilized as matrixes. In the experiments the specimens used were those born in the test aquarium. For each salinity studied three replicates were made with three specimens for each one. The alevins were maintained in salinities of 5, 10, 15, 20, 25, 30 and 35? during a month for adaptation. Gills were extracted in appropriate buffer for isolation of plasma membrane and used for specific dosage of the total enzymatic activity of Na+ K+ - ATPase and Mg++ - ATPase. The relation of alevins to their adaptation towards the salinity variation was also studied. The activity of the two enzymes showed a different result. The major expression of Na+ K+ - ATPase was observed in 20? (35 µmoles Pi.mg protein.h-1, the best salinity to cultivate P. vivipara.

  12. Na+/K+-ATPase α1 subunit, a novel therapeutic target for hepatocellular carcinoma.

    Science.gov (United States)

    Zhuang, Liping; Xu, Litao; Wang, Peng; Jiang, Yan; Yong, Pan; Zhang, Chenyue; Zhang, Haibin; Meng, Zhiqiang; Yang, Peiying

    2015-09-29

    We aimed to identify the expression patterns of Na+/K+-ATPase (NKA) α subunits in human hepatocellular carcinoma (HCC) samples and evaluate these subunits as potential targets for HCC treatment. The mRNA expression profiles of NKA α subunits in human HCC samples were analyzed. We found that the mRNA expression for NKA α1 subunit (ATP1A1) was higher than that for other NKA α subunits. Also, ATP1A1 gene expression was markedly higher in HCC samples than in adjacent nontumor tissue samples. Western blotting data suggested that 6 of 14 (43%) HCC samples had elevated ATP1A1 protein expression. Furthermore, knockdown of ATP1A1 expression in human HCC HepG2 and MHCC97H cells markedly reduced their proliferation in vitro and suppressed the tumorigenicity of MHCC97H cells in vivo. Downregulation of ATP1A1 expression resulted in cell-cycle arrest at G2/M phase and apoptosis in HepG2 cells as well as decreased migration in Hep3B cells. We further validated that ATP1A1 downregulation caused intracellular accumulation of reactive oxygen species. Pretreatment with N-acetyl cysteine blocked cell-growth inhibition induced by ATP1A1 downregulation. Collectively, these data suggested that targeting ATP1A1 is a novel approach to the treatment of HCC.

  13. Seipin promotes adipose tissue fat storage through the ER Ca²⁺-ATPase SERCA.

    Science.gov (United States)

    Bi, Junfeng; Wang, Wei; Liu, Zhonghua; Huang, Xiahe; Jiang, Qingqing; Liu, George; Wang, Yingchun; Huang, Xun

    2014-05-06

    Adipose tissue is central to the regulation of lipid metabolism. Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), one of the most severe lipodystrophy diseases, is caused by mutation of the Seipin gene. Seipin plays an important role in adipocyte differentiation and lipid homeostasis, but its exact molecular functions are still unknown. Here, we show that Seipin physically interacts with the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) in both Drosophila and man. SERCA, an endoplasmic reticulum (ER) calcium pump, is solely responsible for transporting cytosolic calcium into the ER lumen. Like dSeipin, dSERCA cell-autonomously promotes lipid storage in Drosophila fat cells. dSeipin affects dSERCA activity and modulates intracellular calcium homeostasis. Adipose tissue-specific knockdown of the ER-to-cytosol calcium release channel ryanodine receptor (RyR) partially restores fat storage in dSeipin mutants. Our results reveal that Seipin promotes adipose tissue fat storage by regulating intracellular calcium homeostasis. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Regulation of Na(+)/K(+)-ATPase by nuclear respiratory factor 1: implication in the tight coupling of neuronal activity, energy generation, and energy consumption.

    Science.gov (United States)

    Johar, Kaid; Priya, Anusha; Wong-Riley, Margaret T T

    2012-11-23

    NRF-1 regulates mediators of neuronal activity and energy generation. NRF-1 transcriptionally regulates Na(+)/K(+)-ATPase subunits α1 and β1. NRF-1 functionally regulates mediators of energy consumption in neurons. NRF-1 mediates the tight coupling of neuronal activity, energy generation, and energy consumption at the molecular level. Energy generation and energy consumption are tightly coupled to neuronal activity at the cellular level. Na(+)/K(+)-ATPase, a major energy-consuming enzyme, is well expressed in neurons rich in cytochrome c oxidase, an important enzyme of the energy-generating machinery, and glutamatergic receptors that are mediators of neuronal activity. The present study sought to test our hypothesis that the coupling extends to the molecular level, whereby Na(+)/K(+)-ATPase subunits are regulated by the same transcription factor, nuclear respiratory factor 1 (NRF-1), found recently by our laboratory to regulate all cytochrome c oxidase subunit genes and some NMDA and AMPA receptor subunit genes. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, in vivo chromatin immunoprecipitation, promoter mutational analysis, and real-time quantitative PCR, NRF-1 was found to functionally bind to the promoters of Atp1a1 and Atp1b1 genes but not of the Atp1a3 gene in neurons. The transcripts of Atp1a1 and Atp1b1 subunit genes were up-regulated by KCl and down-regulated by tetrodotoxin. Atp1b1 is positively regulated by NRF-1, and silencing of NRF-1 with small interference RNA blocked the up-regulation of Atp1b1 induced by KCl, whereas overexpression of NRF-1 rescued these transcripts from being suppressed by tetrodotoxin. On the other hand, Atp1a1 is negatively regulated by NRF-1. The binding sites of NRF-1 on Atp1a1 and Atp1b1 are conserved among mice, rats, and humans. Thus, NRF-1 regulates key Na(+)/K(+)-ATPase subunits and plays an important role in mediating the tight coupling between

  15. Architecture and characterization of a thermostable MoxR family AAA(+) ATPase from Thermococcus kodakarensis KOD1.

    Science.gov (United States)

    Pham, Bang Phuong; Lee, Sangmin; Jia, Baolei; Kwak, Jae Myeong; Cheong, Gang-Won

    2014-05-01

    AAA(+) ATPases are ubiquitous enzymes that can function as molecular chaperones, employing the energy obtained from ATP hydrolysis to remodel macromolecules. In this report, the MoxR enzyme from Thermococcus kodakarensis KOD1 (TkMoxR) was shown to have two native forms: a two-stack hexameric ring and a hexameric structure, under physiological conditions and cold stress, respectively. TkMoxR was altered to a microtubule-like form in the presence of ATP and tightly interacted with dsDNA molecules of various lengths. In addition, the two-stack hexameric protein catalyzed dsDNA decomposition to form and then release ssDNA, whereas the hexamer TkMoxR structure interacted with but did not release dsDNA. These results suggest that TkMoxR has DNA helicase activity involved in gene expression control.

  16. Altered expression and insulin-induced trafficking of Na+-K+-ATPase in rat skeletal muscle

    DEFF Research Database (Denmark)

    Galuska, Dana; Kotova, Olga; Barres, Romain

    2009-01-01

    . Skeletal muscle insulin resistance was observed after 12 wk of HFD. Na(+)-K(+)-ATPase alpha(1)-subunit protein expression was increased 1.6-fold (P alpha(2)- and beta(1)-subunits and protein expression were decreased twofold (P ...(+)-K(+)-ATPase activity after 4 wk of HFD. Exercise training restored alpha(1)-, alpha(2)-, and beta(1)-subunit expression and Na(+)-K(+)-ATPase activity to control levels and reduced beta(2)-subunit expression 2.2-fold (P alpha(1)-subunit-regulating transcription factor ZEB (AREB6......) and alpha(1) mRNA expression were increased after HFD and restored by ET. DNA binding activity of Sp-1, a transcription factor involved in the regulation of alpha(2)- and beta(1)-subunit expression, was decreased after HFD. ET increased phosphorylation of the Na(+)-K(+)-ATPase regulatory protein...

  17. A conditional mutation affecting localization of the Menkes disease copper ATPase. Suppression by copper supplementation

    National Research Council Canada - National Science Library

    Kim, Byung-Eun; Smith, Kathryn; Meagher, Carisa K; Petris, Michael J

    2002-01-01

    Copper is an essential co-factor for several key metabolic processes. This requirement in humans is underscored by Menkes disease, an X-linked copper deficiency disorder caused by mutations in the copper transporting P-type ATPase, MNK...

  18. The structure of the peripheral stalk of Thermus thermophilus H+-ATPase/synthase

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Lawrence K; Stewart, Alastair G; Donohoe, Mhairi; Bernal, Ricardo A; Stock, Daniela (VCCRI); (New South); (Texas)

    2010-03-22

    Proton-translocating ATPases are ubiquitous protein complexes that couple ATP catalysis with proton translocation via a rotary catalytic mechanism. The peripheral stalks are essential components that counteract torque generated from proton translocation during ATP synthesis or from ATP hydrolysis during proton pumping. Despite their essential role, the peripheral stalks are the least conserved component of the complexes, differing substantially between subtypes in composition and stoichiometry. We have determined the crystal structure of the peripheral stalk of the A-type ATPase/synthase from Thermus thermophilus consisting of subunits E and G. The structure contains a heterodimeric right-handed coiled coil, a protein fold never observed before. We have fitted this structure into the 23 {angstrom} resolution EM density of the intact A-ATPase complex, revealing the precise location of the peripheral stalk and new implications for the function and assembly of proton-translocating ATPases.

  19. Atrial Na,K-ATPase increase and potassium dysregulation accentuate the risk of postoperative atrial fibrillation

    DEFF Research Database (Denmark)

    Tran, Cao Thach; Schmidt, Thomas Andersen; Christensen, John Brochorst

    2009-01-01

    BACKGROUND: Postoperative atrial fibrillation is a common complication to cardiac surgery. Na,K-ATPase is of major importance for the resting membrane potential and action potential. The purpose of the present study was to evaluate the importance of Na,K-ATPase concentrations in human atrial...... biopsies and plasma potassium concentrations for the development of atrial fibrillation. METHODS: Atrial myocardial biopsies were obtained from 67 patients undergoing open chest cardiac surgery. Na,K-ATPase was quantified using vanadate-facilitated 3H-ouabain binding. Plasma potassium concentration...... with postoperative atrial fibrillation. CONCLUSIONS: The present study supports the increasing evidence of dysregulation of the potassium homeostasis as an important factor in the development of cardiac arrhythmias. High atrial Na,K-ATPase and sudden plasma potassium concentration increase may contribute...

  20. Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation

    DEFF Research Database (Denmark)

    Cornelius, Flemming; Mahmmoud, Yasser Ahmed

    2007-01-01

    acids of FXYD10 had been cleaved by mild, controlled trypsin treatment. Several kinetic properties of the Na,K-ATPase reaction cycle as well as the FXYD-regulation of Na,K-ATPase activity were found to be affected by acidic phospholipids like PI, PS, and PG. This takes into consideration the Na+ and K...... with anionic phospholipids. Specifically, the effects of the cytoplasmic domain of FXYD10, which contains the phosphorylation sites for protein kinases, on the kinetics of the Na,K-ATPase reaction were investigated by a comparison of the reconstituted native enzyme and the enzyme where 23 C-terminal amino......+ activation, the K+-deocclusion reaction, and the poise of the E1/E2 conformational equilibrium, whereas the ATP activation was unchanged. Anionic phospholipids increased the intermolecular cross-linking between the FXYD10 C-terminus (Cys74) and the Cys254 in the Na,K-ATPase A-domain. However, neither...

  1. Cryo EM structure of intact rotary H+-ATPase/synthase from Thermus thermophilus.

    Science.gov (United States)

    Nakanishi, Atsuko; Kishikawa, Jun-Ichi; Tamakoshi, Masatada; Mitsuoka, Kaoru; Yokoyama, Ken

    2018-01-08

    Proton translocating rotary ATPases couple ATP hydrolysis/synthesis, which occurs in the soluble domain, with proton flow through the membrane domain via a rotation of the common central rotor complex against the surrounding peripheral stator apparatus. Here, we present a large data set of single particle cryo-electron micrograph images of the V/A type H+-rotary ATPase from the bacterium Thermus thermophilus, enabling the identification of three rotational states based on the orientation of the rotor subunit. Using masked refinement and classification with signal subtractions, we obtain homogeneous reconstructions for the whole complexes and soluble V1 domains. These reconstructions are of higher resolution than any EM map of intact rotary ATPase reported previously, providing a detailed molecular basis for how the rotary ATPase maintains structural integrity of the peripheral stator apparatus, and confirming the existence of a clear proton translocation path from both sides of the membrane.

  2. Effect of Ionophores on Activity of Na+,Cl-(HCO 3- )-ATPase.

    Science.gov (United States)

    Yurkiv, V A; Melikhov, V I; Shubin, V S

    2016-10-01

    We studied the effects of ionophores on activity of Na+,Cl-(HCO 3- )-ATPase. The most significant effect on the activity of this enzyme was produced by protonophore 2,4-dinitrophenol. The effect of this drug largely depended on the cation and anion composition and pH of the incubation medium and its pH. Activity of Na+,Cl-(HCO 3- )-ATPase increased at neutral and weakly alkaline pH and decreased at pH below 6.5-6.7. In control animals (without histamine injection) with very weak or absent Na+,Cl-(HCO 3- )-ATPase activity, the observed effect of the protonophore on ATPase activity was also virtually absent. The stimulatory effect of other ionophores (monensine, valinomycin, and A23187) was significantly weaker and depended on pH of the incubation medium, its cationic and anionic composition, and concentration of these ionophores.

  3. Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy

    Directory of Open Access Journals (Sweden)

    Bondar Alexander

    2011-01-01

    Full Text Available Abstract Background The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. Results With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform in the postsynaptic region of the spine. Conclusions A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

  4. Interaction of phosphatidic acid and phosphatidylserine with the Ca2+-ATPase of sarcoplasmic reticulum and the mechanism of inhibition.

    Science.gov (United States)

    Dalton, K A; East, J M; Mall, S; Oliver, S; Starling, A P; Lee, A G

    1998-02-01

    The sarcoplasmic reticulum of skeletal muscle contains anionic phospholipids as well as the zwitterionic phosphatidylcholine and phosphatidylethanolamine. Here we study the effects of anionic phospholipids on the activity of the Ca2+-ATPase purified from the membrane. Reconstitution of the Ca2+-ATPase into dioleoylphosphatidylserine [di(C18:1)PS] or dioleoylphosphatidic acid [di(C18:1)PA] leads to a decrease in ATPase activity. Measurements of the quenching of the tryptophan fluorescence of the ATPase by brominated phospholipids give a relative binding constant for the anionic lipids compared with dioleoylphosphatidylcholine close to 1 and suggest that phosphatidic acid only binds to the ATPase at the bulk lipid sites around the ATPase. Addition of di(C18:1)PS or di(C18:1)PA to the ATPase in the short-chain dimyristoleoylphosphatidylcholine [di(C14:1)PC] reverse the effects of the short-chain lipid on ATPase activity and on Ca2+ binding, as revealed by the response of tryptophan fluorescence intensity to Ca2+ binding. It is concluded that the lipid headgroup and lipid fatty acyl chains have separate effects on the function of the ATPase. The anionic phospholipids have no significant effect on Ca2+ binding to the ATPase; the level of Ca2+ binding to the ATPase, the affinity of binding and the rate of dissociation of Ca2+ are unchanged by reconstitution into di(C18:1)PA. The major effect of the anionic lipids is a reduction in the maximal level of binding of MgATP. This is attributed to the formation of oligomers of the Ca2+-ATPase, in which only one molecule of the ATPase can bind MgATP dimers in di(C18:1)PS and trimers or tetramers in di(C18:1)PA. The rates of phosphorylation and dephosphorylation for the proportion of the ATPase still able to bind ATP are unaffected by reconstitution. Larger changes were observed in the level of phosphorylation of the ATPase by Pi, which became very low in the anionic phospholipids. The fluorescence response to Mg2+ for the ATPase

  5. [Influence of omeprasole and lansoprasole on Na+, K+ -ATPase and Mg2+ -ATPase activity of the plasmatic membrane of myometrium smooth muscle cells].

    Science.gov (United States)

    Veklich, T O; Shkrabak, O A; Medvediev, V V; Kurs'kyĭ, M D; Kosterin, S O

    2007-01-01

    The paper deals with the influence of the proton pump inhibitors - omeprasole and lansoprasole on the enzymatic activity of the ouabain-sensitive Na+, K+ -ATPase and the ouabain-resistant Mg2+ - ATPase in the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution. It was found, that omeprasole and lansoprasole inhibited Na+, K+ -ATPase in the range from 10 to 100 microM. The maximal effect was observed at a concentration of 100 microM with the percentage of inhibition of 81 and 86% at an average as compared with the control for omeprasole and lansoprasole, respectively. The magnitudes of the inhibition coefficient I(0.5) for omeprasole and lansoprasole were 35.60 +/- 0.81 and 29.40 +/- 1.79 microM respectively. Meanwhile cooperative effects on the Na+, K+ - ATPase activity were not found, as the Hill coefficient n(H) for omeprasole was 1.00 +/- 0.08, while for lansoprasole it was 1.20 +/- 0.03. These substances had also insignificant influence on Mg2+ -ATPase: the enzymatic activity was decreased to 84 and 82% as compared with the control with omeprasole and lansoprasole, respectively, in concentration of 100 microM for each inhibitor. The inhibition of Na+, K+ -ATPase activity can evidence for the possible side effects of omeprasole and lansoprasole when they are used for treatment of acid-dependent diseases of the stomach. In addition, obtained experimental data can be useful for further research of the membrane mechanisms of omeprasole and lansoprasole action on cationic exchange in the smooth muscle cells.

  6. Identification of Domains within the V-ATPase Accessory Subunit Ac45 Involved in V-ATPase Transport and Ca2+-dependent Exocytosis

    Science.gov (United States)

    Jansen, Eric J. R.; van Bakel, Nick. H. M.; Loohuis, Nikkie F. M. Olde; Hafmans, Theo G. M.; Arentsen, Tim; Coenen, Anthon J. M.; Scheenen, Wim J. J. M.; Martens, Gerard J. M.

    2012-01-01

    The vacuolar (H+)-ATPase (V-ATPase) is crucial for maintenance of the acidic microenvironment in intracellular organelles, whereas its membrane-bound V0-sector is involved in Ca2+-dependent membrane fusion. In the secretory pathway, the V-ATPase is regulated by its type I transmembrane and V0-associated accessory subunit Ac45. To execute its function, the intact-Ac45 protein is proteolytically processed to cleaved-Ac45 thereby releasing its N-terminal domain. Here, we searched for the functional domains within Ac45 by analyzing a set of deletion mutants close to the in vivo situation, namely in transgenic Xenopus intermediate pituitary melanotrope cells. Intact-Ac45 was poorly processed and accumulated in the endoplasmic reticulum of the transgenic melanotrope cells. In contrast, cleaved-Ac45 was efficiently transported through the secretory pathway, caused an accumulation of the V-ATPase at the plasma membrane and reduced dopaminergic inhibition of Ca2+-dependent peptide secretion. Surprisingly, removal of the C-tail from intact-Ac45 caused cellular phenotypes also found for cleaved-Ac45, whereas C-tail removal from cleaved-Ac45 still allowed its transport to the plasma membrane, but abolished V-ATPase recruitment into the secretory pathway and left dopaminergic inhibition of the cells unaffected. We conclude that domains located in the N- and C-terminal portions of the Ac45 protein direct its trafficking, V-ATPase recruitment and Ca2+-dependent-regulated exocytosis. PMID:22736765

  7. Neuropharmacological effects of lipoic acid and ubiquinone on δ-aminolevulinic dehydratase, Na(+) , K(+) -ATPase, and Mg(2+) -ATPase activities in rat hippocampus after pilocarpine-induced seizures.

    Science.gov (United States)

    de Freitas, Rivelilson Mendes; Feng, Dejiang; Jordán, Joaquín

    2011-04-01

    In this study, we investigated the effects of lipoic acid (LA) in the hippocampus oxidative stress caused by pilocarpine-induced seizures in adult rats. Wistar rats were treated with 0.9% saline (i.p., control group), LA (10mg/kg, i.p., LA group), ubiquinone [20mg/kg, i.p., ubiquinone (UQ) group], pilocarpine (400mg/kg, i.p., P400 group), and the association of LA (10mg/kg, i.p.) plus pilocarpine (400mg/kg, i.p.) or UQ (20mg/kg, i.p.) plus pilocarpine (400mg/kg, i.p.), 30min before of administration of P400 (LA plus P400 group and UQ plus P400 group, respectively). After the treatments, all groups were observed for 1h. The enzyme activities (δ-aminolevulinic dehydratase (δ-ALA-D), Mg(2+) -ATPase, and Na(+) , K(+) -ATPase) were measured using spectrophotometric methods, and the results compared to values obtained from saline and pilocarpine-treated animals. Protective effects of LA and UQ were also evaluated on the same parameters. We reported here for the first time that Na(+) , K(+) -ATPase and δ-ALA-D activities inhibition and Mg(2+) -ATPase stimulation in the pilocarpine model are probably attributed to the oxidative stress caused by seizures in the rat hippocampus. The addition of the antioxidants LA and UQ may reverses the previously mentioned Na(+) , K(+) -ATPase and δ-ALA-D inhibitions and Mg(2+) -ATPase stimulation. The oxidative stress plays an important signaling role in pilocarpine-induced seizures, and antioxidant drugs might be considered as therapeutical tools in this pathology. © 2010 The Authors Fundamental and Clinical Pharmacology © 2010 Société Française de Pharmacologie et de Thérapeutique.

  8. Na/K-ATPase Signaling and Salt Sensitivity: The Role of Oxidative Stress

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    Jiang Liu

    2017-03-01

    Full Text Available Other than genetic regulation of salt sensitivity of blood pressure, many factors have been shown to regulate renal sodium handling which contributes to long-term blood pressure regulation and have been extensively reviewed. Here we present our progress on the Na/K-ATPase signaling mediated sodium reabsorption in renal proximal tubules, from cardiotonic steroids-mediated to reactive oxygen species (ROS-mediated Na/K-ATPase signaling that contributes to experimental salt sensitivity.

  9. Snakes exhibit tissue-specific variation in cardiotonic steroid sensitivity of Na+/K+-ATPase.

    Science.gov (United States)

    Mohammadi, Shabnam; Petschenka, Georg; French, Susannah S; Mori, Akira; Savitzky, Alan H

    2018-03-01

    Toads are among several groups of organisms chemically defended with lethal concentrations of cardiotonic steroids. As a result, most predators that prey on amphibians avoid toads. However, several species of snakes have gained resistance-conferring mutations of Na+/K+-ATPase, the molecular target of cardiotonic steroids, and can feed on toads readily. Despite recent advances in our understanding of this adaptation at the genetic level, we have lacked functional evidence for how mutations of Na+/K+-ATPase account for cardiotonic steroid resistance in snake tissues. To address this issue, it is necessary to determine how the Na+/K+-ATPases of snakes react to the toxins. Some tissues might have Na+/K+-ATPases that are more susceptible than others and can thus provide clues about how the toxins influence organismal function. Here we provide a mechanistic link between observed Na+/K+-ATPase substitutions and observed resistance using actual snake Na+/K+-ATPases. We used an in vitro approach to determine the tissue-specific levels of sensitivity to cardiotonic steroids in select resistant and non-resistant snakes. We compared the sensitivities of select tissues within and between species. Our results suggest that resistant snakes contain highly resistant Na+/K+-ATPases in their heart and kidney, both of which rely heavily on the enzymes to function, whereas tissues that do not rely as heavily on Na+/K+-ATPases or might be protected from cardiotonic steroids by other means (liver, gut, and brain) contain non-resistant forms of the enzyme. This study reveals functional evidence that tissue-level target-site insensitivity to cardiotonic steroids varies not only among species but also across tissues within resistant taxa. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Structural determinants for the ouabain-stimulated increase in Na-K ATPase activity.

    Science.gov (United States)

    Khundmiri, Syed J; Salyer, Sarah A; Farmer, Brandon; Qipshidze-Kelm, Natia; Murray, Rebecca D; Clark, Barbara J; Xie, Zijian; Pressley, Thomas A; Lederer, Eleanor D

    2014-06-01

    Recent studies suggest that at low concentrations, ouabain increases Na-K ATPase and NHE1 activity and activates the Src signaling cascade in proximal tubule cells. Our laboratory demonstrated that low concentrations of ouabain increase blood pressure in rats. We hypothesize that ouabain-induced increase in blood pressure and Na-K ATPase activity requires NHE1 activity and association. To test this hypothesis we treated rats with ouabain (1μgkg body wt(-1)day(-1)) for 9days in the presence or absence of the NHE1 inhibitor, zoniporide. Ouabain stimulated a significant increase in blood pressure which was prevented by zoniporide. Using NHE1-expressing Human Kidney cells 2 (HK2), 8 (HK8) and 11 (HK11) and Mouse Kidney cells from Wild type (WT) and NHE1 knock-out mice (SWE) cell lines, we show that ouabain stimulated Na-K ATPase activity and surface expression in a Src-dependent manner in NHE1-expressing cells but not in NHE1-deplete cells. Zoniporide prevented ouabain-induced stimulation of (86)Rb uptake in the NHE1-expressing cells. FRET and TIRF microscopy showed that ouabain increased association between GFP-NHE1 and mCherry-Na-K ATPase transfected into NHE1-deficient SWE cells. Mutational analysis demonstrated that the caveolin binding motif (CBM) of Na-K ATPase α1 is required for translocation of both Na-K ATPase α1 and NHE1 to the basolateral membrane. Mutations in activity or scaffold domains of NHE1 resulted in loss of ouabain-mediated regulation of Na-K ATPase. These results support that NHE1 is required for the ouabain-induced increase in blood pressure, and that the caveolin binding motif of Na-K ATPase α1 as well as the activity and scaffolding domains of NHE1 are required for their functional association. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. The Role of the Plasma Membrane H(+)-ATPase in Plant Responses to Aluminum Toxicity.

    Science.gov (United States)

    Zhang, Jiarong; Wei, Jian; Li, Dongxu; Kong, Xiangying; Rengel, Zed; Chen, Limei; Yang, Ye; Cui, Xiuming; Chen, Qi

    2017-01-01

    Aluminum (Al) toxicity is a key factor limiting plant growth and crop production on acid soils. Increasing the plant Al-detoxification capacity and/or breeding Al-resistant cultivars are a cost-effective strategy to support crop growth on acidic soils. The plasma membrane H(+)-ATPase plays a central role in all plant physiological processes. Changes in the activity of the plasma membrane H(+)-ATPase through regulating the expression and phosphorylation of this enzyme are also involved in many plant responses to Al toxicity. The plasma membrane H(+)-ATPase mediated H(+) influx may be associated with the maintenance of cytosolic pH and the plasma membrane gradients as well as Al-induced citrate efflux mediated by a H(+)-ATPase-coupled MATE co-transport system. In particular, modulating the activity of plasma membrane H(+)-ATPase through application of its activators (e.g., magnesium or IAA) or using transgenics has effectively enhanced plant resistance to Al stress in several species. In this review, we critically assess the available knowledge on the role of the plasma membrane H(+)-ATPase in plant responses to Al stress, incorporating physiological and molecular aspects.

  12. Probing determinants of cyclopiazonic acid sensitivity of bacterial Ca2+-ATPases.

    Science.gov (United States)

    Kotšubei, Aljona; Gorgel, Manuela; Morth, Jens P; Nissen, Poul; Andersen, Jacob L

    2013-11-01

    Cyclopiazonic acid (CPA) is a specific and potent inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase 1a (SERCA1a). Despite high sequence similarity to SERCA1a, Listeria monocytogenes Ca(2+)-ATPase 1 (LMCA1) is not inhibited by CPA. To test whether a CPA binding site could be created while maintaining the functionality of the ATPase we targeted four amino acid positions in LMCA1 for mutational studies based on a multiple sequence alignment of SERCA-like Ca(2+)-ATPases and structural analysis of the CPA site. The identification of CPA-sensitive gain-of-function mutants pinpointed key determinants of the CPA binding site. The importance of these determinants was further underscored by the characterization of the CPA sensitivity of two additional bacterial Ca(2+)-ATPases from Lactococcus lactis and Bacillus cereus. The CPA sensitivity was predicted from their sequence compared with the LMCA1 results, and this was experimentally confirmed. Interestingly, a cluster of Lactococcus bacteria applied in the production of fermented cheese display Ca(2+)-ATPases that are predictably CPA insensitive and may originate from their coexistence with CPA-producing Penicillum and Aspergillus fungi in the cheese. The differences between bacterial and mammalian binding pockets encompassing the CPA site suggest that CPA derivatives that are specific for bacteria or other pathogens can be developed. © 2013 FEBS.

  13. Effects of flour bleaching agent on mice liver antioxidant status and ATPases.

    Science.gov (United States)

    Jia, Xiaojing; Wu, Yangxinwei; Liu, Ping

    2011-05-01

    Benzoyl peroxide (BPO) is a strong oxidizing agent and widely used as flour bleaching agent. However their potential risk of liver damage is unknown. The aim of this study was to investigate the effects of BPO on mice liver antioxidant status and ATPases according to the actual amount of BPO in flour from Jinan, China. The results showed that the maximum concentration of BPO reached up to 284.6 mg/kg and content of BPO mainly ranged from 0 to 240 mg/kg. Therefore, four groups of mice were gavaged daily with BPO at doses of 0, 50, 100, 200mg/kg b.w./d for 42 days, respectively. In liver tissue, superoxide dismutase (SOD) activity was significantly decreased, while the content of malondialdehyde (MDA) significantly increased following BPO exposure at 200mg/kg b.w. BPO significantly decreased the Mg(2+)-ATPase and Ca(2+)-ATPase activities of the liver at 200mg/kg b.w. BPO, at all of the doses assayed, produced non-significant effects on glutathione peroxidase (GSH-Px) and Na(+)K(+)-ATPase activities. Experimental results suggested that BPO had certain adverse effects on antioxidant status and the activities of Mg(2+)-ATPase and Ca(2+)-ATPase of liver tissue. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

    Energy Technology Data Exchange (ETDEWEB)

    Miles, Andrew J. [Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX (United Kingdom); Fedosova, Natalya U. [Department of Biomedicine, Aarhus University, DK-8000 Aarhus (Denmark); Hoffmann, Søren V. [ISA, Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus (Denmark); Wallace, B.A. [Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX (United Kingdom); Esmann, Mikael, E-mail: me@biophys.au.dk [Department of Biomedicine, Aarhus University, DK-8000 Aarhus (Denmark)

    2013-05-31

    Highlights: •Ouabain binding to pig and shark Na,K-ATPase enhances thermal stability. •Ouabain stabilises both membrane-bound and solubilised Na,K-ATPase. •Synchrotron radiation circular dichroism is used for structure determination. •Secondary structure in general is not affected by ouabain binding. •Stabilisation is due to re-arrangement of tertiary structure. -- Abstract: Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.

  15. LASS2 inhibits growth and invasion of bladder cancer by regulating ATPase activity

    Science.gov (United States)

    Wang, Haifeng; Zuo, Yigang; Ding, Mingxia; Ke, Changxing; Yan, Ruping; Zhan, Hui; Liu, Jingyu; Wang, Wei; Li, Ning; Wang, Jiansong

    2017-01-01

    Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2) is a novel suppressor of human cancer metastasis, and downregulation of LASS2 has been associated with a poor prognosis in patients with bladder cancer (BC). However, the molecular mechanism underlying LASS2-mediated inhibition of tumor invasion and metastasis in BC remains unclear. LASS2 has been reported to directly bind to subunit C of vacuolar H+-ATPase (V-ATPase) in various types of cancer, suggesting that LASS2 may inhibit cancer invasion and metastasis by regulating the function of V-ATPase. The present study investigated the effect of LASS2-specific small interfering (si)RNA on the invasion and metastasis of the RT4 human BC cell line, which has a low metastatic potential, and its functional interaction with V-ATPase. Silencing of LASS2 in RT4 cells was able to increase V-ATPase activity, the extracellular hydrogen ion concentration and, in turn, the activation of secreted matrix metalloproteinase (MMP)-2 and MMP-9, which occurred simultaneously with enhanced cell proliferation, cell survival and cell invasion in vitro, as well as acceleration of BC growth in vivo. In this process, it was found that siRNA-LASS2 treatment was able to suppress cell apoptosis induced by doxorubicin. These findings suggest that silencing of LASS2 may enhance the growth, invasion and metastasis of BC by regulating ATPase activity. PMID:28356943

  16. Robustness of the rotary catalysis mechanism of F1-ATPase.

    Science.gov (United States)

    Watanabe, Rikiya; Matsukage, Yuki; Yukawa, Ayako; Tabata, Kazuhito V; Noji, Hiroyuki

    2014-07-11

    F1-ATPase (F1) is the rotary motor protein fueled by ATP hydrolysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F1 as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the γ-phosphate of ATP (general base); and an arginine directly contacting the γ-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. In this study, we investigated the role of these charged residues in catalysis and torque generation by analyzing alanine-substituted mutants in the single-molecule rotation assay. Surprisingly, all mutants continuously drove rotary motion, even though the rotational velocity was at least 100,000 times slower than that of wild type. Thus, although these charged residues contribute to highly efficient catalysis, they are not indispensable to chemo-mechanical energy coupling, and the rotary catalysis mechanism of F1 is far more robust than previously thought. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Robustness of the Rotary Catalysis Mechanism of F1-ATPase*

    Science.gov (United States)

    Watanabe, Rikiya; Matsukage, Yuki; Yukawa, Ayako; Tabata, Kazuhito V.; Noji, Hiroyuki

    2014-01-01

    F1-ATPase (F1) is the rotary motor protein fueled by ATP hydrolysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F1 as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the γ-phosphate of ATP (general base); and an arginine directly contacting the γ-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. In this study, we investigated the role of these charged residues in catalysis and torque generation by analyzing alanine-substituted mutants in the single-molecule rotation assay. Surprisingly, all mutants continuously drove rotary motion, even though the rotational velocity was at least 100,000 times slower than that of wild type. Thus, although these charged residues contribute to highly efficient catalysis, they are not indispensable to chemo-mechanical energy coupling, and the rotary catalysis mechanism of F1 is far more robust than previously thought. PMID:24876384

  18. Plasmodium falciparum isolates from southern Ghana exhibit polymorphisms in the SERCA-type PfATPase6 though sensitive to artesunate in vitro.

    Science.gov (United States)

    Kwansa-Bentum, Bethel; Ayi, Irene; Suzuki, Takashi; Otchere, Joseph; Kumagai, Takashi; Anyan, William K; Osei, Joseph H N; Asahi, Hiroko; Ofori, Michael F; Akao, Nobuaki; Wilson, Michael D; Boakye, Daniel A; Ohta, Nobuo

    2011-07-11

    In 2005, Ghana replaced chloroquine with artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. The aim of this work was to determine for the first time, polymorphisms in the putative pfATPase6 and pftctp, pfmdr1, pfcrt genes in Ghanaian isolates, particularly at a time when there is no report on artemisinin resistance in malaria parasites from Ghana. The sensitivity of parasite isolates to anti-malaria drugs were also evaluated for a possible association with polymorphisms in these genes. The prevalence of point mutations in the above Plasmodium falciparum genes were assessed from filter-paper blood blot samples by DNA sequencing. In vitro drug sensitivity test was carried out on some of the blood samples from volunteers visiting hospitals/clinics in southern Ghana using a modified version of the standard WHO Mark III micro-test. All successfully tested parasite isolates were sensitive to artesunate; while 19.4%, 29.0% and 51.6% were resistant to quinine, amodiaquine and chloroquine respectively. The geometric mean of IC50 value for artesunate was 0.73 nM (95% CI, 0.38-1.08), amodiaquine 30.69 nM (95% CI, 14.18-47.20) and chloroquine 58.73 nM (95% CI, 38.08-79.38). Twenty point mutations were observed in pfATPase6 gene, with no L263E and S769N. All mutations found were low in frequency, except D639G which was observed in about half of the isolates but was not associated with artesunate response (p = 0.42). The pftctp gene is highly conserved as no mutation was observed, while CVIET which is chloroquine-resistant genotype at codon 72-76 of the pfcrt gene was identified in about half of the isolates; this was consistent with chloroquine IC50 values (p = 0.001). Mutations were present in pfmdr1 gene but were not associated with artemisinin response (p = 1.00). The pfATPase6 gene is highly polymorphic with D639G appearing to be fixed in Ghanaian isolates. These may just be spontaneous mutations as all parasite isolates that were

  19. Plasmodium falciparum isolates from southern Ghana exhibit polymorphisms in the SERCA-type PfATPase6 though sensitive to artesunate in vitro

    Directory of Open Access Journals (Sweden)

    Ofori Michael F

    2011-07-01

    Full Text Available Abstract Background In 2005, Ghana replaced chloroquine with artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. The aim of this work was to determine for the first time, polymorphisms in the putative pfATPase6 and pftctp, pfmdr1, pfcrt genes in Ghanaian isolates, particularly at a time when there is no report on artemisinin resistance in malaria parasites from Ghana. The sensitivity of parasite isolates to anti-malaria drugs were also evaluated for a possible association with polymorphisms in these genes. Methods The prevalence of point mutations in the above Plasmodium falciparum genes were assessed from filter-paper blood blot samples by DNA sequencing. In vitro drug sensitivity test was carried out on some of the blood samples from volunteers visiting hospitals/clinics in southern Ghana using a modified version of the standard WHO Mark III micro-test. Results All successfully tested parasite isolates were sensitive to artesunate; while 19.4%, 29.0% and 51.6% were resistant to quinine, amodiaquine and chloroquine respectively. The geometric mean of IC50 value for artesunate was 0.73 nM (95% CI, 0.38-1.08, amodiaquine 30.69 nM (95% CI, 14.18-47.20 and chloroquine 58.73 nM (95% CI, 38.08-79.38. Twenty point mutations were observed in pfATPase6 gene, with no L263E and S769N. All mutations found were low in frequency, except D639G which was observed in about half of the isolates but was not associated with artesunate response (p = 0.42. The pftctp gene is highly conserved as no mutation was observed, while CVIET which is chloroquine-resistant genotype at codon 72-76 of the pfcrt gene was identified in about half of the isolates; this was consistent with chloroquine IC50 values (p = 0.001. Mutations were present in pfmdr1 gene but were not associated with artemisinin response (p = 1.00. Conclusion The pfATPase6 gene is highly polymorphic with D639G appearing to be fixed in Ghanaian isolates. These may just

  20. H,K-ATPase and carbonic anhydrase response to chronic systemic rat gastric hypoxia

    Directory of Open Access Journals (Sweden)

    Ulfah Lutfiah

    2015-11-01

    Full Text Available Background: Hypoxia may induce gastric ulcer associated with excessive hidrogen chloride (HCl secretion. Synthesis of HCl involves 2 enzymes, H,K-ATPase and carbonic anhydrase (CA. This study aimed to clarify the underlying cause of gastric ulcer in chronic hypoxic condition, by investigating the H,K-ATPase and CA9 response in rats.Methods: This study was an in vivo experiment, to know the relationship between hypoxia to expression of H,K-ATPase and CA9 mRNA, and H,K-ATPase and total CA specific activity of chronic systemic rat gastric hypoxia. The result was compared to control. Data was analyzed by SPSS. If the data distribution was normal and homogeneous, ANOVA and LSD post-hoc test were used. However, if the distribution was not normal and not homogeneous, and still as such after transformation, data was treated in non-parametric using Kruskal-Wallis and Mann Whitney test. Twenty five male Sprague-Dawley rats were divided into 5 groups: rats undergoing hypoxia for 1, 3, 5, and 7 days placed in hypoxia chamber (10% O2, 90% N2, and one control group. Following this treatment, stomach of the rats was extracted and homogenized. Expression of H,K-ATPase and CA9 mRNA was measured using real time RT-PCR. Specific activity of H,K-ATPase was measured using phosphate standard solution, and specific activity of total CA was measured using p-nitrophenol solution.Results: The expression of H,K-ATPase mRNA was higher in the first day (2.159, and drastically lowered from the third to seventh day (0.289; 0.108; 0.062. Specific activities of H,K-ATPase was slightly higher in the first day (0.765, then was lowered in the third (0.685 and fifth day (0.655, and was higher in the seventh day (0.884. The expression of CA9 mRNA was lowered progressively from the first to seventh day (0.84; 0.766; 0.736; 0.343. Specific activities of total CA was low in the first day (0.083, and was higher from the third to seventh day (0.111; 0.136; 0.144.Conclusion: In hypoxia

  1. Na/K Pump and Beyond: Na/K-ATPase as a Modulator of Apoptosis and Autophagy

    Directory of Open Access Journals (Sweden)

    Cassiano Felippe Gonçalves-de-Albuquerque

    2017-04-01

    Full Text Available Lung cancer is a leading cause of global cancer deaths. Na/K-ATPase has been studied as a target for cancer treatment. Cardiotonic steroids (CS trigger intracellular signalling upon binding to Na/K-ATPase. Normal lung and tumour cells frequently express different pump isoforms. Thus, Na/K-ATPase is a powerful target for lung cancer treatment. Drugs targeting Na/K-ATPase may induce apoptosis and autophagy in transformed cells. We argue that Na/K-ATPase has a role as a potential target in chemotherapy in lung cancer treatment. We discuss the effects of Na/K-ATPase ligands and molecular pathways inducing deleterious effects on lung cancer cells, especially those leading to apoptosis and autophagy.

  2. Na/K Pump and Beyond: Na/K-ATPase as a Modulator of Apoptosis and Autophagy.

    Science.gov (United States)

    Felippe Gonçalves-de-Albuquerque, Cassiano; Ribeiro Silva, Adriana; Ignácio da Silva, Camila; Caire Castro-Faria-Neto, Hugo; Burth, Patrícia

    2017-04-21

    Lung cancer is a leading cause of global cancer deaths. Na/K-ATPase has been studied as a target for cancer treatment. Cardiotonic steroids (CS) trigger intracellular signalling upon binding to Na/K-ATPase. Normal lung and tumour cells frequently express different pump isoforms. Thus, Na/K-ATPase is a powerful target for lung cancer treatment. Drugs targeting Na/K-ATPase may induce apoptosis and autophagy in transformed cells. We argue that Na/K-ATPase has a role as a potential target in chemotherapy in lung cancer treatment. We discuss the effects of Na/K-ATPase ligands and molecular pathways inducing deleterious effects on lung cancer cells, especially those leading to apoptosis and autophagy.

  3. Curcumin modulation of Na,K-ATPase: phosphoenzyme accumulation, decreased K+ occlusion, and inhibition of hydrolytic activity

    DEFF Research Database (Denmark)

    Mahmmoud, Yasser Ahmed

    2005-01-01

    Curcumin, the major constitute of tumeric, is an important nutraceutical that has been shown to be useful in the treatment of many diseases. As an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, curcumin was shown to correct cystic fibrosis (CF) defects in some model systems, whereas others...... have reported no or little effects on CF after curcumin treatment, suggesting that curcumin effect is not due to simple inhibition of the Ca2+-ATPase. We tested the hypothesis that curcumin may modulate other members of the P2-type ATPase superfamily by studying the effects of curcumin on the activity...... and kinetic properties of the Na,K-ATPase. Curcumin treatment inhibited Na,K-ATPase activity in a dose-dependent manner (K0.514.6 M). Curcumin decreased the apparent affinity of Na,K-ATPase for K+ and increased it for Na+ and ATP. Kinetic analyses indicated that curcumin induces a three-fold reduction...

  4. Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids

    NARCIS (Netherlands)

    Qiu, L.; Krieger, E.; Schaftenaar, G.; Swarts, H.G.P.; Willems, P.; Pont, J.J.H.H.M. de; Koenderink, J.B.

    2005-01-01

    Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na, K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we

  5. Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids.

    NARCIS (Netherlands)

    Qiu, L.; Krieger, E.; Schaftenaar, G.; Swarts, H.G.P.; Willems, P.H.G.M.; Pont, J.J.H.H.M. de; Koenderink, J.B.

    2005-01-01

    Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we

  6. Novel ATPase Cu(2+ transporting beta polypeptide mutations in Chinese families with Wilson's disease.

    Directory of Open Access Journals (Sweden)

    Shaojuan Gu

    Full Text Available Wilson's disease (WD is an autosomal recessive inherited disorder caused by mutations in the ATPase Cu(2+ transporting beta polypeptide gene (ATP7B. The detailed metabolism of copper-induced pathology in WD is still unknown. Gene mutations as well as the possible pathways involved in the ATP7B deficiency were documented. The ATP7B gene was analyzed for mutations in 18 Chinese Han families with WD by direct sequencing. Cell viability and apoptosis analysis of ATP7B small interfering RNA (siRNA-treated human liver carcinoma (HepG2 cells were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT assay and Hoechst 33342 staining. Finally, the expression of B-cell CLL/lymphoma 2 (BCL2, BCL2-associated X protein (BAX, sterol regulatory element binding protein 1 (SREBP1, and minichromosome maintenance protein 7 (MCM7 of ATP7B siRNA-treated cells were tested by real-time polymerase chain reaction (real-time PCR and Western blot analysis. Twenty different mutations including four novel mutations (p.Val145Phe, p.Glu388X, p.Thr498Ser and p.Gly837X in the ATP7B gene were identified in our families. Haplotype analysis revealed that founder effects for four mutations (p.Arg778Leu, p.Pro992Leu, p.Ile1148Thr and p.Ala1295Val existed in these families. Transfection of HepG2 cells with ATP7B siRNA resulted in decreased mRNA expression by 86.3%, 93.1% and 90.8%, and decreased protein levels by 58.5%, 85.5% and 82.1% at 24, 48 and 72 hours, respectively (All P<0.01. In vitro study revealed that the apoptotic, cell cycle and lipid metabolism pathway may be involved in the mechanism of WD. Our results revealed that the genetic cause of 18 Chinese families with WD and ATP7B deficiency-induce apoptosis may result from imbalance in cell cycle and lipid metabolism pathway.

  7. INTER-SUBUNIT INTERACTION AND ARRANGEMENT OF THE CENTRAL STALK SUBUNITS OF ENTEROCOCCUS HIRAE V-ATPASE

    Directory of Open Access Journals (Sweden)

    K. M. Mozaffor Hossain1,2

    2017-04-01

    Full Text Available V-ATPases function as ATP-dependent ion pumps in various membrane systems of living organisms. ATP hydrolysis causes rotation of the central rotor complex, which is composed of NtpC, NtpD,and NtpG subunits. We successfully cloned, expressed, and purified the central stalk subunits of Na+- translocating V-type ATPase from E. hirae. Full length DNAs for genes of NtpC, NtpD, and NtpG subunits were amplified by conventional PCR method and cloned into the plasmid vector pET23d. Recombinant DNA transformed E. coli (JM109 cells were grown in LB agar plate containing ampicillin. NtpC and NtpG subunit proteins were expressed as separate His-tagged soluble proteins in E. coli BL21(DE3 cells at 30 °C and NtpD subunit protein was stably expressed in E. coli BL21(DE3 cells at 16 °C. Expressed central stalk subunit proteins were purified by affinity chromatography followed by gel-filtration method. The amount of purified NtpC, NtpD, and NtpG subunit proteins were 13.8 mg/1 liter culture, 16.6 mg/1 liter culture, and 15.2 mg/1 liter culture, respectively. Tobacco etch virus (TEV-protease was also expressed in E. coli BL21(DE3 cells and purified by affinity chromatography. The amount of purified TEV-protease was 8.50 mg/1 liter culture. Here, we report the stable interaction and the complex formation mechanisms of the central stalk subunits of E. hirae V-ATPase. The purified NtpD could interact directly with NtpG and NtpC could bind directly to NtpD-G complex and these three subunits could form NtpD-G-C complex. NtpD could bind to NtpG and formed NtpD-G complex. NtpD or NtpD-G complex could bind to NtpA3-B3 and formed NtpA3-B3-D or NtpA3-B3-D-G complex, respectively. NtpC could bind to NtpA3-B3-D-G and formed NtpA3-B3-D-G-C complex but could not bind to NtpA3-B3, NtpA3-B3-D, NtpD, or NtpG.

  8. Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles.

    Science.gov (United States)

    Datiles, Manuel J; Johnson, Eric A; McCarty, Richard E

    2008-04-01

    Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca2+- and Mg2+-ATPase activities of CF1 deficient in its inhibitory epsilon subunit (CF1-epsilon) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca2+-ATPase activity by CTAB is irreversible. The Ca2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-epsilon or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-epsilon from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the alpha/beta heterohexamer.

  9. Characterization of the sarcoplasmic reticulum Ca-ATPase from rabbit temporalis muscle.

    Science.gov (United States)

    Sánchez, Gabriel Antonio; Di Croce, Daniel Eduardo; Casadoumecq, Ana Clara; Richard, Susana Beatriz; Takara, Delia

    2012-10-01

    The aim of this work was to isolate the sarcoplasmic reticulum (SR) Ca-ATPase from rabbit temporalis muscle and to determine the optimal conditions for calcium transport and enzymatic activity. SR vesicles were isolated from rabbit temporalis muscle by differential centrifugation, the protein composition analyzed by electrophoresis and compared to fast-twitch muscle membrane suspensions. ELISA was used to determine the sarcoendoplasmic reticulum Ca-ATPase (SERCA) isoform. Ca-ATPase activity was determined by a colorimetric method. Calcium-binding to the Ca-ATPase, calcium uptake, calcium efflux and phosphorylation by P(i) were determined with radioisotopic techniques. Sixty five percent of the total protein concentration of SR membranes suspensions from rabbit temporalis corresponded to SERCA. Of the total SERCA protein, 64% was SERCA 2, 35% was SERCA 1 and less than 1% was SERCA 3. The optimal conditions of the SERCA isolated from rabbit temporalis muscle were: pH 7.2, 5 μM Ca(2+), 100 μM EGTA, 90 μM Mg(2+), 3mM ATP and 100mM KCl and did not differ from fast-twitch skeletal muscle. The temporalis maximal calcium uptake and Ca-ATPase activity were lower but the sensitivity to the specific Ca-ATPase inhibitor thapsigargin was higher. Calcium-binding to the enzyme and calcium efflux were similar while the phosphorylation of the enzyme by P(i) was lower. The lower enzymatic activity and calcium transport capability of the Ca-ATPase isolated from rabbit temporalis, and the higher sensitivity to inhibitory drugs are consistent with the presence of a substantial proportion of SERCA 2, which can be expected in other rabbit masticatory muscles. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Active ingredients in Chinese medicines promoting blood circulation as Na+/K+ -ATPase inhibitors.

    Science.gov (United States)

    Chen, Ronald J Y; Jinn, Tzyy-rong; Chen, Yi-ching; Chung, Tse-yu; Yang, Wei-hung; Tzen, Jason T C

    2011-02-01

    The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na(+)/K(+)-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na(+)/K(+)-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na(+)/K(+)-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na(+)/K(+)-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na(+)/K(+)-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na(+)/K(+)-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na(+)/K(+)-ATPase in the brain could be potential drugs for the treatment of ischemic stroke.

  11. Biphasic effect of protein kinase C on rat renal cortical Na+, K+-ATPase.

    Science.gov (United States)

    Bełtowski, J; Górny, D; Marciniak, A

    1998-12-01

    We examined the dependence of rat renal Na+, K+-ATPase activity on protein kinase C (PKC) stimulation. Infusion of either phorbol 12, 13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) into rat abdominal aorta resulted in dose-dependent changes of renal cortical Na+, K+-ATPase activity. Low doses of these esters (3 x 10(-11) mol/kg/min) increased activity of Na+, K+-ATPase whereas high doses (3 x 10(-9) mol/kg/min) decreased it. The changes in Na+, K+-ATPase activity induced by PDBu and PMA were prevented by staurosporine, a PKC inhibitor. 4Alpha phorbol didecanoate (4alpha PDD), phorbol ester which does not activate PKC had no effect on cortical Na+, K+-ATPase. PDBu and PMA did not change Na+, K+-ATPase activity in the renal medulla. The stimulatory effect of PDBu (3 x 10(-11) mol/kg/min) was neither mimicked by amphotericin B, a sodium ionophore nor blocked by amiloride, an inhibitor of Na+/H+-exchanger. The inhibitory effect of 3 x 10(-9) mol/kg/min PDBu was not mimicked by amiloride indicating that the observed effects of PKC stimulation are not secondary to alterations in intracellular sodium concentration. The inhibitory effect of PDBu was prevented by infusion of ethoxyresorufin, an inhibitor of cytochrome P450-dependent arachidonate metabolism. These results suggest that the inhibitory effect of PKC on renal cortical Na+, K+-ATPase is mediated by cytochrome P450-dependent arachidonate metabolites.

  12. The alpha Na-2(+)/K+-ATPase is critical for skeletal and heart muscle function in zebrafish

    DEFF Research Database (Denmark)

    Doganli, Canan; Kjaer-Sorensen, K.; Knoeckel, C.

    2012-01-01

    The Na+/K+-ATPase generates ion gradients across the plasma membrane, essential for multiple cellular functions. In mammals, four different Na+/K+-ATPase alpha-subunit isoforms are associated with characteristic cell-type expression profiles and kinetics. We found the zebrafish alpha Na-2(+)/K......-and downregulation of specific phenotype-related proteins, such as parvalbumin, CaM, GFAP and multiple kinases, thus highlighting a potential proteome change associated with the dynamics of alpha Na-2(+)/K+-ATPase. Taken together, our findings show that zebrafish alpha Na-2(+)/K+-ATPase is important for skeletal...

  13. Insights into the Pathology of the α2-Na(+)/K(+)-ATPase in Neurological Disorders; Lessons from Animal Models.

    Science.gov (United States)

    Isaksen, Toke J; Lykke-Hartmann, Karin

    2016-01-01

    A functional Na(+)/K(+)-ATPase consists of a catalytic α subunit and a regulatory β subunit. Four α isoforms of the Na(+)/K(+)-ATPase are found in mammals, each with a unique expression pattern and catalytic activity. The α2 isoform, encoded by the ATP1A2 gene, is primarily found in the central nervous system (CNS) and in heart-, skeletal- and smooth muscle tissues. In the CNS, the α2 isoform is mainly expressed in glial cells. In particular, the α2 isoform is found in astrocytes, important for astrocytic K(+) clearance and, consequently, the indirect uptake of neurotransmitters. Both processes are essential for proper brain activity, and autosomal dominantly mutations in the ATP1A2 gene cause the neurological disorder Familial hemiplegic migraine type 2 (FHM2). FHM2 is a severe subtype of migraine with aura including temporary numbness or weakness, and affecting only one side of the body. FHM2 patients often suffer from neurological comorbidities such as seizures, sensory disturbances, cognitive impairment, and psychiatric manifestations. The functional consequences of FHM2 disease mutations leads to a partial or complete loss of function of pump activity; however, a clear phenotype-genotype correlation has yet to be elucidated. Gene-modified mouse models targeting the Atp1a2 gene have proved instrumental in the understanding of the pathology of FHM2. Several Atp1a2 knockout (KO) mice targeting different exons have been reported. Homozygous Atp1a2 KO mice die shortly after birth due to respiratory malfunction resulting from abnormal Cl(-) homeostasis in brainstem neurons. Heterozygous KO mice are viable, but display altered behavior and neurological deficits such as altered spatial learning, decreased motor activity and enhanced fear/anxiety compared to wild type mice. FHM2 knock-in (KI) mouse models carrying the human in vivo disease mutations W887R and G301R have also been reported. Both models display altered cortical spreading depression (CSD) and point

  14. INSIGHTS INTO THE PATHOLOGY OF THE α2-Na+/K+-ATPase IN NEUROLOGICAL DISORDERS; LESSONS FROM ANIMAL MODELS

    Directory of Open Access Journals (Sweden)

    Toke Jost Isaksen

    2016-05-01

    Full Text Available A functional Na+/K+-ATPase consists of a catalytic α subunit and a regulatory β subunit. Four α isoforms of the Na+/K+-ATPase are found in mammals, each with a unique expression pattern and catalytic activity. The α2 isoform, encoded by the ATP1A2 gene, is primarily found in the central nervous system (CNS and in heart-, skeletal- and smooth muscle tissues. In the CNS, the α2 isoform is mainly expressed in neuroglial cells. In particular, the α2 isoform is found in astrocytes, and is important for astrocytic K+ clearance and, consequently, the indirect uptake of neurotransmitters. Both processes are essential for proper brain activity, and autosomal dominantly mutations in the ATP1A2 gene cause the neurological disorder Familial hemiplegic migraine type 2 (FHM2. FHM2 is a severe subtype of migraine with aura that involving temporary numbness or weakness, and affecting only one side of the body. FHM2 patients often suffer from neurological comorbidities such as seizures, sensory disturbances, cognitive impairment and psychiatric manifestations. The functional consequences of FHM2 disease mutations leads to a partial or complete loss of function of pump activity; however a clear phenotype-genotype correlation has yet to be elucidated. Gene-modified mouse models targeting the Atp1a2 gene have proved instrumental in the understanding of the pathology of FHM2. Several Atp1a2 knockout (KO mice targeting different exons have been reported. Homozygous Atp1a2 KO mice die shortly after birth due to respiratory malfunction resulting from abnormal Cl- homeostasis in brainstem neurons. Heterozygous KO mice are viable, but display altered behavior and neurological deficits such as altered spatial learning, decreased motor activity and enhanced fear/anxiety compared to wild type mice. FHM2 knock-in (KI mouse models carrying the human in vivo disease mutations W887R and G301R have also been reported. Both models display altered cortical spreading

  15. Host and Pathogen Copper-Transporting P-Type ATPases Function Antagonistically during Salmonella Infection.

    Science.gov (United States)

    Ladomersky, Erik; Khan, Aslam; Shanbhag, Vinit; Cavet, Jennifer S; Chan, Jefferson; Weisman, Gary A; Petris, Michael J

    2017-09-01

    Copper is an essential yet potentially toxic trace element that is required by all aerobic organisms. A key regulator of copper homeostasis in mammalian cells is the copper-transporting P-type ATPase ATP7A, which mediates copper transport from the cytoplasm into the secretory pathway, as well as copper export across the plasma membrane. Previous studies have shown that ATP7A-dependent copper transport is required for killing phagocytosed Escherichia coli in a cultured macrophage cell line. In this investigation, we expanded on these studies by generating Atp7a(LysMcre) mice, in which the Atp7a gene was specifically deleted in cells of the myeloid lineage, including macrophages. Primary macrophages isolated from Atp7a(LysMcre) mice exhibit decreased copper transport into phagosomal compartments and a reduced ability to kill Salmonella enterica serovar Typhimurium compared to that of macrophages isolated from wild-type mice. The Atp7a(LysMcre) mice were also more susceptible to systemic infection by S Typhimurium than wild-type mice. Deletion of the S Typhimurium copper exporters, CopA and GolT, was found to decrease infection in wild-type mice but not in the Atp7a(LysMcre) mice. These studies suggest that ATP7A-dependent copper transport into the phagosome mediates host defense against S Typhimurium, which is counteracted by copper export from the bacteria via CopA and GolT. These findings reveal unique and opposing functions for copper transporters of the host and pathogen during infection. Copyright © 2017 American Society for Microbiology.

  16. Phenylarsine Oxide Inhibits the Fusicoccin-Induced Activation of Plasma Membrane H+-ATPase1

    Science.gov (United States)

    Olivari, Claudio; Albumi, Cristina; Pugliarello, Maria Chiara; De Michelis, Maria Ida

    2000-01-01

    To investigate the mechanism by which fusicoccin (FC) induces the activation of the plasma membrane (PM) H+-ATPase, we used phenylarsine oxide (PAO), a known inhibitor of protein tyrosine-phosphatases. PAO was supplied in vivo in the absence or presence of FC to radish (Raphanus sativus L.) seedlings and cultured Arabidopsis cells prior to PM extraction. Treatment with PAO alone caused a slight decrease of PM H+-ATPase activity and, in radish, a decrease of PM-associated 14-3-3 proteins. When supplied prior to FC, PAO drastically inhibited FC-induced activation of PM H+-ATPase, FC binding to the PM, and the FC-induced increase of the amount of 14-3-3 associated with the PM. On the contrary, PAO was completely ineffective on all of the above-mentioned parameters when supplied after FC. The H+-ATPase isolated from PAO-treated Arabidopsis cells maintained the ability to respond to FC if supplied with exogenous, nonphosphorylated 14-3-3 proteins. Altogether, these results are consistent with a model in which the dephosphorylated state of tyrosine residues of a protein(s), such as 14-3-3 protein, is required to permit FC-induced association between the 14-3-3 protein and the PM H+-ATPase. PMID:10677439

  17. Bidirectional regulation of renal cortical Na+,K+-ATPase by protein kinase C.

    Science.gov (United States)

    Bełtowski, Jerzy; Marciniak, Andrzej; Jamroz-Wiśniewska, Anna; Borkowska, Ewelina; Wójcicka, Grazyna

    2004-01-01

    We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10(-11) mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10(-9) and 10(-8) mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K(0.5) for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and Gö 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.

  18. Enhancement of the hydrolysis activity of F0F1-ATPases using 60 Hz magnetic fields.

    Science.gov (United States)

    Chen, Chuanfang; Cui, Yuanbo; Yue, Jiachang; Huo, Xiaolin; Song, Tao

    2009-12-01

    The effects of extremely low frequency (ELF) magnetic fields on membrane F(0)F(1)-ATPase activity have been studied. When the F(0)F(1)-ATPase was exposed to 60 Hz magnetic fields of different magnetic intensities, 0.3 and 0.5 mT magnetic fields enhanced the hydrolysis activity, whereas 0.1 mT exposure caused no significant changes. Even if the F(0)F(1)-ATPase was inhibited by N,N-dicyclohexylcarbodiimide, its hydrolysis activity was enhanced by a 0.5 mT 60 Hz magnetic field. Moreover, when the chromatophores which were labeled with F-DHPE were exposed to a 0.5 mT, 60 Hz magnetic field, it was found that the pH of the outer membrane of the chromatophore was unchanged, which suggested that the magnetic fields used in this work did not affect the activity of F0. Taken together, our results show that the effects of magnetic fields on the hydrolysis activity of the membrane F(0)F(1)-ATPases were dependent on magnetic intensity and the threshold intensity is between 0.1 and 0.3 mT, and suggested that the F1 part of F(0)F(1)-ATPase may be an end-point affected by magnetic fields.

  19. Cryo-EM studies of the structure and dynamics of vacuolar-type ATPases.

    Science.gov (United States)

    Mazhab-Jafari, Mohammad T; Rubinstein, John L

    2016-07-01

    Electron cryomicroscopy (cryo-EM) has significantly advanced our understanding of molecular structure in biology. Recent innovations in both hardware and software have made cryo-EM a viable alternative for targets that are not amenable to x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Cryo-EM has even become the method of choice in some situations where x-ray crystallography and NMR spectroscopy are possible but where cryo-EM can determine structures at higher resolution or with less time or effort. Rotary adenosine triphosphatases (ATPases) are crucial to the maintenance of cellular homeostasis. These enzymes couple the synthesis or hydrolysis of adenosine triphosphate to the use or production of a transmembrane electrochemical ion gradient, respectively. However, the membrane-embedded nature and conformational heterogeneity of intact rotary ATPases have prevented their high-resolution structural analysis to date. Recent application of cryo-EM methods to the different types of rotary ATPase has led to sudden advances in understanding the structure and function of these enzymes, revealing significant conformational heterogeneity and characteristic transmembrane α helices that are highly tilted with respect to the membrane. In this Review, we will discuss what has been learned recently about rotary ATPase structure and function, with a particular focus on the vacuolar-type ATPases.

  20. Differential effects of inhibitors and detergents on the Ca/sup 2 +/-ATPase and Mg/sup 2 +/-ATPase activities of the plasma membrane of a human oat cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Knowles, A.F.; Lawrence, C.M.

    1986-05-01

    Plasma membranes of human oat cell carcinoma possess Mg/sup 2 +/- and Ca/sup 2 +/-dependent ATPase activities of similar magnitude. These activities exhibit the unusual characteristic of being inactiviated by prolonged incubation of the membrane with 1-2 mM dithiothreitol (DTT). Inactivation by DTT was prevented by lowering the incubation temperature, elevation of the membrane protein concentration, and addition of ATP. Fluorosulfonylbenzoyl adenosine (FSBA), an affinity ATP analog, also inactivates these activities. The Ca/sup 2 +/-ATPase activity appears to be more sensitive to both DTT and FSBA. The Ca/sup 2 +/-ATPase activity is more easily inactivated by Triton X-100, while the Mg/sup 2 +/-ATPase is preferentially activated by digitonin. These differential effects of inhibitors and detergents suggest that the Ca/sup 2 +/-ATPase and Mg/sup 2 +/-ATPase are separate enzymes. Incubation of oat cell carcinoma plasma membrane with (/sup 3/H)FSBA resulted in the labeling of several proteins. A labelled 35,000 dalton protein corresponds to the molecular weight of the oat cell carcinoma plasma membrane Ca/sup 2 +/-ATPase previously purified in this laboratory. The identity of one or more of the other labelled proteins with the Mg/sup 2 +/-ATPase has not been demonstrated, but is presently under investigation.

  1. ε subunit of Bacillus subtilis F1-ATPase relieves MgADP inhibition.

    Directory of Open Access Journals (Sweden)

    Junya Mizumoto

    Full Text Available MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1, which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF1. We conclude that the ε subunit relieves BF1 from MgADP inhibition.

  2. Single-molecule, structural and functional studies of Listeria monocytogenes Ca2+-ATPase

    DEFF Research Database (Denmark)

    Dyla, Mateusz

    -ion transport (e.g. H+ for Ca2+-ATPases). P-type ATPases undergo major conformational changes during their functional cycle, as has been learned from a wealth of atomic-resolution X-ray crystallographic structures (4). In this work, single-molecule, structural and functional studies were employed to investigate...... of Cy3 and Cy5 in an optimized form of LMCA1 with reduced background labeling. LMCA1 was found to reside in the high-FRET E1 conformational state through most of its functional cycle, even in the absence of Ca2+. Binding of Ca2+ brought the cytoplasmic domains of LMCA1 closer together, whereas...... of single vesicles, providing complementary read-out of the single-molecule dynamics. Furthermore, the effects of metal fluorides on the ATPase activity of the pump were characterized to validate the possibility of trapping LMCA1 in specific functional states analogous to the well-studied sarco...

  3. Understanding the inhibitory effect of highly potent and selective archazolides binding to the vacuolar ATPase.

    Science.gov (United States)

    Dreisigacker, Sandra; Latek, Dorota; Bockelmann, Svenja; Huss, Markus; Wieczorek, Helmut; Filipek, Slawomir; Gohlke, Holger; Menche, Dirk; Carlomagno, Teresa

    2012-08-27

    Vacuolar ATPases are a potential therapeutic target because of their involvement in a variety of severe diseases such as osteoporosis or cancer. Archazolide A (1) and related analogs have been previously identified as selective inhibitors of V-ATPases with potency down to the subnanomolar range. Herein we report on the determination of the ligand binding mode by a combination of molecular docking, molecular dynamics simulations, and biochemical experiments, resulting in a sound model for the inhibitory mechanism of this class of putative anticancer agents. The binding site of archazolides was confirmed to be located in the equatorial region of the membrane-embedded V(O)-rotor, as recently proposed on the basis of site-directed mutagenesis. Quantification of the bioactivity of a series of archazolide derivatives, together with the docking-derived binding mode of archazolides to the V-ATPase, revealed favorable ligand profiles, which can guide the development of a simplified archazolide analog with potential therapeutic relevance.

  4. Glutamate transporter activity promotes enhanced Na+/K+-ATPase-mediated extracellular K+ management during neuronal activity

    DEFF Research Database (Denmark)

    Larsen, Brian Roland; Holm, Rikke; Vilsen, Bente

    2016-01-01

    Neuronal activity is associated with transient [K+]o increases. The excess K+ is cleared by surrounding astrocytes, partly by the Na+/K+-ATPase of which several subunit isoform combinations exist. The astrocytic Na+/K+-ATPase α2β2 isoform constellation responds directly to increased [K+]o but...... Na+ affinity of isoform constellations involving the astrocytic β2 has remained elusive as a result of inherent expression of β1 in most cell systems, as well as technical challenges involved in measuring intracellular affinity in intact cells. We therefore expressed the different astrocytic isoform...... constellations in Xenopus oocytes and determined their apparent Na+ affinity in intact oocytes and isolated membranes. The Na+/K+-ATPase was not fully saturated at basal astrocytic [Na+]i, irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na+ affinity to the α1 and α2...

  5. Quantity of Na/K-ATPase and glucose transporters in the plasma membrane of rat adipocytes is reduced by in vivo triiodothyronine

    DEFF Research Database (Denmark)

    Voldstedlund, M.; Tranum-Jensen, Jørgen; Handberg, Aa.

    1995-01-01

    Anatomi, Na/K-ATPase, glucose transporters, adipocyt, immuno-gold labelling, electron microscopy......Anatomi, Na/K-ATPase, glucose transporters, adipocyt, immuno-gold labelling, electron microscopy...

  6. Presence of cardiac alpha-myosin correlates with histochemical myosin Ca2+ ATPase activity in rabbit masseter muscle

    NARCIS (Netherlands)

    Bredman, J. J.; Weijs, W. A.; Moorman, A. F.

    1992-01-01

    A combined enzyme-histochemical (ATPase reactivity) and immunohistochemical study has been performed on sections of rabbit masseter muscle. The majority of the fibres previously designated as type IIC and/or type I according to their ATPase activity were found to contain 'cardiac' alpha-myosin heavy

  7. Diphyllin, a novel and naturally potent V-ATPase inhibitor, abrogates acidification of the osteoclastic resorption lacunae and bone resorption

    DEFF Research Database (Denmark)

    Sørensen, Mette G; Henriksen, Kim; Neutzsky-Wulff, Anita V

    2007-01-01

    Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays...

  8. Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-ATPase

    NARCIS (Netherlands)

    Duarte, A.M.; Wolfs, C.J.A.M.; Nuland, van N.A.J.; Harrison, M.A.; Findlay, J.B.C.; Mierlo, van C.P.M.; Hemminga, M.A.

    2007-01-01

    Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific

  9. Proton pump inhibitors decrease eotaxin-3/CCL26 expression in patients with chronic rhinosinusitis with nasal polyps: Possible role of the nongastric H,K-ATPase.

    Science.gov (United States)

    Min, Jin-Young; Ocampo, Christopher J; Stevens, Whitney W; Price, Caroline P E; Thompson, Christopher F; Homma, Tetsuya; Huang, Julia H; Norton, James E; Suh, Lydia A; Pothoven, Kathryn L; Conley, David B; Welch, Kevin C; Shintani-Smith, Stephanie; Peters, Anju T; Grammer, Leslie C; Harris, Kathleen E; Hulse, Kathryn E; Kato, Atsushi; Modyanov, Nikolai N; Kern, Robert C; Schleimer, Robert P; Tan, Bruce K

    2017-01-01

    Chronic rhinosinusitis with nasal polyps (CRSwNP) is often characterized by tissue eosinophilia that is associated with poor prognosis. Recent findings that proton pump inhibitors (PPIs) directly modulate the expression of eotaxin-3, an eosinophil chemoattractant, in patients with eosinophilic diseases suggest therapeutic potential for PPIs in those with CRSwNP. We assessed the effect of type 2 mediators, particularly IL-13 and eotaxin-3, on tissue eosinophilia and disease severity in patients with chronic rhinosinusitis (CRS). Further investigation focused on PPI suppression of eotaxin-3 expression in vivo and in vitro, with exploration of underlying mechanisms. Type 2 mediator levels in nasal tissues and secretions were measured by using a multiplex immunoassay. Eotaxin-3 and other chemokines expressed in IL-13-stimulated human sinonasal epithelial cells (HNECs) and BEAS-2B cells with or without PPIs were assessed by using ELISA, Western blotting, real-time PCR, and intracellular pH imaging. Nasal tissues and secretions from patients with CRSwNP had increased IL-13, eotaxin-2, and eotaxin-3 levels, and these were positively correlated with tissue eosinophil cationic protein levels and radiographic scores in patients with CRS (P H(+),K(+)-exchange, which was blocked by PPIs and the mechanistically unrelated H,K-ATPase inhibitor, SCH-28080. Furthermore, knockdown of ATP12A (gene for the nongastric H,K-ATPase) significantly attenuated IL-13-induced eotaxin-3 expression in HNECs. PPIs also had effects on accelerating IL-13-induced eotaxin-3 mRNA decay. Our results demonstrated that PPIs reduce IL-13-induced eotaxin-3 expression by airway epithelial cells. Furthermore, mechanistic studies suggest that the nongastric H,K-ATPase is necessary for IL-13-mediated epithelial responses, and its inhibitors, including PPIs, might be of therapeutic value in patients with CRSwNP by reducing epithelial production of eotaxin-3. Copyright © 2016 American Academy of Allergy

  10. Towards defining the substrate of orphan P5A-ATPases

    DEFF Research Database (Denmark)

    Sørensen, Danny Mollerup; Holen, Henrik Waldal; Holemans, Tine

    2015-01-01

    of tail-anchored proteins in the ER membrane. A role for P5A-ATPases in vesicle formation would explain why sterol transport and distribution are affected in knock out cells, which in turn has a negative impact on the spontaneous insertion of tail-anchored proteins. It would also explain why secretory...... significance Identification of the substrate of P5A-ATPases would throw light on an important general process in the ER that is still not fully understood. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins....

  11. Effect of TGFβ on Na{sup +}/K{sup +} ATPase activity in megakaryocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina [Department of Physiology, University of Tübingen (Germany); Laufer, Stefan [Pharmaceutical Chemistry, University of Tübingen (Germany); Borst, Oliver; Gawaz, Meinrad [Cardiology and Cardiovascular Medicine, University of Tübingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tübingen (Germany)

    2014-09-26

    Highlights: • TGFß1 markedly up-regulates Na{sup +}/K{sup +} ATPase in megakaryocytes. • The effect is abrogated by p38-MAP kinase inhibitor skepinone. • The effect is abrogated by SGK inhibitor EMD638683. • The effect is abrogated by NF-κB inhibitor wogonin. - Abstract: The Na{sup +}/K{sup +} ATPase generates the Na{sup +} and K{sup +} concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na{sup +}/K{sup +} ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ influences Na{sup +}/K{sup +} ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na{sup +}/K{sup +} ATPase activity determined from K{sup +} induced current utilizing whole cell patch clamp. The pump current (I{sub pump}) was determined in the absence and presence of Na{sup +}/K{sup +} ATPase inhibitor ouabain (100 μM). TGFß1 (60 ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) or NF-κB inhibitor wogonin (50 nM). As a result, the I{sub pump} was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24 h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion

  12. Structure and function of Cu(I)- and Zn(II)-ATPases

    DEFF Research Database (Denmark)

    Sitsel, Oleg; Grønberg, Christina; Autzen, Henriette

    2015-01-01

    Copper and zinc are micronutrients essential for the function of many enzymes while also being toxic at elevated concentrations. Cu(I)- and Zn(II)-transporting P-type ATPases of subclass 1B are of key importance for the homeostasis of these transition metals, allowing ion transport across cellular...... membranes at the expense of ATP. Recent biochemical studies and crystal structures have significantly improved our understanding of the transport mechanisms of these proteins, but many details about their structure and function remain elusive. Here we compare the Cu(I)- and Zn(II)-ATPases, scrutinizing...

  13. Membrane Anchoring and Ion-Entry Dynamics in P-type ATPase Copper Transport

    DEFF Research Database (Denmark)

    Grønberg, Christina; Sitsel, Oleg; Lindahl, Erik

    2016-01-01

    Cu(+)-specific P-type ATPase membrane protein transporters regulate cellular copper levels. The lack of crystal structures in Cu(+)-binding states has limited our understanding of how ion entry and binding are achieved. Here, we characterize the molecular basis of Cu(+) entry using molecular-dynamics...... simulations, structural modeling, and in vitro and in vivo functional assays. Protein structural rearrangements resulting in the exposure of positive charges to bulk solvent rather than to lipid phosphates indicate a direct molecular role of the putative docking platform in Cu(+) delivery. Mutational analyses...... and provide a molecular understanding of ion entry in Cu(+)-transporting P-type ATPases....

  14. Regulatory assembly of the vacuolar proton pump VoV1-ATPase in yeast cells by FLIM-FRET

    Science.gov (United States)

    Ernst, Stefan; Batisse, Claire; Zarrabi, Nawid; Böttcher, Bettina; Börsch, Michael

    2010-02-01

    We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca2+ homoeostasis in the cell. The activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.

  15. MsmK, an ATPase, Contributes to Utilization of Multiple Carbohydrates and Host Colonization of Streptococcus suis.

    Science.gov (United States)

    Tan, Mei-Fang; Gao, Ting; Liu, Wan-Quan; Zhang, Chun-Yan; Yang, Xi; Zhu, Jia-Wen; Teng, Mu-Ye; Li, Lu; Zhou, Rui

    2015-01-01

    Acquisition and metabolism of carbohydrates are essential for host colonization and pathogenesis of bacterial pathogens. Different bacteria can uptake different lines of carbohydrates via ABC transporters, in which ATPase subunits energize the transport though ATP hydrolysis. Some ABC transporters possess their own ATPases, while some share a common ATPase. Here we identified MsmK, an ATPase from Streptococcus suis, an emerging zoonotic bacterium causing dead infections in pigs and humans. Genetic and biochemistry studies revealed that the MsmK was responsible for the utilization of raffinose, melibiose, maltotetraose, glycogen and maltotriose. In infected mice, the msmK-deletion mutant showed significant defects of survival and colonization when compared with its parental and complementary strains. Taken together, MsmK is an ATPase that contributes to multiple carbohydrates utilization and host colonization of S. suis. This study gives new insight into our understanding of the carbohydrates utilization and its relationship to the pathogenesis of this zoonotic pathogen.

  16. Quaternary structure of the ATPase complex of human 26S proteasomes determined by chemical cross-linking

    DEFF Research Database (Denmark)

    Hartmann-Petersen, R; Tanaka, K; Hendil, K B

    2001-01-01

    and substrate specificity. Among the approximately 18 subunits of PA700 regulator, six are ATPases. The ATPases presumably recognize, unfold, and translocate substrates into the interior of the 26S proteasome. It is generally believed that the ATPases form a hexameric ring. By means of chemical cross-linking......, immunoprecipitation, and blotting, we have determined that the ATPases are organized in the order S6-S6'-S10b-S8-S4-S7. Additionally, we found cross-links between the ATPase S10b and the 20S proteasome subunit alpha6. Together with the previously known interaction between S8 and alpha1 and between S4 and alpha7...

  17. Immunocytochemical localization of V-H(+) -ATPase, Na(+) /K(+) -ATPase, and carbonic anhydrase in gill lamellae of adult freshwater euryhaline shrimp Macrobrachium acanthurus (Decapoda, Palaemonidae).

    Science.gov (United States)

    Maraschi, Anieli Cristina; Freire, Carolina Arruda; Prodocimo, Viviane

    2015-08-01

    Physiological (organismal), biochemical, and molecular biological contributions to the knowledge of the osmoregulatory plasticity of palaemonid freshwater shrimps has provided a fairly complete model of transporter localization in their branchial epithelium. Direct immunological demonstration of the main enzymes in the gill epithelia of adult palaemonids is, however, still incipient. The diadromous freshwater shrimp Macrobrachium acanthurus was exposed to increased salinity (25‰ for 24 hr), and its responses at the systemic level were evaluated through the assays of hemolymph osmolality and muscle hydration, and at cellular and subcellular levels through the activity and localization of the V-H(+) -ATPase, the Na(+) /K(+) -ATPase, and the carbonic anhydrase. Results showed an increase in hemolymph osmolality (629 ± 5.3 mOsm/kg H2 O) and a decrease in muscle hydration (73.8 ± 0.5%), comparing values after 24 hr in 25‰ with control shrimps in freshwater (respectively 409.5 ± 15.8 mOsm/kg H2 O and 77.5 ± 0.4%). V-H(+) -ATPase was localized in pillar cells, whereas Na(+) /K(+) -ATPase in the septal cells. The main novelty of this study was that carbonic anhydrase was localized in the whole branchial tissue, in pillar and septal cells. Exposure to high salinity for 24 hr led to no detectable changes in their localization or in vitro activity. Immunolocalization data corroborated the literature and current models of palaemonid gill ion transport. The absence of changes reinforces the need for the constant expression of these enzymes to account for the euryhalinity of these shrimps. © 2015 Wiley Periodicals, Inc.

  18. Increased oxidative stress and decreased activities of Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase in the red blood cells of the hibernating black bear.

    Science.gov (United States)

    Chauhan, Ved P S; Tsiouris, John A; Chauhan, Abha; Sheikh, Ashfaq M; Brown, W Ted; Vaughan, Michael

    2002-05-31

    During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P hibernation (P hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.

  19. Increased oxidative stress and decreased activities of Ca2+/Mg2+-ATPase and Na+/K+-ATPase in the red blood cells of the hibernating black bear

    Science.gov (United States)

    Chauhan, V.P.S.; Tsiouris, J.A.; Chauhan, A.; Sheikh, A.M.; Brown, W. Ted; Vaughan, M.

    2002-01-01

    During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P hibernation (P hibernating state as compared to the active state. Na+/K+-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca2+/Mg2+-ATPase and Na+/K+-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression. ?? 2002 Elsevier Science Inc. All rights reserved.

  20. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy; (IIT); (Penn)

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  1. Plasmodium falciparum from Pará state (Brazil) shows satisfactory in vitro response to artemisinin derivatives and absence of the S769N mutation in the SERCA-type PfATPase6.

    Science.gov (United States)

    Ferreira, Isabel D; Martinelli, Axel; Rodrigues, Louise A; do Carmo, Ediclei L; do Rosário, Virgílio E; Póvoa, Marinete M; Cravo, Pedro

    2008-02-01

    To evaluate the in vitro efficacy of artesunate (ATN) and artemether (ATH) against Plasmodium falciparum isolates from the Brazilian Amazon state of Pará and to search for mutations and/or altered copy numbers in the putative resistance-associated pfcrt, pfmdr1 and pfATPase6 genes. In vitro efficacy of ATN and ATH was successfully measured in 56 freshly collected P. falciparum isolates, using a conventional WHO microtest with minor modifications. Single nucleotide polymorphisms (SNPs) in the same isolates were inspected using DNA sequencing and/or PCR-RFLP. We used real-time quantitative PCR to assess gene copy numbers. ATN and ATH geometric mean IC(50)s were 0.85 nm, 95% CI (0.55-1.15) and 3.0 nm, 95% CI (1.5-4.5), respectively. There was extremely limited diversity of pfcrt and pfmdr1 genotypes and three SNPs were identified in the pfATPase6 gene: one T to A synonymous mutation at nucleotide 2694 and two non-synonymous (both G to A) mutations at nucleotides 110 and 1916, causing predicted aminoacid shifts of arginine to lysine and of glycine to aspartate, respectively. The previously reported S769N mutation was not detected in any of the isolates inspected. In addition, no gene amplifications were detected in a subset of eight isolates. Artemisinin derivatives display satisfactory in vitro activity locally and the pfATPase6 gene is distinct from that reported in French Guiana, suggesting that those haplotypes have not been introduced regionally.

  2. Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

    Science.gov (United States)

    Zhang, Junjie; He, Shanping; Wang, Yi; Brulois, Kevin; Lan, Ke; Jung, Jae U; Feng, Pinghui

    2015-03-01

    G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi's sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of "constitutive" NFAT activation by viral GPCRs.

  3. Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

    Directory of Open Access Journals (Sweden)

    Junjie Zhang

    2015-03-01

    Full Text Available G protein-coupled receptors (GPCRs constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi's sarcoma-associated herpesvirus (kGPCR and cytomegalovirus (US28 shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA, which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of "constitutive" NFAT activation by viral GPCRs.

  4. Potassium as an intrinsic uncoupler of the plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Palmgren, Michael Gjedde; Buch-Pedersen, Morten Jeppe

    The plant plasma membrane proton pump (H(+)-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K(+) is bound to the proton pump at a site involving Asp(617) in the cytoplasmic...

  5. Biochemical studies on the origin of the ATPase of the avian ...

    Indian Academy of Sciences (India)

    tribpo

    also been demonstrated in herpes virus (Epstein and Hott, 1963) and influenza virus. (Ada and Lind, 1961). De-The (1964a) has presented evidence based on electron microscopic studies that the ATPase of the AMV originates from the cell membrane of the myeloblasts. The studies presented in this paper support the ...

  6. Quantification of renal Na-K-ATPase activity by image analysing system.

    Science.gov (United States)

    Laborde, K; Bussieres, L; De Smet, A; Dechaux, M; Sachs, C

    1990-01-01

    The localisation of renal Na-K-ATPase activity along the rat nephron by a cytochemical method, and its quantification by an image analysis system, are described in this paper. Frozen kidney sections were exposed to a trapping agent, the lead ammoniac-citrate-acetate complex (LACA), and to all the substrates necessary to the enzyme activity. The absorbance of the histochemical reaction product (precipitated in situ), proportional to the enzymatic activity, was then measured through the analysis of the grey levels of the transmitted image of the kidney section. This method was both sufficiently sensitive and technically simple to permit measurements of the enzyme in large numbers of tubules and to determine its activity in each region of the nephron. The Na-K-ATPase activity has been determined in the proximal convoluted tubule (PCT), the medullary thick ascending limb of the Henle's loop (mTAL), and the distal convoluted tubules (DCT) of the rat nephron. The Na-K-ATPase distribution shows an activity per millimeter tubule length higher in the DCT than in the mTAL and the PCT: 1,406 +/- 33, 823 +/- 64, and 350 +/- 71 pmoles Pi/tubule mm/h, respectively. In conclusion, the described method allows the segmental quantification of Na-K-ATPase activity at a cellular level and offers a precise approach to the analysis of this enzyme along the length of nephrons.

  7. Raman Spectroscopy of Conformational Changes in Membrane-Bound Sodium Potassium ATPase

    DEFF Research Database (Denmark)

    Helix Nielsen, Claus; Abdali, Salim; Lundbæk, Jens August

    2007-01-01

    In this investigation we assess the potential of Raman spectroscopy as a tool for probing conformational changes in membrane-spanning proteins — in this case, the sodium potassium adenosine triphosphatase (Na+,K+-ATPase). Spectral analysis of protein-lipid complexes is complicated by the presence...

  8. Identification of Na+/K+-ATPase inhibitors in bovine plasma as fatty acids and hydrocarbons

    DEFF Research Database (Denmark)

    Tal, D M; Yanuck, M D; Van Hall, Gerrit

    1989-01-01

    ouabain, and in addition it enhanced ouabain binding at high dilutions. These properties are indicative of nonspecific interactions with the Na+/K+-ATPase. The active fraction was identified by TLC, HPLC, NMR, GLC and GC-MS, to be a mixture of three unesterified fatty acids, mainly oleic acid (72...

  9. Mechanism of the asymmetric activation of the MinD ATPase by MinE

    Science.gov (United States)

    Park, Kyung-Tae; Wu, Wei; Lovell, Scott; Lutkenhaus, Joe

    2012-01-01

    Summary MinD is a component of the Min system involved in the spatial regulation of cell division. It is an ATPase in the MinD/ParA/Mrp deviant Walker A motif family which is within the P loop GTPase superfamily. Its ATPase activity is stimulated by MinE, however, the mechanism of this activation is unclear. MinD forms a symmetric dimer with two binding sites for MinE, however, a recent model suggested that MinE occupying one site was sufficient for ATP hydrolysis. By generating heterodimers with one binding site for MinE we show that one binding site is sufficient for stimulation of the MinD ATPase. Furthermore, comparison of structures of MinD and related proteins led us to examine the role of N45 in the switch I region. An asparagine at this position is conserved in four of the deviant Walker A motif subfamilies (MinD, chromosomal ParAs, Get3 and FleN) and we find that N45 in MinD is essential for MinE stimulated ATPase activity and suggest that it is a key residue affected by MinE binding. PMID:22651575

  10. V-ATPase, ScNhx1p and yeast vacuole fusion.

    Science.gov (United States)

    Qiu, Quan-Sheng

    2012-04-20

    Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos. It is a central cellular reaction that plays important roles in signal transduction, protein sorting and subcellular compartmentation. Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summarized in this article. It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhx1p are key components of the vacuole fusion machinery in yeast. Yeast ScNhx1p regulates vacuole fusion by controlling the luminal pH. V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast. Fission defects are epistatic to fusion defects. Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast, the fusion reaction does not need the transport activity but requires the physical presence of the proton pump. V0, the membrane-integral sector of the V-ATPase, forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the V0trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion. Copyright © 2012. Published by Elsevier Ltd.

  11. Occurrence of NaK-ATPase isoforms during rat inner ear development and functional implications.

    NARCIS (Netherlands)

    Peters, T.A.; Kuijpers, W.; Curfs, J.H.A.J.

    2001-01-01

    This study examined the presence of NaK-ATPase isoforms in the developing inner ear of the rat and studied the importance of functional subunit combinations in endolymph homeostasis. The findings were: (a) the combination alpha 1 beta 1 is found in epithelial, mesenchymal, and neural inner ear cells

  12. Electron cryomicroscopy of two-dimensional crystals of the H+-ATPase from chloroplasts

    NARCIS (Netherlands)

    Böttcher, Bettina; Gräber, Peter; Boekema, Egbert J.; Lücken, Uwe

    1995-01-01

    The H+-ATPase from spinach chloroplasts was isolated and purified. Two-dimensional crystals were obtained from the protein/lipid/detergent micelles by treatment with phospholipase and simultaneous removal of detergent and fatty acids by Biobeads. The resulting two-dimensionally ordered arrays were

  13. ELECTRON CRYOMICROSCOPY OF 2-DIMENSIONAL CRYSTALS OF THE H+-ATPASE FROM CHLOROPLASTS

    NARCIS (Netherlands)

    BOTTCHER, B; GRABER, P; BOEKEMA, EJ; LUCKEN, U

    1995-01-01

    The H+-ATPase from spinach chloroplasts was isolated and purified, Two-dimensional crystals were obtained from the protein/lipid/detergent micelles by treatment with phospholipase and simultaneous removal of detergent and fatty acids by Biobeads. The resulting two-dimensionally ordered arrays were

  14. Increased daylength stimulates plasma growth hormone and gill Na+, K+ and -ATPase Atlantic salmon (Salmo salar )

    Science.gov (United States)

    McCormick, S.D.; Bjornsson, Bjorn Thrandur; Sheridan, M.; Eilertson, C.; Carey, J.B.; O'Dea, M.

    1995-01-01

    Atlantic salmon juveniles reared at constant temperature (9–10°C) were exposed to four photoperiod treatment and sampled every 2 weeks from January through May. Fish reared under normal photoperiod exhibited eight-and three fold increases in plasma growth hormone and gill Na+, K+-ATPase activity, respectively, between January and April. Fish exposed to abrupt increases in daylength (LD 15:9) in February or March responded with earlier increases in plasma growth hormone and gill Na+, K+-ATPase activity, and earlier decreases in condition factor relative to fish in the normal photoperiod group. Fish maintained under short daylength (LD 9:15) from January to May exhibited delayed and muted increases in plasma growth hormone and gill Na+, K+-ATPase activity. Plasma thyroxine exhibited a 2.5-fold increase from February to late March in the normal photoperiod group, was generally lower in the LD 9:15 group, but exhibited no obvious response to abrupt increases in daylength. There was an increase in plasma 3,5,3′-triiodo-l-thyronine with time in all groups (43–80%) but no significant response to photoperiod. Plasma levels of somatostatin-25 were highest in the LD 9:15 group, but there was no detectable response to increased daylength in any of the photoperiod treatments. The results indicate that plasma growth hormone is responsive to increased daylength and may be causally related to subsequent increases in gill Na+, K+-ATPase.

  15. [Peroxidative vulnerability of synaptosomal high affinity Ca++-ATPase and pharmacologic effects].

    Science.gov (United States)

    Blaschke, M; Fischer, H D; Schmidt, J

    1988-01-01

    The high affinity Ca++-ATPase participates essentially in the regulation of intrasynaptosomal calcium homeostasis. Related to posthypoxically restricted transmitter release, we examined the influence of newly-generated free radicals (ascorbic acid-ferric salt mixture) or sodium dodecyl sulfate in vitro and of a mild hypobaric hypoxia in vivo on the activity of synaptosomal high affinity Ca++-ATPase. Moreover we tested the effectiveness of piracetam, meclofenoxate hydrochloride, pyritinol and verapamil on the changed enzyme activity subsequent to a hypoxic exposure. The activity of synaptosomal high affinity Ca++-ATPase (1.04 +/- 0.03 mumol Pi/mg.h) is reduced by not more than 40% depending on the concentration of the ascorbic acid-ferric salt mixture used but is nearly totally inhibited by sodium dodecyl sulfate (0.2 mg/ml). Hypobaric hypoxia (18 h, 8.7 kPa) decreases the enzyme activity to 0.79 +/- 0.03 mumol Pi/mg.h. Piracetam, meclofenoxate hydrochloride and pyritinol are protectively effective on the decrease of enzyme activity induced by hypoxia. The results emphasize the importance of intact protein-phospholipid interactions for the enzyme activity and support relations between synaptosomal high affinity Ca++-ATPase and transmitter release.

  16. Electrostatic Stabilization Plays a Central Role in Autoinhibitory Regulation of the Na+,K+-ATPase

    DEFF Research Database (Denmark)

    Jiang, Qiucen; Garcia, Alvaro; Han, Minwoo

    2017-01-01

    The Na+,K+-ATPase is present in the plasma membrane of all animal cells. It plays a crucial role in maintaining the Na+ and K+ electrochemical potential gradients across the membrane, which are essential in numerous physiological processes, e.g., nerve, muscle, and kidney function. Its cellular a...

  17. Neurological disease mutations compromise a C-terminal ion pathway in the Na(+)/K(+)-ATPase

    DEFF Research Database (Denmark)

    Poulsen, Hanne; Khandelia, Himanshu; Morth, J Preben

    2010-01-01

    The Na(+)/K(+)-ATPase pumps three sodium ions out of and two potassium ions into the cell for each ATP molecule that is split, thereby generating the chemical and electrical gradients across the plasma membrane that are essential in, for example, signalling, secondary transport and volume regulat...

  18. Glutamate Water Gates in the Ion Binding Pocket of Na(+) Bound Na(+), K(+)-ATPase

    DEFF Research Database (Denmark)

    Han, Minwoo; Kopec, Wojciech; Solov'yov, Ilia A

    2017-01-01

    The dynamically changing protonation states of the six acidic amino acid residues in the ion binding pocket of the Na(+), K(+) -ATPase (NKA) during the ion transport cycle are proposed to drive ion binding, release and possibly determine Na(+) or K(+) selectivity. We use molecular dynamics (MD) a...

  19. Tuning of the Na,K-ATPase by the beta subunit

    DEFF Research Database (Denmark)

    Hilbers, Florian; Kopec, Wojciech; Isaksen, Toke Jost

    2016-01-01

    The vital gradients of Na(+) and K(+) across the plasma membrane of animal cells are maintained by the Na,K-ATPase, an αβ enzyme complex, whose α subunit carries out the ion transport and ATP hydrolysis. The specific roles of the β subunit isoforms are less clear, though β2 is essential for motor...

  20. Na(+), K(+)-ATPase dysfunction causes cerebrovascular endothelial cell degeneration in rat prefrontal cortex slice cultures.

    Science.gov (United States)

    Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Katsuki, Hiroshi

    2016-08-01

    Cerebrovascular endothelial cell dysfunction resulting in imbalance of cerebral blood flow contributes to the onset of psychiatric disorders such as depression, schizophrenia and bipolar disorder. Although decrease in Na(+), K(+)-ATPase activity has been reported in the patients with schizophrenia and bipolar disorder, the contribution of Na(+), K(+)-ATPase to endothelial cell dysfunction remains poorly understood. Here, by using rat neonatal prefrontal cortex slice cultures, we demonstrated that pharmacological inhibition of Na(+), K(+)-ATPase by ouabain induced endothelial cell injury. Treatment with ouabain significantly decreased immunoreactive area of rat endothelial cell antigen-1 (RECA-1), a marker of endothelial cells, in a time-dependent manner. Ouabain also decreased Bcl-2/Bax ratio and phosphorylation level of glycogen synthase kinase 3β (GSK3β) (Ser9), which were prevented by lithium carbonate. On the other hand, ouabain-induced endothelial cell injury was exacerbated by concomitant treatment with LY294002, an inhibitor of phosphoinositide 3- (PI3-) kinase. We also found that xestospongin C, an inhibitor of inositol triphosphate (IP3) receptor, but not SEA0400, an inhibitor of Na(+), Ca(2+) exchanger (NCX), protected endothelial cells from cytotoxicity of ouabain. These results suggest that cerebrovascular endothelial cell degeneration induced by Na(+), K(+)-ATPase inhibition resulting in Ca(2+) release from endoplasmic reticulum (ER) and activation of GSK3β signaling underlies pathogenesis of these psychiatric disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. The sensitivity of Na + , K + ATPase as an indicator of blood diseases

    African Journals Online (AJOL)

    Background: Blood-related hereditary diseases are widespread in Eastern and SouthWestern regions of Saudi Arabia until recently. In this study, we used Na+, K+ATPase as an enzymatic indicator for the diagnosis of the diseases. Materials and methods: Individuals with different blood diseases (iron deficiency (n=13), ...

  2. Inhibition of Na(+)/K(+) -ATPase by antcins, unique steroid-like compounds in Antrodia camphorate.

    Science.gov (United States)

    Chung, Tse-Yu; Li, Feng-Yin; Chang, Chi-I; Jinn, Tzyy-Rong; Tzen, Jason T C

    2012-01-01

    The inhibition of Na(+)/K(+) -ATPase by versatile steroid-like compounds contributes to the putative therapeutic effects of many Chinese medicinal cardiac products via the same molecular mechanism triggered by cardiac glycosides. Five major steroid-like compounds, antcin A, B, C, H, and K were isolated from Niuchangchih (Antrodia camphorata), a unique Taiwan mushroom, and all inhibited Na(+)/K(+) -ATPase. Antcin A exhibited significantly higher inhibitory potency than the other four antcins, though weaker than ginsenoside Rh2 . In contrast, cortisone (an analogous steroid with anti-inflammatory effects stronger than antcin A) showed no detectable inhibitory potency. Molecular modeling has shown that antcins bind to Na(+)/K(+) -ATPase with the steroidal skeleton structurally upside-down in comparison with ginsenoside Rh2 . The inhibitory potency of antcin A is attributed to steroidal hydrophobic interaction within the binding pocket and the formation of three hydrogen bonds between its carboxyl group and two cationic residues around the cavity entrance of Na(+)/K(+) -ATPase. The presence of an additional carbonyl or hydroxyl group at C7 of the other four antcins leads to severe repulsion in the hydrophobic pocket, and thus significantly reduces inhibitory potency. It is proposed that antcin A is a bi-functional compound that exerts anti-inflammatory effects and that enhances blood circulation via two different molecular mechanisms.

  3. Biochemical Characterization of P4-ATPase Mutations Associated with Intrahepatic Cholestatic Disease

    DEFF Research Database (Denmark)

    Gantzel, Rasmus; Vestergaard, Anna Lindeløv; Mikkelsen, Stine

    The cholestatic disorders progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1) are caused by mutation of the P4-ATPase ATP8B1 that flips phospholipid from the exoplasmic leaflet to the cytoplasmic leaflet of canalicular membrane...

  4. Biochemical characterization of P4-ATPase mutations associated with Intrahepatic Cholestatic Disease

    DEFF Research Database (Denmark)

    Gantzel, Rasmus; Vestergaard, Anna Lindeløv; Mikkelsen, Stine

    Progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1) are caused by mutation of the P4-ATPase ATP8B1 that flips phospholipid from the exoplasmic leaflet to the cytoplasmic leaflet of canalicular membranes. It is hypothesized that...

  5. Biochemical Characterization of P4-ATPase Mutations Associated with Intrahepatic Cholestatic Disease

    DEFF Research Database (Denmark)

    Gantzel, Rasmus; Vestergaard, Anna Lindeløv; Mikkelsen, Stine

    The cholestatic disorders progressive familial intrahepatic cholestasis type 1 (PFIC1, also referred to as Byler’s disease) and benign recurrent intrahepatic cholestasis type 1 (BRIC1) are caused by mutation of the P4-ATPase ATP8B1. The substrate of ATP8B1 is very likely to be phosphatidylserine ...

  6. Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs.

    Directory of Open Access Journals (Sweden)

    Yong-Qiang Zhang

    2010-06-01

    Full Text Available Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H(+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma(- phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca(2+ and H(+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca(2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.

  7. Ubxd1 is a novel co-factor of the human p97 ATPase

    DEFF Research Database (Denmark)

    Madsen, Louise; Andersen, Katrine M; Prag, Søren

    2008-01-01

    The AAA ATPase complex known as p97 or VCP in mammals and Cdc48 in yeast is connected to a multitude of cellular pathways, including membrane fusion, protein folding, protein degradation and activation of membrane-bound transcription factors. The mechanism by which p97 participates in such a broa...

  8. Sodium potassium adenosine triphosphatase (Na/K-ATPase) as a therapeutic target for uremic cardiomyopathy.

    Science.gov (United States)

    Wang, Xiaoliang; Liu, Jiang; Drummond, Christopher A; Shapiro, Joseph I

    2017-05-01

    Clinically, patients with significant reductions in renal function present with cardiovascular dysfunction typically termed, uremic cardiomyopathy. It is a progressive series of cardiac pathophysiological changes, including left ventricular diastolic dysfunction and hypertrophy (LVH) which sometimes progress to left ventricular dilation (LVD) and systolic dysfunction in the setting of chronic kidney disease (CKD). Uremic cardiomyopathy is almost ubiquitous in patients afflicted with end stage renal disease (ESRD). Areas covered: This article reviews recent epidemiology, pathophysiology of uremic cardiomyopathy and provide a board overview of Na/K-ATPase research with detailed discussion on the mechanisms of Na/K-ATPase/Src/ROS amplification loop. We also present clinical and preclinical evidences as well as molecular mechanism of this amplification loop in the development of uremic cardiomyopathy. A potential therapeutic peptide that targets on this loop is discussed. Expert opinion: Current clinical treatment for uremic cardiomyopathy remains disappointing. Targeting the ROS amplification loop mediated by the Na/K-ATPase signaling function may provide a novel therapeutic target for uremic cardiomyopathy and related diseases. Additional studies of Na/K-ATPase and other strategies that regulate this loop will lead to new therapeutics.

  9. The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura

    The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues Rosa Laura López-Marqués1, Lisbeth Rosager Poulsen1, Katharina Meffert2, Thomas Pomorski2, Michael Gjedde Palmgren1 1Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation...

  10. The nonlinear chemo-mechanic coupled dynamics of the F 1 -ATPase molecular motor.

    Science.gov (United States)

    Xu, Lizhong; Liu, Fang

    2012-03-01

    The ATP synthase consists of two opposing rotary motors, F0 and F1, coupled to each other. When the F1 motor is not coupled to the F0 motor, it can work in the direction hydrolyzing ATP, as a nanomotor called F1-ATPase. It has been reported that the stiffness of the protein varies nonlinearly with increasing load. The nonlinearity has an important effect on the rotating rate of the F1-ATPase. Here, considering the nonlinearity of the γ shaft stiffness for the F1-ATPase, a nonlinear chemo-mechanical coupled dynamic model of F1 motor is proposed. Nonlinear vibration frequencies of the γ shaft and their changes along with the system parameters are investigated. The nonlinear stochastic response of the elastic γ shaft to thermal excitation is analyzed. The results show that the stiffness nonlinearity of the γ shaft causes an increase of the vibration frequency for the F1 motor, which increases the motor's rotation rate. When the concentration of ATP is relatively high and the load torque is small, the effects of the stiffness nonlinearity on the rotating rates of the F1 motor are obvious and should be considered. These results are useful for improving calculation of the rotating rate for the F1 motor and provide insight about the stochastic wave mechanics of F1-ATPase.

  11. Control analysis of the dependence of Escherichia coli physiology on the H+ -ATPase

    DEFF Research Database (Denmark)

    Jensen, Peter Ruhdal; Michelsen, Ole; Westerhoff, Hans V.

    1993-01-01

    The H+-ATPase plays a central role in Escherichia coli free-energy transduction and hence in E. coli physiology. We here investigate the extent to which this enzyme also controls the growth rate, growth yield, and respiratory rate of E. coli. We modulate the expression of the atp operon and deter...

  12. The drug diazaborine blocks ribosome biogenesis by inhibiting the AAA-ATPase Drg1

    NARCIS (Netherlands)

    Loibl, M.; Klein, I; Prattes, M.; Schmidt, C.; Kappel, L.; Zisser, G.; Gungl, A.; Krieger, E.; Pertschy, B.; Bergler, H.

    2014-01-01

    The drug diazaborine is the only known inhibitor of ribosome biogenesis and specifically blocks large subunit formation in eukaryotic cells. However, the target of this drug and the mechanism of inhibition were unknown. Here we identify the AAA-ATPase Drg1 as a target of diazaborine. Inhibitor

  13. Photoinactivation of Detergent-Solubilized Plasma Membrane ATPase from Rosa damascena: Action Spectra.

    Science.gov (United States)

    Imbrie, C W; Murphy, T M

    1984-03-01

    The photochemistry of vesicular and detergent-solubilized preparations of plasma membrane-associated ATPase was investigated in Rosa damascena. The cholate-solubilized ATPase activity fractionated into two peaks on a Sephadex G-150 column with simple, but different ultraviolet (UV) sensitivities. The larger enzyme was UV sensitive; the smaller enzyme was relatively insensitive. The activity of both ATPase fractions depended on environment: both were inactive in cholate, relatively inactive in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and active in phosphatidylglycerol and phosphatidylserine. The UV sensitivities of both fractions also depended on their environment. For the UV sensitive fraction, the action spectrum differed in the 300 to 400 nanometers range when the fraction was irradiated with and without lipids. For the resistant fraction, UV sensitivity at 290 nanometers differed (up to 6-fold) in different lipids. The resistant fraction solubilized in octylglucoside had an action spectrum very different from that in cholate or in lipid vesicles. The absorption spectra of the different preparations reflected the action spectra. For both UV sensitive and insensitive fractions, the action spectra for photoinactivation had peaks at 290 nanometers, suggesting that the chromophores were tryptophanyl residues. The loss of ATPase activity was strictly correlated with the loss of fluorescence from tryptophan in the partially purified enzymes. Cs(+) protected the UV sensitive activity but not the insensitive one. We propose a model which explains the difference in UV sensitivities based on the positions of the tryptophan residues in the two proteins.

  14. Photoinactivation of Detergent-Solubilized Plasma Membrane ATPase from Rosa damascena

    Science.gov (United States)

    Imbrie, Catherine W.; Murphy, Terence M.

    1984-01-01

    The photochemistry of vesicular and detergent-solubilized preparations of plasma membrane-associated ATPase was investigated in Rosa damascena. The cholate-solubilized ATPase activity fractionated into two peaks on a Sephadex G-150 column with simple, but different ultraviolet (UV) sensitivities. The larger enzyme was UV sensitive; the smaller enzyme was relatively insensitive. The activity of both ATPase fractions depended on environment: both were inactive in cholate, relatively inactive in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and active in phosphatidylglycerol and phosphatidylserine. The UV sensitivities of both fractions also depended on their environment. For the UV sensitive fraction, the action spectrum differed in the 300 to 400 nanometers range when the fraction was irradiated with and without lipids. For the resistant fraction, UV sensitivity at 290 nanometers differed (up to 6-fold) in different lipids. The resistant fraction solubilized in octylglucoside had an action spectrum very different from that in cholate or in lipid vesicles. The absorption spectra of the different preparations reflected the action spectra. For both UV sensitive and insensitive fractions, the action spectra for photoinactivation had peaks at 290 nanometers, suggesting that the chromophores were tryptophanyl residues. The loss of ATPase activity was strictly correlated with the loss of fluorescence from tryptophan in the partially purified enzymes. Cs+ protected the UV sensitive activity but not the insensitive one. We propose a model which explains the difference in UV sensitivities based on the positions of the tryptophan residues in the two proteins. PMID:16663470

  15. The F- or V-Type Na+-ATPase of the Thermophilic Bacterium Clostridium fervidus

    NARCIS (Netherlands)

    SPEELMANS, G; POOLMAN, B; ABEE, T; KONINGS, WN

    Clostridium fervidus is a thermophilic, anaerobic bacterium which uses solely Na+ as a coupling ion for energy transduction. Important features of the primary Na+ pump (ATPase) that generates the sodium motive force are presented. The advantage of using a sodium rather than a proton motive force at

  16. Sepsis impairs alveolar epithelial function by downregulating Na-K-ATPase pump.

    Science.gov (United States)

    Berger, Gidon; Guetta, Julia; Klorin, Geula; Badarneh, Reem; Braun, Eyal; Brod, Vera; Saleh, Niroz Abu; Katz, Adriana; Bitterman, Haim; Azzam, Zaher S

    2011-07-01

    Widespread vascular endothelial injury is the major mechanism for multiorgan dysfunction in sepsis. Following this process, the permeability of the alveolar capillaries is augmented with subsequent increase in water content and acute respiratory distress syndrome (ARDS). Nevertheless, the role of alveolar epithelium is less known. Therefore, we examined alveolar fluid clearance (AFC) using isolated perfused rat lung model in septic rats without ARDS. Sepsis was induced by ligating and puncturing the cecum with a 21-gauge needle. AFC was examined 24 and 48 h later. The expression of Na-K-ATPase proteins was examined in type II alveolar epithelial cells (ATII) and basolateral membrane (BLM). The rate of AFC in control rats was 0.51 ± 0.02 ml/h (means ± SE) and decreased to 0.3 ± 0.02 and 0.33 ± 0.03 ml/h in 24 and 48 h after sepsis induction, respectively (P sodium channel and Na-K-ATPase proteins in whole ATII cells was not different in both cecal ligation and puncture and control groups; however, the abundance of Na-K-ATPase proteins was significantly decreased in BLMs of ATII cells in sepsis. Early decrease in AFC in remote sepsis is probably related to endocytosis of the Na-K-ATPase proteins from the cell plasma membrane into intracellular pools, with resultant inhibition of active sodium transport in ATII cells.

  17. bcpmr1 encodes a P-type Ca(2+)/Mn(2+)-ATPase mediating cell-wall integrity and virulence in the phytopathogen Botrytis cinerea.

    Science.gov (United States)

    Plaza, Verónica; Lagües, Yanssuy; Carvajal, Mauro; Pérez-García, Luis A; Mora-Montes, Hector M; Canessa, Paulo; Larrondo, Luis F; Castillo, Luis

    2015-03-01

    The cell wall of fungi is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. These usually contain both N- and O-linked oligosaccharides, coupled to the proteins by stepwise addition of mannose residues by mannosyltransferases in the endoplasmic reticulum and the Golgi apparatus. In yeast, an essential luminal cofactor for these mannosyltransferases is Mn(2+) provided by the Ca(2+)/Mn(2+)-ATPase known as Pmr1. In this study, we have identified and characterized the Botrytis cinerea pmr1 gene, the closest homolog of yeast PMR1. We hypothesized that bcpmr1 also encodes a Ca(2+)/Mn(2+)-ATPase that plays an important role in the protein glycosylation pathway. Phenotypic analysis showed that bcpmr1 null mutants displayed a significant reduction in conidial production, radial growth and diameter of sclerotia. Significant alterations in hyphal cell wall composition were observed including a 83% decrease of mannan levels and an increase in the amount of chitin and glucan. These changes were accompanied by a hypersensitivity to cell wall-perturbing agents such as Calcofluor white, Congo red and zymolyase. Importantly, the Δbcpmr1 mutant showed reduced virulence in tomato (leafs and fruits) and apple (fruits) and reduced biofilm formation. Together, our results highlight the importance of bcpmr1 for protein glycosylation, cell wall structure and virulence of B. cinerea. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Function and evolution of a MicroRNA that regulates a Ca2+-ATPase and triggers the formation of phased small interfering RNAs in tomato reproductive growth.

    Science.gov (United States)

    Wang, Ying; Itaya, Asuka; Zhong, Xuehua; Wu, Yang; Zhang, Jianfeng; van der Knaap, Esther; Olmstead, Richard; Qi, Yijun; Ding, Biao

    2011-09-01

    MicroRNAs (miRNAs) regulate a wide variety of biological processes in most eukaryotes. We investigated the function and evolution of miR4376 in the family Solanaceae. We report that the 22-nucleotide miR4376 regulates the expression of an autoinhibited Ca(2+)-ATPase, tomato (Solanum lycopersicum) ACA10, which plays a critical role in tomato reproductive growth. Deep phylogenetic mapping suggested (1) an evolution course of MIR4376 loci and posttranscriptional processing of pre-miR4376 as a likely limiting step for the evolution of miR4376, (2) an independent phylogenetic origin of the miR4376 target site in ACA10 homologs, and (3) alternative splicing as a possible mechanism of eliminating such a target in some ACA10 homologs. Furthermore, miR4376 triggers the formation of phased small interfering RNAs (siRNAs) from Sl ACA10 and its Solanum tuberosum homolog. Together, our data provide experimental evidence of miRNA-regulated expression of universally important Ca(2+)-ATPases. The miR4376-regulated expression of ACA10 itself, and possibly also the associated formation of phased siRNAs, may function as a novel layer of molecular mechanisms underlying tomato reproductive growth. Finally, our data suggest that the stochastic emergence of a miRNA-target gene combination involves multiple molecular events at the genomic, transcriptional, and posttranscriptional levels that may vary drastically in even closely related species.

  19. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase

    DEFF Research Database (Denmark)

    Jahn, T.; Fuglsang, A.T.; Olsson, A.

    1997-01-01

    Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-AT...... plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase....

  20. The effect of exercise and beta2-adrenergic stimulation on glutathionylation and function of the Na,K-ATPase in human skeletal muscle

    DEFF Research Database (Denmark)

    Juel, Carsten; Hostrup, Morten; Bangsbo, Jens

    2015-01-01

    subunits are further glutathionylated by exercise and beta2-adrenergic stimulation. Our data suggest that glutathionylation contributes to the complex regulation of Na,K-ATPase function in human skeletal muscle. Glutathionylation of the Na,K-ATPase may explain reductions in maximal Na,K-ATPase activity......Potassium and sodium displacements across the skeletal muscle membrane during exercise may cause fatigue and are in part controlled by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for muscle functioning. We investigated the effect of oxidative stress (glutathionylation...

  1. Surface Plasmon Resonance Biosensor Method for Palytoxin Detection Based on Na+,K+-ATPase Affinity

    Directory of Open Access Journals (Sweden)

    Amparo Alfonso

    2013-12-01

    Full Text Available Palytoxin (PLTX, produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (kobs. From the representation of kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (KD for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods.

  2. Surface Plasmon Resonance Biosensor Method for Palytoxin Detection Based on Na+,K+-ATPase Affinity

    Science.gov (United States)

    Alfonso, Amparo; Pazos, María-José; Fernández-Araujo, Andrea; Tobio, Araceli; Alfonso, Carmen; Vieytes, Mercedes R.; Botana, Luis M.

    2013-01-01

    Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (kobs). From the representation of kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (KD) for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods. PMID:24379088

  3. Toward an understanding of the Cdc48/p97 ATPase [version 1; referees: 4 approved

    Directory of Open Access Journals (Sweden)

    Nicholas Bodnar

    2017-08-01

    Full Text Available A conserved AAA+ ATPase, called Cdc48 in yeast and p97 or VCP in metazoans, plays an essential role in many cellular processes by segregating polyubiquitinated proteins from complexes or membranes. For example, in endoplasmic reticulum (ER-associated protein degradation (ERAD, Cdc48/p97 pulls polyubiquitinated, misfolded proteins out of the ER and transfers them to the proteasome. Cdc48/p97 consists of an N-terminal domain and two ATPase domains (D1 and D2. Six Cdc48 monomers form a double-ring structure surrounding a central pore. Cdc48/p97 cooperates with a number of different cofactors, which bind either to the N-terminal domain or to the C-terminal tail. The mechanism of Cdc48/p97 action is poorly understood, despite its critical role in many cellular systems. Recent in vitro experiments using yeast Cdc48 and its heterodimeric cofactor Ufd1/Npl4 (UN have resulted in novel mechanistic insight. After interaction of the substrate-attached polyubiquitin chain with UN, Cdc48 uses ATP hydrolysis in the D2 domain to move the polypeptide through its central pore, thereby unfolding the substrate. ATP hydrolysis in the D1 domain is involved in substrate release from the Cdc48 complex, which requires the cooperation of the ATPase with a deubiquitinase (DUB. Surprisingly, the DUB does not completely remove all ubiquitin molecules; the remaining oligoubiquitin chain is also translocated through the pore. Cdc48 action bears similarities to the translocation mechanisms employed by bacterial AAA ATPases and the eukaryotic 19S subunit of the proteasome, but differs significantly from that of a related type II ATPase, the NEM-sensitive fusion protein (NSF. Many questions about Cdc48/p97 remain unanswered, including how it handles well-folded substrate proteins, how it passes substrates to the proteasome, and how various cofactors modify substrates and regulate its function.

  4. Radiation inactivation analysis of oligomeric structure of the H,K-ATPase.

    Science.gov (United States)

    Rabon, E C; Gunther, R D; Bassilian, S; Kempner, E S

    1988-11-05

    The oligomeric size of the H,K-ATPase was determined in frozen gastric microsomal vesicles irradiated with high energy electrons. Target sizes of various catalytic activities associated with H,K-ATPase function fell into two distinct groups. The lower group of target sizes described the radiation-induced loss of steady-state phosphoenzyme and structural monomer: the MgATP-dependent formation of a beta-aspartyl phosphate exhibited a size range of 133-147 kDa; the size range for the structural measurement (i.e. loss of H,K-ATPase monomer on sodium dodecyl sulfate-polyacrylamide gels) was 92-143 kDa. In contrast, a larger group of target sizes described the loss of full cycle catalytic activities (i.e. K+-dependent stimulation of p-nitrophenyl phosphate and ATP hydrolysis). The K+-phosphatase and K+-stimulated ATPase exhibited target sizes fo 200 +/- 13 and 232 +/- 23 kDa, respectively. The lower target size group represents the first evidence that a monomer of the catalytic subunit maintains partial enzyme function. The larger group of target sizes describing K+-phosphatase and ATPase activities suggest that subunit interactions contribute to full cycle catalytic activity. Subunit interactions appear to be involved in all ion transport activities. Passive Rb+ exchange and active H+ transport in reconstituted proteoliposomes exhibited target sizes of 233n = 2 and 388 +/- 48 kDa, respectively. H+ transport appears to require a subunit arrangement more complex than that associated with catalytic activity or passive ion transport.

  5. Inhibition of duodenal enterocyte Mg2+-ATPase by arachidonic acid is not mediated by an effect on protein kinase C.

    Science.gov (United States)

    Haag, M; Leonard, F; Magada, O N; Kruger, M C

    2000-03-01

    Active absorption processes in the duodenal enterocyte are driven by various ATPases. It is known that the activity of Na+,K+-ATPase, Ca2+-ATPase and Mg2+-ATPase can be modulated by polyunsaturated fatty acids of the n-6 series, for example by linoleic and gamma-linolenic acids. These effects may be achieved by protein phosphorylation via protein kinase C. The present study was undertaken to determine the effect of arachidonic acid on Mg2+-ATPase (measured colorimetrically) activity in basolateral membranes prepared from rat duodenum. It shows, for the first time, significant dose-dependent inhibition of Mg2+-ATPase (26-62%) by arachidonic acid (10-50 microg/ml) which already takes place after one minute of exposure, indicating involvement of a rapid signal transduction mechanism. Addition of the protein kinase C inhibitors bisimidolylmaleimide (2.5 microM) and calphostin (0.5 microM) did not influence the action of arachidonic acid on Mg2+-ATPase; protein kinase C involvement in this process is thus not indicated.

  6. Chronic nicotine modifies skeletal muscle Na,K-ATPase activity through its interaction with the nicotinic acetylcholine receptor and phospholemman.

    Directory of Open Access Journals (Sweden)

    Alexander V Chibalin

    Full Text Available Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21-31 days. Chronic nicotine produced a steady membrane depolarization of ∼3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV while the activity of the α1 isoform decreased (-4.4 mV. Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM, measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser(63 and Ser(68. Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM.

  7. H(+) -ATPase-defective variants of Lactobacillus delbrueckii subsp. bulgaricus contribute to inhibition of postacidification of yogurt during chilled storage.

    Science.gov (United States)

    Wang, Xinhui; Ren, Hongyang; Liu, Dayu; Wang, Bing; Zhu, Wenyou; Wang, Wei

    2013-02-01

    Continued acid production by Lactobacillus delbrueckii subsp. bulgaricus during the chilled storage of yogurt is the major cause of postacidification, resulting in a short shelf life. Two H(+) -ATPase defective variants of L. delbrueckii subsp. bulgaricus were successfully isolated and their H(+) -ATPase activities were reduced by 51.3% and 34.3%, respectively. It was shown that growth and acid production of variants were remarkably inhibited. The variants were more sensitive to acidic condition and had a significant rate for inactivation of H(+) -ATPase by N, N-dicyclohexylcarbodiimide (DCCD), along with a low H(+) -extrusion, suggesting that H(+) -ATPase is direct response for H(+) -extrusion. In addition, the variants were also more sensitive to NaCl, while H(+) -ATPase activities of variants and parent strain were significantly enhanced by NaCl stress. Obviously, H(+) -ATPase might be involved in Na(+) transportation. Furthermore, variants were inoculated in fermented milk to ferment yogurt. There was no significant difference in flavor, whereas the postacidification of yogurt during chilled storage was remarkably inhibited. It is suggested that application of L. delbrueckii subsp. bulgaricus with reduced H(+) -ATPase activity in yogurt fermentation is one of effect, economic and simple avenues of inhibiting postacidification of yogurt during refrigerated storage, giving a longer shelf life. During yogurt fermentation, continued acid production by Lactobacillus delbrueckii subsp. bulgaricus during the chilled storage of yogurt leads to milk fermentation with high postacidification, resulting in a short shelf life. In this work, 2 acid-sensitive variant strains of L. delbrueckii subsp. bulgaricus were isolated. The characteristics related to H(+) -ATPase were compared and it was observed that milk fermented by the variants had lower postacidification, giving a longer shelf life. Application of L. delbrueckii subsp. bulgaricus with reduced H(+) -ATPase activity

  8. Proton Pumping and Slippage Dynamics of a Eukaryotic P-Type ATPase Studied at the Single-Molecule Level

    DEFF Research Database (Denmark)

    Veshaguri, Salome

    In all eukaryotes the plasma membrane potential and secondary transport systems are energized by P-type ATPases whose regulation however remains poorly understood. Here we monitored at the single-molecule level the activity of the prototypic proton pumping P-type ATPase Arabidopsis thaliana isoform...... pumping rates remained constant. Titration of ATP down to ~1% of apparent Km for ATPase activity exclusively affected the distributions of the durations the pump spends in active and inactive states. The dramatic consequence of our findings is that ATP reduction decreased ATP/H+ stoichiometry of the pump...

  9. Phylogenetic incongruence and the evolutionary origins of cardenolide-resistant forms of Na(+) ,K(+) -ATPase in Danaus butterflies.

    Science.gov (United States)

    Aardema, Matthew L; Andolfatto, Peter

    2016-08-01

    Many distantly related insect species are specialized feeders of cardenolide-containing host plants such as milkweed (Asclepias spp.). Previous studies have revealed frequent, parallel substitution of a functionally important amino acid substitution (N122H) in the alpha subunit of Na(+) ,K(+) -ATPase in a number of these species. This substitution facilitates the ability of these insects to feed on their toxic hosts and sequester cardenolides for their own use in defense. Among milkweed butterflies of the genus Danaus, the previously established phylogeny for this group suggests that N122H arose independently and fixed in two distinct lineages. We reevaluate this conclusion by examining Danaus phylogenetic relationships using >400 orthologous gene sequences assembled from transcriptome data. Our results indicate that the three Danaus species known to harbor the N122H substitution are more closely related than previously thought, consistent with a single, common origin for N122H. However, we also find evidence of both incomplete lineage sorting and post-speciation genetic exchange among these butterfly species, raising the possibility of collateral evolution of cardenolide-insensitivity in this species group. © 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.

  10. Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells.

    Science.gov (United States)

    Seo, Jeong-Ah; Kim, Boyun; Dhanasekaran, Danny N; Tsang, Benjamin K; Song, Yong Sang

    2016-02-01

    Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca(2+) homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca(2+) concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca(2+) flux was evident after the curcumin treatment (15 µM). Treatment with Ca(2+) chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca(2+) concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca(2+) elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca(2+) homeostasis and thereby promotes apoptosis in ovarian cancer cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  11. Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression

    Science.gov (United States)

    Zatyka, Malgorzata; Da Silva Xavier, Gabriela; Bellomo, Elisa A.; Leadbeater, Wendy; Astuti, Dewi; Smith, Joel; Michelangeli, Frank; Rutter, Guy A.; Barrett, Timothy G.

    2015-01-01

    Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca2+ imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca2+ concentration ([Ca2+]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis. PMID:25274773

  12. Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders.

    Science.gov (United States)

    Kang, Soojeong; Dahl, Russell; Hsieh, Wilson; Shin, Andrew; Zsebo, Krisztina M; Buettner, Christoph; Hajjar, Roger J; Lebeche, Djamel

    2016-03-04

    Dysregulation of endoplasmic reticulum (ER) Ca(2+) homeostasis triggers ER stress leading to the development of insulin resistance in obesity and diabetes. Impaired function of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) has emerged as a major contributor to ER stress. We pharmacologically activated SERCA2b in a genetic model of insulin resistance and type 2 diabetes (ob/ob mice) with a novel allosteric activator, CDN1163, which markedly lowered fasting blood glucose, improved glucose tolerance, and ameliorated hepatosteatosis but did not alter glucose levels or body weight in lean controls. Importantly, CDN1163-treated ob/ob mice maintained euglycemia comparable with that of lean mice for >6 weeks after cessation of CDN1163 administration. CDN1163-treated ob/ob mice showed a significant reduction in adipose tissue weight with no change in lean mass, assessed by magnetic resonance imaging. They also showed an increase in energy expenditure using indirect calorimetry, which was accompanied by increased expression of uncoupling protein 1 (UCP1) and UCP3 in brown adipose tissue. CDN1163 treatment significantly reduced the hepatic expression of genes involved in gluconeogenesis and lipogenesis, attenuated ER stress response and ER stress-induced apoptosis, and improved mitochondrial biogenesis, possibly through SERCA2-mediated activation of AMP-activated protein kinase pathway. The findings suggest that SERCA2b activation may hold promise as an effective therapy for type-2 diabetes and metabolic dysfunction. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. An ENA ATPase, MaENA1, of Metarhizium acridum influences the Na(+)-, thermo- and UV-tolerances of conidia and is involved in multiple mechanisms of stress tolerance.

    Science.gov (United States)

    Ma, Qinsi; Jin, Kai; Peng, Guoxiong; Xia, Yuxian

    2015-10-01

    In fungi, ENA ATPases play key roles in osmotic and alkaline pH tolerance, although their functions in thermo- and UV-tolerances have not been explored. Entomopathogenic fungi are naturally widespread and have considerable potential in pest control. An ENA ATPase gene, MaENA1, from the entomopathogenic fungus Metarhizium acridum was functionally analyzed by deletion. MaENA1-disruption strain (ΔMaENA1) was less tolerant to NaCl, heat, and UV radiation than a wild-type strain (WT). Digital Gene Expression profiling of conidial RNAs resulted in 281 differentially expressed genes (DEGs) between the WT and ΔMaENA1 strains. Eighty-five DEGs, 56 of which were down-regulated in the ΔMaENA1 strain, were shown to be associated with heat/UV tolerance, including six cytochrome P450 superfamily genes, 35 oxidoreductase genes, 24 ion-binding genes, seven DNA repair genes, and five other genes. In addition, eight genes were components of stress responsive pathways, including the Ras-cAMP PKA pathway, the RIM101 pathway, the Ca(2+)/calmodulin pathway, the TOR pathway, and the HOG/Spc1/Sty1/JNK pathway. These results demonstrated that MaENA1 influences fungal tolerances to Na(+), heat, and UV radiation in M. acridum, and is involved in multiple mechanisms of stress tolerance. Therefore, MaENA1 is required for the adaptation and survival of entomopathogenic fungi in stressful conditions in the environment and in their hosts. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase.

    Science.gov (United States)

    Song, Jianliang; Zhang, Xue-Qian; Wang, JuFang; Cheskis, Ellina; Chan, Tung O; Feldman, Arthur M; Tucker, Amy L; Cheung, Joseph Y

    2008-10-01

    Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct

  15. Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis.

    Directory of Open Access Journals (Sweden)

    Nathália Rocco-Machado

    Full Text Available Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2 generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite.

  16. Molecular Cloning and Characterization of Porcine Na+/K+-ATPase Isoforms α1, α2, α3 and the ATP1A3 Promoter

    DEFF Research Database (Denmark)

    Henriksen, Carina; Kjaer-Sorensen, Kasper; Einholm, Anja Pernille

    2013-01-01

    in studies of human diseases caused by Na+/K+-ATPase mutations, we have determined the porcine coding sequences of the α1–α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where...... was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis...... for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations...

  17. Does Increased Expression of the Plasma Membrane Calcium-ATPase Isoform 2 Confer Resistance to Apoptosis on Breast Cancer Cells?

    National Research Council Canada - National Science Library

    VanHouten, Joshua N

    2008-01-01

    The plasma membrane calcium ATPase isoform 2 (PMCA2) is highly expressed on the apical membrane of mammary epithelial cells during lactation, and is the predominant pump responsible for calcium transport into milk...

  18. Increased membrane lipid peroxidation and decreased Na+/K+-ATPase activity in erythrocytes of patients with stable coronary artery disease.

    Science.gov (United States)

    Namazi, Gholamreza; Jamshidi Rad, Sabieh; Attar, Ahmad Movahedian; Sarrafzadegan, Nizal; Sadeghi, Masoumeh; Naderi, Gholamali; Pourfarzam, Morteza

    2015-05-01

    We aimed to determine erythrocyte membrane lipid peroxidation levels and Na/K-ATPase activity in patients with and without coronary artery disease (CAD) documented by coronary angiography. A total of 144 patients who had undergone coronary angiography were divided into a CAD group (n=82) and a non-CAD group (control group, n=62) according to the results of coronary angiography. Lipid peroxide levels in plasma and the erythrocyte membrane were measured using a fluorimetric method. Total antioxidant status and Na/K-ATPase activity in plasma were determined using spectrophotometric methods. Lipid peroxidation levels were significantly higher in the erythrocyte membrane of CAD patients compared with controls, whereas Na/K-ATPase activity was significantly lower in the erythrocyte membrane of CAD patients compared with controls. The coronary artery scores were correlated positively with membrane lipid peroxidation (r=0.324, Plipid peroxidation and Na/K-ATPase activity are correlated with the severity of CAD.

  19. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein

    DEFF Research Database (Denmark)

    Fuglsang, Anja Thoe; Guo, Yan; Cuin, Tracey A.

    2007-01-01

    that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM Hþ-ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM Hþ-ATPase AHA2 at a novel...... site, Ser-931, in the C-terminal regulatory domain. Phosphorylation at this site inhibits interaction between the PM Hþ-ATPase and an activating 14-3-3 protein in a yeast expression system. We show that PKS5 interacts with the calcium binding protein SCaBP1 and that high external pH can trigger...... an increase in the concentration of cytosolic-free calcium. These results suggest that PKS5 is part of a calcium-signaling pathway mediating PM Hþ-ATPase regulation....

  20. RIN4 functions with plasma membrane H+-ATPases to regulate stomatal apertures during pathogen attack

    DEFF Research Database (Denmark)

    Liu, Jun; Elmore, James M.; Fuglsang, Anja Thoe

    2009-01-01

    Abstract Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4- mediated immune signal transduction, we...... exhibit differential PM H+-ATPase activity. PM H+-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H+-ATPase activity and, importantly, its...... stomata could not be re-opened by virulent Pseudomonas syringae. We also demonstrate that RIN4 is expressed in guard cells, highlighting the importance of this cell type in innate immunity. These results indicate that the Arabidopsis protein RIN4 functions with the PM H+-ATPase to regulate stomatal...

  1. Crystal Structure of the Sodium-Potassium Pump (Na⁺, K⁺-ATPase) with Bound Potassium and Ouabain

    National Research Council Canada - National Science Library

    Haruo Ogawa; Takehiro Shinoda; Flemming Cornelius; Chikashi Toyoshima

    2009-01-01

    The sodium-potassium pump (Na⁺,K⁺-ATPase) is responsible for establishing Na⁺ and K⁺ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials...

  2. Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoforms α1, α2, α3 and the ATP1A3 promoter.

    Directory of Open Access Journals (Sweden)

    Carina Henriksen

    Full Text Available Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1-α4 encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by

  3. Membrane Structure of CtrA3, a Copper-transporting P-type-ATPase from Aquifex aeolicus

    NARCIS (Netherlands)

    Chintalapati, Sivaram; Kurdi, Rana Al; Terwisscha van Scheltinga, Anke C.; Kühlbrandt, Werner

    2008-01-01

    We have produced and characterized two new copper-transporting ATPases, CtrA2 and CtrA3 from Aquifex aeolicus, that belong to the family of heavy metal ion-transporting PIB-type ATPases. CtrA2 has a CPC metal-binding sequence in TM6 and a CxxC metal-binding N-terminal domain, while CtrA3 has a CPH

  4. Compensatory Internalization of Pma1 in V-ATPase Mutants in Saccharomyces cerevisiae Requires Calcium- and Glucose-Sensitive Phosphatases.

    Science.gov (United States)

    Velivela, Swetha Devi; Kane, Patricia M

    2017-12-18

    Loss of V-ATPase activity in organelles, whether through V-ATPase inhibition or V-ATPase ( vma) mutations, triggers a compensatory downregulation of the essential plasma membrane proton pump, Pma1 in S. cerevisiae We have previously determined that the a-arrestin Rim8 and ubiquitin ligase Rsp5 are essential for Pma1 ubiquination and endocytosis in response to loss of V-ATPase activity. Here, we show that Pma1 endocytosis in V-ATPase mutants does not require Rim101 pathway components upstream and downstream of Rim8, indicating that Rim8 is acting independently in Pma1 internalization. We find that two phosphatases, the calcium-responsive phosphatase, calcineurin and glucose-sensitive phosphatase, Glc7 (PP1) and one of the Glc7 regulatory subunits, Reg1, exhibit negative synthetic genetic interactions with vma mutants, and demonstrate that both phosphatases are essential for ubiquitination and endocytic downregulation of Pma1 in these mutants.  Although both acute and chronic loss of V-ATPase activity trigger internalization of approximately 50% of surface Pma1, a comparable reduction in Pma1 expression in a pma1-007 mutant neither compensates for loss of V-ATPase activity nor stops further Pma1 endocytosis. The results indicate that cell surface level of Pma1 is not directly sensed and that internalized Pma1 may play a role in compensating for loss of V-ATPase-dependent acidification.  Taken together, these results provide new insights into crosstalk between two major proton pumps central to cellular pH control. Copyright © 2017, Genetics.

  5. Relationship between intracellular Na+ concentration and reduced Na+ affinity in Na+,K+-ATPase mutants causing neurological disease

    DEFF Research Database (Denmark)

    Toustrup-Jensen, Mads Schak; Einholm, Anja P.; Schack, Vivien

    2014-01-01

    The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na+,K+-ATPase α2- and α3-isoforms, expressed in glial and neuronal cells, respectively. Although these disorders...... mutations that increase Na+ affinity were found to reduce [Na+]i. It is concluded that the Na+ affinity of the Na+,K+-ATPase is an important determinant of [Na+]i....

  6. Isoform-Specific Na,K-ATPase Alterations Precede Disuse-Induced Atrophy of Rat Soleus Muscle

    Directory of Open Access Journals (Sweden)

    Violetta V. Kravtsova

    2015-01-01

    Full Text Available This study examines the isoform-specific effects of short-term hindlimb suspension (HS on the Na,K-ATPase in rat soleus muscle. Rats were exposed to 24–72 h of HS and we analyzed the consequences on soleus muscle mass and contractile parameters; excitability and the resting membrane potential (RMP of muscle fibers; the electrogenic activity, protein, and mRNA content of the α1 and α2 Na,K-ATPase; the functional activity and plasma membrane localization of the α2 Na,K-ATPase. Our results indicate that 24–72 h of HS specifically decreases the electrogenic activity of the Na,K-ATPase α2 isozyme and the RMP of soleus muscle fibers. This decrease occurs prior to muscle atrophy or any change in contractile parameters. The α2 mRNA and protein content increased after 24 h of HS and returned to initial levels at 72 h; however, even the increased content was not able to restore α2 enzyme activity in the disused soleus muscle. There was no change in the membrane localization of α2 Na,K-ATPase. The α1 Na,K-ATPase electrogenic activity, protein and mRNA content did not change. Our findings suggest that skeletal muscle use is absolutely required for α2 Na,K-ATPase transport activity and provide the first evidence that Na,K-ATPase alterations precede HS-induced muscle atrophy.

  7. Overproduction in yeast and rapid and efficient purification of the rabbit SERCA1a Ca(2+)-ATPase.

    Science.gov (United States)

    Lenoir, Guillaume; Menguy, Thierry; Corre, Fabienne; Montigny, Cédric; Pedersen, Per A; Thinès, Denyse; le Maire, Marc; Falson, Pierre

    2002-02-18

    Large amounts of heterologous C-terminally his-tagged SERCA1a Ca(2+)-ATPase were expressed in yeast using a galactose-regulated promoter and purified by Ni(2+) affinity chromatography followed by Reactive red chromatography. Optimizing the number of galactose inductions and increasing the amount of Gal4p transcription factor improved expression. Lowering the temperature from 28 degrees C to 18 degrees C during expression enhanced the recovery of solubilized and active Ca(2+)-ATPase. In these conditions, a 4 l yeast culture produced 100 mg of Ca(2+)-ATPase, 60 and 22 mg being pelleted with the heavy and light membrane fractions respectively, representing 7 and 1.7% of total proteins. The Ca(2+)-ATPase expressed in light membranes was 100% solubilized with L-alpha-lysophosphatidylcholine (LPC), 50% with n-dodecyl beta-D-maltoside (DM) and 25% with octaethylene glycol mono-n-dodecyl ether (C(12)E(8)). Compared to LPC, DM preserved specific activity of the solubilized Ca(2+)-ATPase during the chromatographic steps. Starting from 1/6 (3.8 mg) of the total amount of Ca(2+)-ATPase expressed in light membranes, 800 microg could be routinely purified to 50% purity by metal affinity chromatography and then 200 microg to 70% with Reactive red chromatography. The purified Ca(2+)-ATPase displayed the same K(m) for calcium and ATP as the native enzyme but a reduced specific activity ranging from 4.5 to 7.3 micromol ATP hydrolyzed/min/mg Ca(2+)-ATPase. It was stable and active for several days at 4 degrees C or after removal of DM with Bio-beads and storage at -80 degrees C.

  8. The Putative Role of the Non-Gastric H+/K+-ATPase ATP12A (ATP1AL1 as Anti-Apoptotic Ion Transporter: Effect of the H+/K+ ATPase Inhibitor SCH28080 on Butyrate-Stimulated Myelomonocytic HL-60 Cells

    Directory of Open Access Journals (Sweden)

    Martin Jakab

    2014-10-01

    Full Text Available Background/Aims: The ATP12A gene codes for a non-gastric H+/K+ ATPase, which is expressed in a wide variety of tissues. The aim of this study was to test for the molecular and functional expression of the non-gastric H+/K+ ATPase ATP12A/ATP1AL1 in unstimulated and butyrate-stimulated (1 and 10 mM human myelomonocytic HL-60 cells, to unravel its potential role as putative apoptosis-counteracting ion transporter as well as to test for the effect of the H+/K+ ATPase inhibitor SCH28080 in apoptosis. Methods: Real-time reverse-transcription PCR (qRT-PCR was used for amplification and cloning of ATP12A transcripts and to assess transcriptional regulation. BCECF microfluorimetry was used to assess changes of intracellular pH (pHi after acute intracellular acid load (NH4Cl prepulsing. Mean cell volumes (MCV and MCV-recovery after osmotic cell shrinkage (Regulatory Volume Increase, RVI were assessed by Coulter counting. Flow-cytometry was used to measure MCV (Coulter principle, to assess apoptosis (phosphatidylserine exposure to the outer leaflet of the cell membrane, caspase activity, 7AAD staining and differentiation (CD86 expression. Results: We found by RT-PCR, intracellular pH measurements, MCV measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL-60 cells. Treatment of HL-60 cells with 1 mM butyrate leads to monocyte-directed differentiation whereas higher concentrations (10 mM induce apoptosis as assessed by flow-cytometric determination of CD86 expression, caspase activity, phosphatidylserine exposure on the outer leaflet of the cell membrane and MCV measurements. Transcriptional up-regulation of ATP12A and CD86 is evident in 1 mM butyrate-treated HL-60 cells. The H+/K+ ATPase inhibitor SCH28080 (100 µM diminishes K+-dependent pHi recovery after intracellular acid load and blocks RVI after osmotic cell shrinkage. After seeding, HL-60 cells increase their MCV within the first 24 h in culture, and subsequently

  9. Endogenous plasma Na,K-ATPase inhibitory activity and digoxin like immunoreactivity after acute myocardial infarction.

    Science.gov (United States)

    Bagrov AYa; Fedorova, O V; Maslova, M N; Roukoyatkina, N I; Ukhanova, M V; Zhabko, E P

    1991-05-01

    The aim was to look for the presence of circulating factor(s) with Na,K-ATPase inhibitory properties and digoxin like immunoreactivity in patients after acute myocardial infarction. Venous blood samples were obtained when the patients were admitted and different methods were used to monitor the plasma concentrations of factor(s) with properties of digitalis. SUBJECTS - These were 26 patients of both sexes (mean age 57.7 years, range 40-72) during the first 24 h of a first transmural acute myocardial infarct, 11 male patients with unstable angina pectoris (52.5 years, 45-67), and 18 healthy male controls (25 to 50 years). There was significant inhibition of ouabain sensitive Na,K-ATPase in intact erythrocytes in patients with myocardial infarction [1.4(SEM 0.15)mumol Pi.mg-1.h-1] compared with patients with unstable angina pectoris [3.1(0.4), p less than 0.01] and healthy controls [3.4(0.25), p less than 0.01]. In myocardial infarction complicated by ventricular fibrillation (n = 5) Na,K-ATPase activity was significantly lower than in the other 21 patients [0.95(0.2) and 1.55(0.11) mumol Pi.mg-1.h-1 respectively, p less than 0.05]. There was no change in erythrocyte Na,K-ATPase activity in myocardial infarction complicated by acute pulmonary oedema, nor was there any difference in activity in erythrocyte ghosts obtained from the patients with myocardial infarction v healthy controls, at 0.47(0.13) v 0.50(0.02) mumol Pi.mg-1.h-1. Boiled plasma supernatants obtained from the patients with myocardial infarction inhibited Na,K-ATPase in erythrocytes from healthy subjects. This inhibitory effect was antagonised by antidigoxin antibody. Plasma inhibitory potency was correlated with erythrocyte Na,K-ATPase activity in the patients with myocardial infarction (r = -0.65, p less than 0.001, n = 23). There was a 2.5-fold increase in plasma digoxin like immunoreactivity in the patients with myocardial infarction [1.65(0.5) ng.ml-1] using DELFIA fluoroimmunoassay as compared

  10. Structure and mechanism of Zn2+-transporting P-type ATPases

    DEFF Research Database (Denmark)

    Wang, Kaituo; Sitsel, Oleg; Meloni, Gabriele

    2014-01-01

    Zinc is an essential micronutrient for all living organisms. It is required for signalling and proper functioning of a range of proteins involved in, for example, DNA binding and enzymatic catalysis. In prokaryotes and photosynthetic eukaryotes, Zn(2+)-transporting P-type ATPases of class IB (Znt......A) are crucial for cellular redistribution and detoxification of Zn(2+) and related elements. Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2·Pi) of ZntA from Shigella sonnei, determined at 3.2 Å and 2.7 Å resolution, respectively....... The structures reveal a similar fold to Cu(+)-ATPases, with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn(2+) ions by the transporter. The E2P structure...

  11. Ammonium transport and the role of the Na,K-ATPase.

    Science.gov (United States)

    Wall, S M

    1996-01-01

    Ammonium (NH4+) excretion varies appropriately with changes in acid-base balance and represents the major regulatable component of net acid excretion. The transport of ammonium can occur by 'diffusion trapping', or active H+ secretion in parallel with passive NH3 diffusion. In addition, direct NH4+ transport is important in many nephron segments. Since NH4+ and K+ have a similar hydrated radius, these ions share common transport pathways in many renal and nonrenal cell types. For example, these ions compete for a common binding site on the Na,K-ATPase. In addition to Na+ pump-mediated NH4+ transport, the Na,K-ATPase generates an electrochemical gradient across the cell membrane which affects other H+ and NH4+ transport pathways. In this review, the role of the Na+ pump on each of these renal ammonium transport mechanisms will be reviewed.

  12. Na,K-ATPase is a target of cigarette smoke and reduced expression predicts poor patient outcome of smokers with lung cancer

    Science.gov (United States)

    Huynh, Thu P.; Mah, Vei; Sampson, Valerie B.; Chia, David; Fishbein, Michael C.; Horvath, Steve; Alavi, Mohammad; Wu, Debbie C.; Harper, Jeffrey; Sarafian, Ted; Dubinett, Steven M.; Langhans, Sigrid A.; Goodglick, Lee

    2012-01-01

    Diminished Na,K-ATPase expression has been reported in several carcinomas and has been linked to tumor progression. However, few studies have determined whether Na,K-ATPase function and expression are altered in lung malignancies. Because cigarette smoke (CS) is a major factor underlying lung carcinogenesis and progression, we investigated whether CS affects Na,K-ATPase activity and expression in lung cell lines. Cells exposed to CS in vitro showed a reduction of Na,K-ATPase activity. We detected the presence of reactive oxygen species (ROS) in cells exposed to CS before Na,K-ATPase inhibition, and neutralization of ROS restored Na,K-ATPase activity. We further determined whether Na,K-ATPase expression correlated with increasing grades of lung adenocarcinoma and survival of patients with smoking history. Immunohistochemical analysis of lung adenocarcinoma tissues revealed reduced Na,K-ATPase expression with increasing tumor grade. Using tissue microarray containing lung adenocarcinomas of patients with known smoking status, we found that high expression of Na,K-ATPase correlated with better survival. For the first time, these data demonstrate that CS is associated with loss of Na,K-ATPase function and expression in lung carcinogenesis, which might contribute to disease progression. PMID:22345575

  13. Ca-ATPase and ALPase activities at the initial calcification sites of dentin and enamel in the rat incisor.

    Science.gov (United States)

    Takano, Y; Ozawa, H; Crenshaw, M A

    1986-01-01

    Enzymatic activities of calcium-magnesium dependent adenosine triphosphatase (Ca-ATPase) and nonspecific alkaline phosphatase (ALPase) were localized at the initial calcification sites of dentin and enamel of rat incisor teeth using electron-microscopic cytochemistry. Ca-ATPase was localized in the Golgi cisternae, cytoplasmic vesicles and along the outer surface of the presecretory and secretory ameloblasts, whereas it was totally absent from the odontoblasts in the pulp. Inversely, ALPase reaction was localized along the outer surface of the odontoblasts, but almost completely absent from the ameloblasts. Diffuse extracellular reactions of both enzymes were distributed throughout the unmineralized fibrous matrix of mantle dentin in which a large number of matrix vesicles were scattered. Both Ca-ATPase and ALPase reactions, which appeared in the matrix vesicles in the process of formation of mantle dentin, became most conspicuous at the site of initial dentin calcification. At this stage, an intense Ca-ATPase reaction also appeared along some of the collagen fibrils adjacent to the reactive matrix vesicles. No ALPase reaction was localized along these Ca-ATPase reactive collagen fibrils. Our observations suggest strongly that Ca-ATPase in the matrix vesicles originates from the inner enamel epithelium and/or preameloblasts whereas ALPase originates from the odontoblasts in the pulp. The importance of the coexistence of both enzymes for the control of initial calcification of dental hard tissues is suggested.

  14. Dependence of myosin-ATPase on structure bound creatine kinase in cardiac myfibrils from rainbow trout and freshwater turtle

    DEFF Research Database (Denmark)

    Haagensen, L.; Jensen, D.H.; Gesser, Hans

    2008-01-01

    The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP by the pyr......The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP...... by the pyruvate kinase reaction alone or together with the amount of creatine formed, when myofibrillar bound creatine kinase was activated with phosphocreatine. The steady-state concentration of ADP in the solution was varied through the activity of pyruvate kinase added to the solution. For rainbow trout...... myofibrils at a high pyruvate kinase activity, creatine kinase competed for ADP but did not influence the total ATPase activity. When the ADP concentration was elevated within the physiological range by lowering the pyruvate kinase activity, creatine kinase competed efficiently and increased the ATPase...

  15. Ecto-F1-ATPase/P2Y pathways in metabolic and vascular functions of high density lipoproteins.

    Science.gov (United States)

    Martinez, Laurent O; Najib, Souad; Perret, Bertrand; Cabou, Cendrine; Lichtenstein, Laeticia

    2015-01-01

    The atheroprotective property of High Density Lipoprotein (HDL) is supported by many epidemiological studies and cellular and in vivo approaches on animal models. While the anti-atherogenic effects of HDL are thought to derive primarily from its role in reverse cholesterol transport, together with anti-inflammatory, anti-oxidant, anti-thrombotic and cytoprotective properties, the mechanisms that support these effects are still not completely understood. However, many advances in identifying the cellular partners involved in HDL functions have been made over the last two decades. This review highlights the diverse roles of the HDL receptor ecto-F1-ATPase coupled to purinergic P2Y receptors in the modulation of important metabolic and vascular functions of HDL. On hepatocytes, the ecto-F1-ATPase is coupled to P2Y13 receptor and contributes to HDL holoparticle endocytosis. On endothelial cells, ecto-F1-ATPase/P2Ys pathway is involved in HDL-mediated endothelial protection and HDL transcytosis. The clinical relevance of this F1-ATPase/P2Ys axis in humans has recently been supported by the identification of serum F1-ATPase inhibitor (IF1) as an independent determinant of HDL-Cholesterol (HDL-C) and coronary heart disease risk. Therapeutic strategies targeting F1-ATPase/P2Y pathways for the treatment of atherosclerosis are currently being explored. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. HNF-1B specifically regulates the transcription of the {gamma}a-subunit of the Na{sup +}/K{sup +}-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Ferre, Silvia [Department of Physiology, Radboud University Nijmegen Medical Centre (Netherlands); Veenstra, Gert Jan C. [Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen (Netherlands); Bouwmeester, Rianne; Hoenderop, Joost G.J. [Department of Physiology, Radboud University Nijmegen Medical Centre (Netherlands); Bindels, Rene J.M., E-mail: r.bindels@fysiol.umcn.nl [Department of Physiology, Radboud University Nijmegen Medical Centre (Netherlands)

    2011-01-07

    Research highlights: {yields} Defects in HNF-1B transcription factor affect Mg{sup 2+} handling in the distal kidney. {yields} {gamma}a- and {gamma}b- subunits of the Na{sup +}/K{sup +}-ATPase colocalize in the distal convoluted tubule of the nephron. {yields} HNF-1B specifically activates {gamma}a expression. {yields} HNF-1B mutants have a dominant negative effect on wild type HNF-1B activity. {yields} Defective transcription of {gamma}a may promote renal Mg{sup 2+} wasting. -- Abstract: Hepatocyte nuclear factor-1B (HNF-1B) is a transcription factor involved in embryonic development and tissue-specific gene expression in several organs, including the kidney. Recently heterozygous mutations in the HNF1B gene have been identified in patients with hypomagnesemia due to renal Mg{sup 2+} wasting. Interestingly, ChIP-chip data revealed HNF-1B binding sites in the FXYD2 gene, encoding the {gamma}-subunit of the Na{sup +}/K{sup +}-ATPase. The {gamma}-subunit has been described as one of the molecular players in the renal Mg{sup 2+} reabsorption in the distal convoluted tubule (DCT). Of note, the FXYD2 gene can be alternatively transcribed into two main variants, namely {gamma}a and {gamma}b. In the present study, we demonstrated via two different reporter gene assays that HNF-1B specifically acts as an activator of the {gamma}a-subunit, whereas the {gamma}b-subunit expression was not affected. Moreover, the HNF-1B mutations H69fsdelAC, H324S325fsdelCA, Y352finsA and K156E, previously identified in patients with hypomagnesemia, prevented transcription activation of {gamma}a-subunit via a dominant negative effect on wild type HNF1-B. By immunohistochemistry, it was shown that the {gamma}a- and {gamma}b-subunits colocalize at the basolateral membrane of the DCT segment of mouse kidney. On the basis of these data, we suggest that abnormalities involving the HNF-1B gene may impair the relative abundance of {gamma}a and {gamma}b, thus affecting the transcellular Mg{sup 2

  17. Effects of γ-irradiation on Na,K-ATPase in cardiac sarcolemma.

    Science.gov (United States)

    Mézešová, L; Vlkovičová, J; Kaločayová, B; Jendruchová, V; Barančík, M; Fülöp, M; Slezák, J; Babál, P; Janega, P; Vrbjar, N

    2014-03-01

    Previous studies showed that adverse effect of ionizing radiation on the cardiovascular system is beside other factors mostly mediated by reactive oxygen and nitrogen species, which deplete antioxidant stores. One of the structures highly sensitive to radicals is the Na,K-ATPase the main system responsible for extrusion of superfluous Na(+) out of the cell which utilizes the energy derived from ATP. The aim of present study was the investigation of functional properties of cardiac Na,K-ATPase in 20-week-old male rats 6 weeks after γ-irradiation by a dose 25 Gy (IR). Irradiation induced decrease of systolic blood pressure from 133 in controls to 85 mmHg in IR group together with hypertrophy of right ventricle (RV) and hypotrophy of left ventricle (LV). When activating the cardiac Na,K-ATPase with substrate, its activity was lower in IR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed a decrease of the maximum velocity (V max) by 40 % with no changes in the value of Michaelis-Menten constant (K m). During activation with Na(+), we observed a decrease of the enzyme activity in hearts from IR at all tested Na(+) concentrations. The value of V max decreased by 38 %, and the concentration of Na(+) that gives half maximal reaction velocity (K Na) increased by 62 %. This impairment in the affinity of the Na(+)-binding site together with decreased number of active Na,K-ATPase molecules, as indicated by lowered V max values, are probably responsible for the deteriorated efflux of the excessive Na(+) from the intracellular space in hearts of irradiated rats.

  18. Nanoparticulate vacuolar ATPase blocker exhibits potent host-targeted antiviral activity against feline coronavirus

    OpenAIRE

    Hu, Che-Ming Jack; Chang, Wei-Shan; Fang, Zih-Syun; Chen, You-Ting; Wang, Wen-Lin; Tsai, Hsiao-Han; Chueh, Ling-Ling; Takano, Tomomi; Hohdatsu, Tsutomu; Chen, Hui-Wen

    2017-01-01

    Feline infectious peritonitis (FIP), caused by a mutated feline coronavirus, is one of the most serious and fatal viral diseases in cats. The disease remains incurable, and there is no effective vaccine available. In light of the pathogenic mechanism of feline coronavirus that relies on endosomal acidification for cytoplasmic entry, a novel vacuolar ATPase blocker, diphyllin, and its nanoformulation are herein investigated for their antiviral activity against the type II feline infectious per...

  19. The Na, K-ATPase β-Subunit Isoforms Expression in Glioblastoma Multiforme: Moonlighting Roles

    Science.gov (United States)

    Rotoli, Deborah; Cejas, Mariana-Mayela; Maeso, María-del-Carmen; Pérez-Rodríguez, Natalia-Dolores; Morales, Manuel; Ávila, Julio

    2017-01-01

    Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Recent studies point out that gliomas exploit ion channels and transporters, including Na, K-ATPase, to sustain their singular growth and invasion as they invade the brain parenchyma. Moreover, the different isoforms of the β-subunit of Na, K-ATPase have been implicated in regulating cellular dynamics, particularly during cancer progression. The aim of this study was to determine the Na, K-ATPase β subunit isoform subcellular expression patterns in all cell types responsible for microenvironment heterogeneity of GBM using immunohistochemical analysis. All three isoforms, β1, β2/AMOG (Adhesion Molecule On Glia) and β3, were found to be expressed in GBM samples. Generally, β1 isoform was not expressed by astrocytes, in both primary and secondary GBM, although other cell types (endothelial cells, pericytes, telocytes, macrophages) did express this isoform. β2/AMOG and β3 positive expression was observed in the cytoplasm, membrane and nuclear envelope of astrocytes and GFAP (Glial Fibrillary Acidic Protein) negative cells. Interestingly, differences in isoforms expression have been observed between primary and secondary GBM: in secondary GBM, β2 isoform expression in astrocytes was lower than that observed in primary GBM, while the expression of the β3 subunit was more intense. These changes in β subunit isoforms expression in GBM could be related to a different ionic handling, to a different relationship between astrocyte and neuron (β2/AMOG) and to changes in the moonlighting roles of Na, K-ATPase β subunits as adaptor proteins and transcription factors. PMID:29117147

  20. Mitochondrial ultrastructural and atpase changes during the life cycle of Ascaris Suum

    Directory of Open Access Journals (Sweden)

    G. E. Rodrick

    1982-06-01

    Full Text Available Ultrastructural morphology and ATPase specific activities of mitochondria isolated from 1-celled fertilized egg, 10-day embryo, 21-day infective larvae and adult body wall muscle of Ascaris suum and rat liver were determined and compared. Although cristae of both muscle and egg mitochondria contained numerous elementary particles with head pieces of conventional diameter (85 A, each muscle mitochondrion contained relatively few, short cristae with a diminished frequency of elementary particles and associated ATPase activity. These morphological relationships are related to the previous conclusion that the transition from an aerobic to an essentially anaerobic metabolism is intimately associated with the mitochondrion and is a normal and mandatory feature of development.Foram determinadas e comparadas a morfologia estrutural e as atividades específicas da ATPase de mitocôndrias do Ascaris suum (isoladas do ovo unicelular fertilizado, do embrião de 10 dias, da larva infectante de 21 dias e do músculo da parede do corpo do adulto e do fígado do rato. Embora as cristas das mitocôndrias do músculo e do ovo contivessem numerosas partículas elementares com cabeças de diâmetro convencional (85 A, cada mitocôndria do músculo continha cristas curtas e em número relativamente pequeno, com diminuição da frequência das partículas elementares e da respectiva atividade de ATPase. Estas relações morfol[ogicas são vinculadas à conclusão prévia de que a transição do metabolismo aeróbio para o metabolismo essencialmente anaeróbio está intimamente associada à mitocôndria e constitui um característico normal e obrigatório do desenvolvimento.

  1. Influence of silver nanoparticles on the activity of rat liver mitochondrial ATPase

    Science.gov (United States)

    Chichova, Mariela; Shkodrova, Milena; Vasileva, Penka; Kirilova, Katerina; Doncheva-Stoimenova, Diliana

    2014-02-01

    Mitochondria are one of the most sensitive targets for the toxicity of silver nanoparticles (AgNPs). Limited studies have demonstrated nanoparticle-induced impairment of mitochondrial oxidative phosphorylation. Reduced adenosine triphosphate (ATP) production can be due to inhibition of the respiratory chain and/or to direct effects of AgNPs on the activity of mitochondrial ATP synthase/ATPase. In this regard, we synthesized and evaluated the in vitro effects of two types of AgNPs with various environmental friendly coatings—polysaccharide starch (AgNPs/Starch, D av = 15.4 ± 3.9 nm) and trisaccharide raffinose (AgNPs/Raff, D av = 24.8 ± 6.8 nm), with an emphasis on their potential action on rat liver mitochondrial ATPase. Both types of AgNPs showed decoupling effect on intact mitochondria. Unlike AgNPs/Raff, AgNPs/Starch reduced 2,4-dinitrophenol-stimulated ATPase activity of intact mitochondria, which suggests that they are able to penetrate the inner mitochondrial membrane. Both types of AgNPs inhibited ATPase activity of freeze/thawed mitochondria and submitochondrial particles as the effects of AgNPs/Starch were more pronounced. UV-Visible absorption measurements showed changes in the absorption spectrum of AgNPs/Raff added to the reaction medium. This suggests nanoparticle aggregation and thus a possible reduction in their reactivity. The distinction in the effects of the two types AgNPs studied may be due to their different sizes and/or to the stabilizing agents used for their synthesis, which determine AgNPs colloidal stability in the assay media. This study suggests the need for further research into the importance of surface modifications of AgNPs for their interaction with cellular components. Our findings could contribute to the elucidation of the mechanisms underlying AgNPs toxicity.

  2. Salt stress reduces kernel number of corn by inhibiting plasma membrane H(+)-ATPase activity.

    Science.gov (United States)

    Jung, Stephan; Hütsch, Birgit W; Schubert, Sven

    2017-04-01

    Salt stress affects yield formation of corn (Zea mays L.) at various physiological levels resulting in an overall grain yield decrease. In this study we investigated how salt stress affects kernel development of two corn cultivars (cvs. Pioneer 3906 and Fabregas) at and shortly after pollination. In an earlier study, we found an accumulation of hexoses in the kernel tissue. Therefore, it was hypothesized that hexose uptake into developing endosperm and embryo might be inhibited. Hexoses are transported into the developing endosperm by carriers localized in the plasma membrane (PM). The transport is driven by the pH gradient which is built up by the PM H(+)-ATPase. It was investigated whether the PM H(+)-ATPase activity in developing corn kernels was inhibited by salt stress, which would cause a lower pH gradient resulting in impaired hexose import and finally in kernel abortion. Corn grown under control and salt stress conditions was harvested 0 and 2 days after pollination (DAP). Under salt stress sucrose and hexose concentrations in kernel tissue were higher 0 and 2 DAP. Kernel PM H(+)-ATPase activity was not affected at 0 DAP, but it was reduced at 2 DAP. This is in agreement with the finding, that kernel growth and thus kernel setting was not affected in the salt stress treatment at pollination, but it was reduced 2 days later. It is concluded that inhibition of PM H(+)-ATPase under salt stress impaired the energization of hexose transporters into the cells, resulting in lower kernel growth and finally in kernel abortion. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  3. Breast cancer associated a2 isoform vacuolar ATPase immunomodulates neutrophils: potential role in tumor progression

    Science.gov (United States)

    Ibrahim, Safaa A.; Katara, Gajendra K.; Kulshrestha, Arpita; Jaiswal, Mukesh K.; Amin, Magdy A.; Beaman, Kenneth D.

    2015-01-01

    In invasive breast cancer, tumor associated neutrophils (TAN) represent a significant portion of the tumor mass and are associated with increased angiogenesis and metastasis. Identifying the regulatory factors that control TAN behavior will help in developing ideal immunotherapies. Vacuolar ATPases (V-ATPases), multi-subunit proton pumps, are highly expressed in metastatic breast cancer cells. A cleaved peptide from a2 isoform V-ATPase (a2NTD) has immunomodulatory role in tumor microenvironment. Here, we report for the first time the role of V-ATPase in neutrophils modulation. In invasive breast cancer cells, a2NTD was detected and a2V was highly expressed on the surface. Immunohistochemical analysis of invasive breast cancer tissues revealed that increased neutrophil recruitment and blood vessel density correlated with increased a2NTD levels. In order to determine the direct regulatory role of a2NTD on neutrophils, recombinant a2NTD was used for the treatment of neutrophils isolated from the peripheral blood of healthy volunteers. Neutrophils treated with a2NTD (a2Neuɸ) showed increased secretion of IL-1RA, IL-10, CCL-2 and IL-6 that are important mediators in cancer related inflammation. Moreover, a2Neuɸ exhibited an increased production of protumorigenic factors including IL-8, matrix metaloprotinase-9 and vascular endothelial growth factor. Further, functional characterization of a2Neuɸ revealed that a2Neuɸ derived products induce in vitro angiogenesis as well as increase the invasiveness of breast cancer cells. This study establishes the modulatory effect of breast cancer associated a2V on neutrophils, by the action of a2NTD, which has a positive impact on tumor progression, supporting that a2V can be a potential selective target for breast cancer therapy. PMID:26460736

  4. Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

    Directory of Open Access Journals (Sweden)

    D.V. Vassallo

    2008-09-01

    Full Text Available Lead (Pb2+ poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 µM to the bath. Changes in rate of stimulation (0.1-1.5 Hz, relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM, and the effect of isoproterenol (20 ng/mL were determined before and after the addition of 100 µM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 µM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 µM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 µM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 µM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

  5. Demethoxycurcumin is a potent inhibitor of P-type ATPases from diverse kingdoms of life

    DEFF Research Database (Denmark)

    Dao, Trong Tuan; Sehgal, Pankaj; Thanh Tung, Truong

    2016-01-01

    as drugs. In this work a library of natural compounds was screened and we first identified curcuminoids as plasma membrane H+-ATPases inhibitors in plant and fungal cells. We also found that some of the commercial curcumins contain several curcuminoids. Three of these were purified and, among...... of these pumps. Future research on biological effects of commercial preparations of curcumin should consider the heterogeneity of the material....

  6. Endothelin, a peptide inhibitor of Na(+)-K(+)-ATPase in intact renaltubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Zeidel, M.L.; Brady, H.R.; Kone, B.C.; Gullans, S.R. (Brigham and Women' s Hospital, Boston, MA (USA))

    1989-12-01

    Endothelin, a potent vasoconstrictor released by vascular endothelial cells, can induce natriuresis in vivo. These studies examined the regulation of Na+ transport by endothelin in suspensions of rabbit proximal tubule (PT) and inner medullary collecting duct (IMCD) cells. Endothelin reduced oxygen consumption (QO2) by 18 +/- 1% in IMCD cells but did not alter QO2 in PT cells. In IMCD cells, endothelin inhibited QO2 half maximally at approximately 5 x 10(-12) M. Several lines of evidence indicate that endothelin reduces QO2 by inhibiting the Na(+)-K(+)-ATPase. (1) Endothelin gave no further inhibition of QO2 after ouabain and blunted the stimulatory effect of amphotericin B on QO2 (+29 +/- 4% in absence of endothelin, 0 +/- 5% in presence of endothelin; n = 6 preparations, P less than 0.001). (2) Endothelin inhibited ouabain-sensitive 86Rb+ uptake by 46.6 +/- 8.6% at 10 s and by 35.4 +/- 5.3% at 30 s without altering uptake at (60 min. 3) Addition of endothelin to IMCD cells induced a net K+ efflux with an initial rate of 32.2 +/- 4.8 nmol.min-1.mg protein-1, consistent with inhibition of the Na(+)-K(+)-ATPase. In contrast to the response observed in intact cells, in permeabilized IMCD cells endothelin did not inhibit ouabain-sensitive ATPase. Several observations indicated that prostaglandin E2 (PGE2) mediates endothelin inhibition of Na(+)-K(+)-ATPase activity. (1) The response to endothelin was blocked by ibuprofen in assays of QO2, net K+ flux, and 86Rb+ uptake. (2) Endothelin and PGE2 gave equivalent, nonadditive inhibition of ouabain-sensitive 86Rb+ uptake.

  7. The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells

    Directory of Open Access Journals (Sweden)

    Martin Jakab

    2017-10-01

    Full Text Available Background/Aims: Glucose-stimulated insulin secretion (GSIS of pancreatic β-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem, activation of voltage-activated Ca2+ currents (ICav and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi or cell volume, which also affect β-cell viability, can elicit or modify insulin release. In β-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs. To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. Methods: In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability and cytotoxicity assays. The mean cell volume (MCV, cell granularity (side-scatter; SSC, phosphatidylserine (PS exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨm were measured by flow cytometry. Results: We found that the α-subunit of the non-gastric H+/K+ ATPase (HKα2 is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release

  8. The H+/K+ ATPase Inhibitor SCH-28080 Inhibits Insulin Secretion and Induces Cell Death in INS-1E Rat Insulinoma Cells.

    Science.gov (United States)

    Jakab, Martin; Ketterl, Nina; Fürst, Johannes; Beyreis, Marlena; Kittl, Michael; Kiesslich, Tobias; Hauser-Kronberger, Cornelia; Gaisberger, Martin; Ritter, Markus

    2017-10-02

    Glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells involves glucose uptake and metabolism, closure of KATP channels and depolarization of the cell membrane potential (Vmem), activation of voltage-activated Ca2+ currents (ICav) and influx of Ca2+, which eventually triggers hormone exocytosis. Beside this classical pathway, KATP-independent mechanisms such as changes in intracellular pH (pHi) or cell volume, which also affect β-cell viability, can elicit or modify insulin release. In β-cells the regulation of pHi is mainly accomplished by Na+/H+ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H+/K+ ATPase in rat insulinoma cells and assessed effects of the H+/K+ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. In INS-1E cell cultures, H+/K+ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pHi) recovery after acute acidic load was measured by NH4Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. Vmem, K+ and Ca2+ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume (MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. We found that the α-subunit of the non-gastric H+/K+ ATPase (HKα2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH4Cl prepulsing experiments no K+-dependent pHi recovery was observed under Na+-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release was unaffected. The L-type ICav blocker

  9. Evidence for a role in growth and salt resistance of a plasma membrane H+-ATPase in the root endodermis

    Science.gov (United States)

    Vitart, V.; Baxter, I.; Doerner, P.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2001-01-01

    The plasma membrane of plant cells is energized by an electrochemical gradient produced by P-type H+-ATPases (proton pumps). These pumps are encoded by at least 12 genes in Arabidopsis. Here we provide evidence that isoform AHA4 contributes to solute transport through the root endodermis. AHA4 is expressed most strongly in the root endodermis and flowers, as suggested by promoter-GUS reporter assays. A disruption of this pump (aha4-1) was identified as a T-DNA insertion in the middle of the gene (after VFP(574)). Truncated aha4-1 transcripts accumulate to approximately 50% of the level observed for AHA4 mRNA in wild-type plants. Plants homozygous for aha4-1 (-/-) show a subtle reduction in root and shoot growth compared with wild-type plants when grown under normal conditions. However, a mutant phenotype is very clear in plants grown under salt stress (e.g., 75 or 110 mM NaCl). In leaves of mutant plants subjected to Na stress, the ratio of Na to K increased 4-5-fold. Interestingly, the aha4-1 mutation appears to be semidominant and was only partially complemented by the introduction of additional wild-type copies of AHA4. These results are consistent with the hypothesis that aha4-1 may produce a dominant negative protein or RNA that partially disrupts the activity of other pumps or functions in the root endodermal tissue, thereby compromising the function of this cell layer in controlling ion homeostasis and nutrient transport.

  10. The transjugation machinery of Thermus thermophilus: Identification of TdtA, an ATPase involved in DNA donation.

    Science.gov (United States)

    Blesa, Alba; Baquedano, Ignacio; Quintáns, Nieves G; Mata, Carlos P; Castón, José R; Berenguer, José

    2017-03-01

    In addition to natural competence, some Thermus thermophilus strains show a high rate of DNA transfer via direct cell-to-cell contact. The process is bidirectional and follows a two-step model where the donor cell actively pushes out DNA and the recipient cell employs the natural competence system to take up the DNA, in a hybrid transformation-dependent conjugation process (transjugation). While the DNA uptake machinery is well known as in other bacterial species that undergo transformation, the pushing step of transjugation remains to be characterized. Here we have searched for hypothetical DNA translocases putatively involved in the pushing step of transjugation. Among candidates encoded by T. thermophilus HB27, the TdtA protein was found to be required for DNA pushing but not for DNA pulling during transjugation, without affecting other cellular processes. Purified TdtA shows ATPase activity and oligomerizes as hexamers with a central opening that can accommodate double-stranded DNA. The tdtA gene was found to belong to a mobile 14 kbp-long DNA element inserted within the 3' end of a tRNA gene, flanked by 47 bp direct repeats. The insertion also encoded a homolog of bacteriophage site-specific recombinases and actively self-excised from the chromosome at high frequency to form an apparently non-replicative circular form. The insertion also encoded a type II restriction endonuclease and a NurA-like nuclease, whose activities were required for efficient transjugation. All these data support that TdtA belongs to a new type of Integrative and Conjugative Element which promotes the generalized and efficient transfer of genetic traits that could facilitate its co-selection among bacterial populations.

  11. The transjugation machinery of Thermus thermophilus: Identification of TdtA, an ATPase involved in DNA donation.

    Directory of Open Access Journals (Sweden)

    Alba Blesa

    2017-03-01

    Full Text Available In addition to natural competence, some Thermus thermophilus strains show a high rate of DNA transfer via direct cell-to-cell contact. The process is bidirectional and follows a two-step model where the donor cell actively pushes out DNA and the recipient cell employs the natural competence system to take up the DNA, in a hybrid transformation-dependent conjugation process (transjugation. While the DNA uptake machinery is well known as in other bacterial species that undergo transformation, the pushing step of transjugation remains to be characterized. Here we have searched for hypothetical DNA translocases putatively involved in the pushing step of transjugation. Among candidates encoded by T. thermophilus HB27, the TdtA protein was found to be required for DNA pushing but not for DNA pulling during transjugation, without affecting other cellular processes. Purified TdtA shows ATPase activity and oligomerizes as hexamers with a central opening that can accommodate double-stranded DNA. The tdtA gene was found to belong to a mobile 14 kbp-long DNA element inserted within the 3' end of a tRNA gene, flanked by 47 bp direct repeats. The insertion also encoded a homolog of bacteriophage site-specific recombinases and actively self-excised from the chromosome at high frequency to form an apparently non-replicative circular form. The insertion also encoded a type II restriction endonuclease and a NurA-like nuclease, whose activities were required for efficient transjugation. All these data support that TdtA belongs to a new type of Integrative and Conjugative Element which promotes the generalized and efficient transfer of genetic traits that could facilitate its co-selection among bacterial populations.

  12. Role of Ca++, CaATPase and alkaline protease in cardiac contraction band formation.

    Science.gov (United States)

    Smith, A N; Marquet, E; Sobel, H J

    1982-07-01

    Cardiac cells' total homogenates and subcellular components Ca++, CaATPase and alkaline protease levels were matched with their respective microscopic samples in order to ascertain their role in the formation of contraction bands. It was seen that those samples having contraction bands also had a high incidence of intracellular Ca++, low CaATPase and high alkaline protease levels. This study confirms reports that contraction bands are not limited to pathological hearts (Adomian et al., 1977), but neither are they limited to artifacts related to autopsy procedures. They were seen in varying degrees of severity in hearts of healthy rats sacrificed by decapitation and less frequently with only sarcomeric involvement to absence of in animals sacrificed by diethyl ether overdose. It is postulated that the elevated Ca++ activated alkaline protease (pH 8.5) by inhibiting its inhibitor (Waxman and Krebs, 1978). It may have inhibited total CaATPase activity (Hasselbach, 1974) as well. We believe that the activated alkaline protease produced irreversible contraction bands by lysing the microhinge between light and heavy meromyosin.

  13. [-Na(+)-K+ ATPase activity in vesicles of plasmatic membrane of breast cancer].

    Science.gov (United States)

    Salazar Esquivel, Edith Lucía; Calzada Sánchez, Leobardo

    2005-03-01

    -Na(+)-K+ ATPase is a useful marker which determines the origin of breast cancer cells. -Na(+)-K+ ATPase activity, as well as viability of plasma membrane vesicles isolated from breast carcinoma tissues were demonstrated by histochemical detection. Breast carcinoma tissue samples of patients who attended consultation in the oncology service at Hospital de Ginecoobstetricia Núm. 4 Dr. Luis Castelazo Ayala, IMSS were examined. Tissue samples from adenocarcinoma were homogenized in 4 volumes of TED solution at 4 degrees C (Tris-HCI 0.01 M, EDTA 0.0015 M, dithiothreitol 0.001 M, pH 7.4) and subsequently centrifuged. The collected sample was homogenized and stratified in a discontinuous sucrose gradient (20 to 50%) and then centrifuged for 60 min at 30,000 xg. In order to determine -Na(+)-K+ ATPase activity in plasma membrane vesicles, suspension was incubated at Tris-maleate 0.04 M, adenosine triphosphate (ATP) 0.004 M, Mgcl 20.004 M, NaCI 0.1 M y Pb (NO3) 20.005 M, pH 7.0. Reactions were carried out for 15 min at 37 degrees C in prefixed vesicles in 3% glutaraldehyde in 0.1 M, cacodylate buffer stock, pH7.4 for 60 min. Histochemical detection demonstrated membrane vesicles from breast carcinoma tissues and proved their viability after tumoral progression.

  14. The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues

    DEFF Research Database (Denmark)

    Poulsen, Lisbeth Rosager

    physiological function.   1 Poulsen, L.R; López-Marqués, R.L et al. (2008) The Arabidopsis P4-ATPase ALA3 localizes to the Golgi and requires a ß-subunit to function in lipid translocation and secretory vesicle formation. The Plant Cell, vol. 20, 658-676. 2 Gomès, E. et al. (2000) Chilling tolerance......  The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues Rosa Laura López-Marqués1, Lisbeth Rosager Poulsen1, Katharina Meffert2, Thomas Pomorski2, Michael Gjedde Palmgren1 1Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation......, Department of Plant Biology and Biotechnology, University of Copenhagen, DK-1871 Frederiksberg C, Denmark 2Humboldt-University Berlin, Faculty of Mathematics and Natural Science I, Institute of Biology, 10115 Berlin, Germany In the model plant Arabidopsis thaliana 12 P4-ATPases are present, named ALA1 to 12...

  15. Architecture and Molecular Mechanism of PAN, the Archaeal Proteasome Regulatory ATPase*

    Science.gov (United States)

    Medalia, Noa; Beer, Avital; Zwickl, Peter; Mihalache, Oana; Beck, Martin; Medalia, Ohad; Navon, Ami

    2009-01-01

    In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5′-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the “exit port” of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide. PMID:19363223

  16. Localization and irregular distribution of Na,K-ATPase in myelin sheath from rat sciatic nerve.

    Science.gov (United States)

    Alberti, Sandra; Gregório, Elisa Aparecida; Spadella, César Tadeu; Cojocel, Constantin

    2007-06-01

    Sodium, potassium adenosine triphosphatase (Na,K-ATPase) is a membrane-bound enzyme that maintains the Na(+) and K(+) gradients used in the nervous system for generation and transmission of bioelectricity. Recently, its activity has also been demonstrated during nerve regeneration. The present study was undertaken to investigate the ultrastructural localization and distribution of Na,K-ATPase in peripheral nerve fibers. Small blocks of the sciatic nerves of male Wistar rats weighing 250-300g were excised, divided into two groups, and incubated with and without substrate, the para-nitrophenyl phosphate (pNPP). The material was processed for transmission electron microscopy, and the ultra-thin sections were examined in a Philips CM 100 electron microscope. The deposits of reaction product were localized mainly on the axolemma, on axoplasmic profiles, and irregularly dispersed on the myelin sheath, but not in the unmyelinated axons. In the axonal membrane, the precipitates were regularly distributed on the cytoplasmic side. These results together with published data warrant further studies for the diagnosis and treatment of neuropathies with compromised Na,K-ATPase activity.

  17. Zinc and ATP Binding of the Hexameric AAA-ATPase PilF from Thermus thermophilus

    Science.gov (United States)

    Salzer, Ralf; Herzberg, Martin; Nies, Dietrich H.; Joos, Friederike; Rathmann, Barbara; Thielmann, Yvonne; Averhoff, Beate

    2014-01-01

    The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions. PMID:25202014

  18. Reaction mechanism of the membrane-bound ATPase of submitochondrial particles from beef heart.

    Science.gov (United States)

    Penefsky, H S

    1985-11-05

    Submitochondrial particles from beef heart, washed with dilute solutions of KCl so as to activate the latent, membrane-bound ATPase, F1, may be used to study single site catalysis by the enzyme. [gamma-32P]ATP, incubated with a molar excess of catalytic sites, a condition which favors binding of substrate in only a single catalytic site on the enzyme, is hydrolyzed via a four-step reaction mechanism. The mechanism includes binding in a high affinity catalytic site, Ka = 10(12)M-1, a hydrolytic step for which the equilibrium constant is near unity, and two product release steps in which Pi dissociates from catalytic sites about 10 times more rapidly than ADP. Catalysis by the membrane-bound ATPase also is characterized by a 10(6)-fold acceleration in the rate of net hydrolysis of [gamma-32P]ATP, bound in the high affinity catalytic site, that occurs when substrate is made available to additional catalytic sites on the enzyme. These aspects of the reaction mechanism of the ATPase of submitochondrial particles closely parallel the reaction mechanism determined for solubilized, homogeneous F1 (Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100). The finding that removal of the enzyme from the membrane does not significantly alter the properties of single site catalysis lends support to models of ATP synthesis in oxidative phosphorylation, catalyzed by membrane-bound F1, that have been based on the study of the soluble enzyme.

  19. Purification of the synaptosomal plasma membrane (Ca(2+) + Mg(2+))-ATPase from pig brain.

    Science.gov (United States)

    Salvador, J M; Mata, A M

    1996-04-01

    The Ca(2+)-ATPase from the synaptosomal plasma membrane has been purified nearly to homogeneity from pig brain by a new procedure involving the calmodulin-affinity-chromatography technique. This is a convenient alternative to the standard methods for the purification of the plasma membrane Ca(2+)-ATPase from different sources that were unsuitable to purify the enzyme from pig brain. The main feature of this procedure is the use of 15% (v/v) glycerol as stabilizing agent, instead of acidic phospholipid. By using this protocol the enzyme was purified 36-fold with respect to the plasma membrane vesicle fraction, showing a specific activity of 2.3 i.u. in the presence of acidic phospholipid. In SDS/PAGE, it appears as a single protein band around Mr140 000 that can be phosphorylated by [gamma-(32)P]ATP in the presence of La(3+) and recognized by specific antibodies against the plasma membrane Ca(2+)-ATPase from pig antral smooth muscle. Calmodulin activates the enzyme 1.5-1.8-fold in the presence of phosphatidylcholine but not in the presence of phosphatidylserine.

  20. The RIG-I ATPase domain structure reveals insights into ATP-dependent antiviral signalling.

    Science.gov (United States)

    Civril, Filiz; Bennett, Matthew; Moldt, Manuela; Deimling, Tobias; Witte, Gregor; Schiesser, Stefan; Carell, Thomas; Hopfner, Karl-Peter

    2011-10-28

    RIG-I detects cytosolic viral dsRNA with 5' triphosphates (5'-ppp-dsRNA), thereby initiating an antiviral innate immune response. Here we report the crystal structure of superfamily 2 (SF2) ATPase domain of RIG-I in complex with a nucleotide analogue. RIG-I SF2 comprises two RecA-like domains 1A and 2A and a helical insertion domain 2B, which together form a 'C'-shaped structure. Domains 1A and 2A are maintained in a 'signal-off' state with an inactive ATP hydrolysis site by an intriguing helical arm. By mutational analysis, we show surface motifs that are critical for dsRNA-stimulated ATPase activity, indicating that dsRNA induces a structural movement that brings domains 1A and 2A/B together to form an active ATPase site. The structure also indicates that the regulatory domain is close to the end of the helical arm, where it is well positioned to recruit 5'-ppp-dsRNA to the SF2 domain. Overall, our results indicate that the activation of RIG-I occurs through an RNA- and ATP-driven structural switch in the SF2 domain.

  1. Functions of nucleotide binding subunits in the tonoplast ATPase from Beta vulgaris L

    Energy Technology Data Exchange (ETDEWEB)

    Manolson, M.F.; Poole, R.J.

    1986-04-01

    Partial purification of NO/sub 3/ sensitive H/sup +/-ATPases from the vacuolar membranes of high plants reveal two prominent polypeptides of approximately 60 and 70 kDa. Both polypeptides appear to contain nucleotide binding sites. The photoactive affinity analog of ATP, BzATP, cannot be hydrolyzed by the tonoplast ATPase but is a potential inhibitor (apparent K/sub I/ = 11 ..mu..M). /sup 32/P-BzATP was shown to specifically photolabel the 60 kDa polypeptide. In contrast, Mandala and Taiz have shown the photoincorporation of /sup 32/P-azidoATP to the 70 kDa polypeptide. This sterically different photoaffinity probe can be hydrolyzed although with a low affinity. Azido and benzophenone derivatives of the product, ADP, are currently being examined with respect to their inhibition kinetics of, and their photoincorporation into, the tonoplast ATPase from Beta vulgaris L. Kinetic data will be integrated with patterns of photoincorporation using analogs of both substrate and product, in order to illuminate the functions of the two nucleotide binding subunits.

  2. Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp

    2013-04-05

    Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

  3. F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements.

    Science.gov (United States)

    Sugawa, Mitsuhiro; Okazaki, Kei-Ichi; Kobayashi, Masaru; Matsui, Takashi; Hummer, Gerhard; Masaike, Tomoko; Nishizaka, Takayuki

    2016-05-24

    Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F1-ATPase (F1) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α3β3, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three β-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two β-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F1 structure and the rotation angle of the motor. Remarkably, a structure of F1 in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αβ-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of β and the loosening/tightening motions at the αβ-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F1-ATPase.

  4. Nucleotide-induced asymmetry within ATPase activator ring drives σ54-RNAP interaction and ATP hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B. Tracy [IIT; (Penn)

    2013-12-10

    It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54–RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.

  5. Multivalent Chromatin Engagement and Inter-domain Crosstalk Regulate MORC3 ATPase

    Directory of Open Access Journals (Sweden)

    Forest H. Andrews

    2016-09-01

    Full Text Available MORC3 is linked to inflammatory myopathies and cancer; however, the precise role of MORC3 in normal cell physiology and disease remains poorly understood. Here, we present detailed genetic, biochemical, and structural analyses of MORC3. We demonstrate that MORC3 is significantly upregulated in Down syndrome and that genetic abnormalities in MORC3 are associated with cancer. The CW domain of MORC3 binds to the methylated histone H3K4 tail, and this interaction is essential for recruitment of MORC3 to chromatin and accumulation in nuclear bodies. We show that MORC3 possesses intrinsic ATPase activity that requires DNA, but it is negatively regulated by the CW domain, which interacts with the ATPase domain. Natively linked CW impedes binding of the ATPase domain to DNA, resulting in a decrease in the DNA-stimulated enzymatic activity. Collectively, our studies provide a molecular framework detailing MORC3 functions and suggest that its modulation may contribute to human disease.

  6. Disrupted Hydrogen-Bond Network and Impaired ATPase Activity in an Hsc70 Cysteine Mutant.

    Science.gov (United States)

    O'Donnell, John P; Marsh, Heather M; Sondermann, Holger; Sevier, Carolyn S

    2018-02-20

    The ATPase domain of members of the 70 kDa heat shock protein (Hsp70) family shows a high degree of sequence, structural, and functional homology across species. A broadly conserved residue within the Hsp70 ATPase domain that captured our attention is an unpaired cysteine, positioned proximal to the site of nucleotide binding. Prior studies of several Hsp70 family members show this cysteine is not required for Hsp70 ATPase activity, yet select amino acid replacements of the cysteine can dramatically alter ATP hydrolysis. Moreover, post-translational modification of the cysteine has been reported to limit ATP hydrolysis for several Hsp70s. To better understand the underlying mechanism for how perturbation of this noncatalytic residue modulates Hsp70 function, we determined the structure for a cysteine-to-tryptophan mutation in the constitutively expressed, mammalian Hsp70 family member Hsc70. Our work reveals that the steric hindrance produced by a cysteine-to-tryptophan mutation disrupts the hydrogen-bond network within the active site, resulting in a loss of proper catalytic magnesium coordination. We propose that a similarly altered active site is likely observed upon post-translational oxidation. We speculate that the subtle changes we detect in the hydrogen-bonding network may relate to the previously reported observation that cysteine oxidation can influence Hsp70 interdomain communication.

  7. Do electromagnetic fields interact with electrons in the Na,K-ATPase?

    Science.gov (United States)

    Blank, Martin

    2005-12-01

    The effects of low frequency electric and magnetic fields on several biochemical systems, including the Na,K-ATPase, indicate that electromagnetic (EM) fields interact with electrons. The frequency optima for two enzymes in response to EM fields are very close to their turnover numbers, suggesting that these interactions directly affect reaction rates. Nevertheless, generally accepted ideas about Na,K-ATPase function and ion transport mechanisms do not consider interactions with electrons. To resolve the clash of paradigms, we hypothesize interaction with transient electrons and protons that arise from flickering of H-bonds in the hydrated protein. These transient charges in the enzyme could provide a trigger for the sequence of conformation changes that are part of the ion transport mechanism. If the distributions of transient electrons and protons in the membrane are affected by their concentration and the membrane potential, as expected from electric double layer theory, this can account for the different effects of low frequency electric and magnetic fields, as well as for the observation that membrane hyperpolarization reverses the ATPase reaction to generate ATP. Bioelectromagnetics (c) 2005 Wiley-Liss, Inc.

  8. Effect of salinity on the biosynthesis of amines in Litopenaeus vannamei and the expression of gill related ion transporter genes

    Science.gov (United States)

    Pan, Luqing; Liu, Hongyu; Zhao, Qun

    2014-06-01

    This study examined the effect of salinity on the expression of Na+/K+-ATPase (NKA) α-subunit and vacuolar-type H+-ATPase (V-ATPase) β-subunit gene in the gill of Litopenaeus vannamei. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay showed that the expression of NKA α-subunit and V-ATPase β-subunit gene was significantly influenced by salinity. It was found that the NKA activity significantly varied with salinity in time and dose dependent manner; whereas the V-ATPase activity did not. The abundance of NKA α-subunit gene transcript increased rapidly when the salinity decreased from 26b to 21, and slowly when the salinity decreased from 26 to 31 within the first 24 h. When the salinity decreased from 26 to 21, the transcription of NKA α-subunit gene in gill epithelium was higher at 12 h than that at 0 h, which was consistent with the result of immunoblotting assay of NKA α-subunit. In addition, salinity had a significant time- and dose-dependent effect on the concentration of biogenic amines in both hemolymph and gill. As compared to other parameters, the concentration of dopamine (DA) and 5-hydroxytryptamine (5-HT) varied in different patterns when the salinity decreased from 26 to 21 or increased from 26 to 31, suggesting that DA and 5-HT played different regulatory roles in osmotic adaption and modulation of shrimp when salinity varies.

  9. SoxR-dependent response to oxidative stress and virulence of Erwinia chrysanthemi: the key role of SufC, an orphan ABC ATPase.

    Science.gov (United States)

    Nachin, L; El Hassouni, M; Loiseau, L; Expert, D; Barras, F

    2001-02-01

    Erwinia chrysanthemi causes soft-rot disease in a great variety of plants. In addition to the depolymerizing activity of plant cell wall-degrading enzymes, iron acquisition and resistance to oxidative stress contribute greatly to the virulence of this pathogen. Here, we studied the pin10 locus originally thought to encode new virulence factors. The sequence analysis revealed six open reading frames that were homologous to the Escherichia coli sufA, sufB, sufC, sufD, sufS and sufE genes. Sequence similarity searching predicted that (i) SufA, SufB, SufD, SufS and SufE proteins are involved in iron metabolism and possibly in Fe-S cluster assembly; and (ii) SufC is an ATPase of an ABC transporter. The reverse transcription-polymerase chain reaction procedure showed that the sufABCDSE genes constitute an operon. Expression of a sufB:uidA fusion was found to be induced in iron-deficient growth conditions and to be repressed by the iron-sensing Fur repressor. Each of the six suf genes was inactivated by the insertion of a cassette generating a non-polar mutation. The intracellular iron level in the sufA, sufB, sufC, sufS and sufE mutants was higher than in the wild type, as assessed by increased sensitivity to the iron-activated antibiotic streptonigrin. In addition, inactivation of sufC and sufD led to increased sensitivity to paraquat. Virulence tests showed that sufA and sufC mutants exhibited reduced ability to cause maceration of chicory leaves, whereas a functional sufC gene was necessary for the bacteria to cause systemic invasion of Saintpaulia ionantha. The E. coli sufC homologue was inactivated by reverse genetic. This mutation was found to modify the soxR-dependent induction of soxS gene expression. We discuss the possibility that SufC is a versatile ATPase that can associate either with the other Suf proteins to form a Fe-S cluster-assembling machinery or with membrane proteins encoded elsewhere in the chromosome to form an Fe-S ABC exporter. Overall, these

  10. Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly.

    Science.gov (United States)

    Chan, Chun-Yuan; Parra, Karlett J

    2014-07-11

    V-ATPases are conserved ATP-driven proton pumps that acidify organelles. Yeast V-ATPase assembly and activity are glucose-dependent. Glucose depletion causes V-ATPase disassembly and its inactivation. Glucose readdition triggers reassembly and resumes proton transport and organelle acidification. We investigated the roles of the yeast phosphofructokinase-1 subunits Pfk1p and Pfk2p for V-ATPase function. The pfk1Δ and pfk2Δ mutants grew on glucose and assembled wild-type levels of V-ATPase pumps at the membrane. Both phosphofructokinase-1 subunits co-immunoprecipitated with V-ATPase in wild-type cells; upon deletion of one subunit, the other subunit retained binding to V-ATPase. The pfk2Δ cells exhibited a partial vma growth phenotype. In vitro ATP hydrolysis and proton transport were reduced by 35% in pfk2Δ membrane fractions; they were normal in pfk1Δ. In vivo, the pfk1Δ and pfk2Δ vacuoles were alkalinized and the cytosol acidified, suggestive of impaired V-ATPase proton transport. Overall the pH alterations were more dramatic in pfk2Δ than pfk1Δ at steady state and after readdition of glucose to glucose-deprived cells. Glucose-dependent reassembly was 50% reduced in pfk2Δ, and the vacuolar lumen was not acidified after reassembly. RAVE-assisted glucose-dependent reassembly and/or glucose signals were disturbed in pfk2Δ. Binding of disassembled V-ATPase (V1 domain) to its assembly factor RAVE (subunit Rav1p) was 5-fold enhanced, indicating that Pfk2p is necessary for V-ATPase regulation by glucose. Because Pfk1p and Pfk2p are necessary for V-ATPase proton transport at the vacuole in vivo, a role for glycolysis at regulating V-ATPase proton transport is discussed. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane.

    Science.gov (United States)

    Efendiev, R; Bertorello, A M; Pressley, T A; Rousselot, M; Féraille, E; Pedemonte, C H

    2000-08-15

    Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.

  12. Virtual prototyping study shows increased ATPase activity of Hsp90 to be the key determinant of cancer phenotype.

    Science.gov (United States)

    Vali, Shireen; Pallavi, Rani; Kapoor, Shweta; Tatu, Utpal

    2010-03-01

    Hsp90 is an ATP-dependent molecular chaperone that regulates key signaling proteins and thereby impacts cell growth and development. Chaperone cycle of Hsp90 is regulated by ATP binding and hydrolysis through its intrinsic ATPase activities, which is in turn modulated by interaction with its co-chaperones. Hsp90 ATPase activity varies in different organisms and is known to be increased in tumor cells. In this study we have quantitatively analyzed the impact of increasing Hsp90 ATPase activity on the activities of its clients through a virtual prototyping technology, which comprises a dynamic model of Hsp90 interaction with clients involved in proliferation pathways. Our studies highlight the importance of increased ATPase activity of Hsp90 in cancer cells as the key modulator for increased proliferation and survival. A tenfold increase in ATPase activity of Hsp90 often seen in cancer cells increases the levels of active client proteins such as Akt-1, Raf-1 and Cyclin D1 amongst others to about 12-, 8- and 186-folds respectively. Additionally we studied the effect of a competitive inhibitor of Hsp90 activity on the reduction in the client protein levels. Virtual prototyping experiments corroborate with findings that the drug has almost 10- to 100-fold higher affinity as indicated by a lower IC(50) value (30-100 nM) in tumor cells with higher ATPase activity. The results also indicate a 15- to 25-fold higher efficacy of the inhibitor in reducing client levels in tumor cells. This analysis provides mechanistic insights into the links between increased Hsp90 ATPase activity, tumor phenotype and the hypersensitivity of tumor Hsp90 to inhibition by ATP analogs. The online version of this article (doi:10.1007/s11693-009-9046-3) contains supplementary material, which is available to authorized users.

  13. Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na+/K+ -ATPase.

    Science.gov (United States)

    Chen, Ronald J Y; Chung, Tse-yu; Li, Feng-yin; Yang, Wei-hung; Jinn, Tzyy-rong; Tzen, Jason T C

    2010-06-01

    To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain. The inhibitory potency of ouabain and the identified steroid-like compounds on Na(+)/K(+)-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na(+)/K(+)-ATPase. All the examined steroid-like compounds displayed more or less inhibition on Na(+)/K(+)-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na(+)/K(+)-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K(+) binding sites of Na(+)/K(+)-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na(+)/K(+)-ATPase. Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na(+)/K(+)-ATPase.

  14. Na,K-ATPase β1-subunit is a target of sonic hedgehog signaling and enhances medulloblastoma tumorigenicity.

    Science.gov (United States)

    Lee, Seung Joon; Litan, Alisa; Li, Zhiqin; Graves, Bruce; Lindsey, Stephan; Barwe, Sonali P; Langhans, Sigrid A

    2015-08-19

    The Sonic hedgehog (Shh) signaling pathway plays an important role in cerebellar development, and mutations leading to hyperactive Shh signaling have been associated with certain forms of medulloblastoma, a common form of pediatric brain cancer. While the fundamentals of this pathway are known, the molecular targets contributing to Shh-mediated proliferation and transformation are still poorly understood. Na,K-ATPase is a ubiquitous enzyme that maintains intracellular ion homeostasis and functions as a signaling scaffold and a cell adhesion molecule. Changes in Na,K-ATPase function and subunit expression have been reported in several cancers and loss of the β1-subunit has been associated with a poorly differentiated phenotype in carcinoma but its role in medulloblastoma progression is not known. Human medulloblastoma cell lines and primary cultures of cerebellar granule cell precursors (CGP) were used to determine whether Shh regulates Na,K-ATPase expression. Smo/Smo medulloblastoma were used to assess the Na,K-ATPase levels in vivo. Na,K-ATPase β1-subunit was knocked down in DAOY cells to test its role in medulloblastoma cell proliferation and tumorigenicity. Na,K-ATPase β1-subunit levels increased with differentiation in normal CGP cells. Activation of Shh signaling resulted in reduced β1-subunit mRNA and protein levels and was mimicked by overexpression of Gli1and Bmi1, both members of the Shh signaling cascade; overexpression of Bmi1 reduced β1-subunit promoter activity. In human medulloblastoma cells, low β1-subunit levels were associated with increased cell proliferation and in vivo tumorigenesis. Na,K-ATPase β1-subunit is a target of the Shh signaling pathway and loss of β1-subunit expression may contribute to tumor development and progression not only in carcinoma but also in medulloblastoma, a tumor of neuronal origin.

  15. The secretory response of parathyroid hormone to acute hypocalcemia in vivo is independent of parathyroid glandular sodium/potassium-ATPase activity

    DEFF Research Database (Denmark)

    Martuseviciene, Giedre; Hofman-Bang, Jacob; Clausen, Torben

    2011-01-01

    The involvement of sodium/potassium-ATPase in regulating parathyroid hormone (PTH) secretion is inferred from in vitro studies. Recently, the α-klotho-dependent rapid recruitment of this ATPase to the parathyroid cell plasma membrane in response to low extracellular calcium ion was suggested......-treated parathyroid glands, indicating inhibition of the ATPase. As ouabain induced systemic hyperkalemia, the effect of high potassium on hormone secretion was also examined but was found to have no effect. Thus, inhibition of the parathyroid gland sodium/potassium-ATPase activity in vivo had no effect...

  16. Regulation of Na+-K+-ATPase effected high glucose-induced myocardial cell injury through c-Src dependent NADPH oxidase/ROS pathway.

    Science.gov (United States)

    Yan, Xiaofei; Xun, Meng; Dou, Xiaojuan; Wu, Litao; Han, Yan; Zheng, Jin

    2017-08-15

    Depressed Na+/K+-ATPase activity has long been reported to be involved in diabetic-related cardiomyocyte death and cardiac dysfunction. However, the nature of directly regulating Na+-K+-ATPase in diabetic-related myocardial diseases remains unknown. Hyperglycemia is believed as one of major factors responsible for diabetic-related myocardial apoptosis and dysfunction. In this study, whether inhibiting Na+-K+-ATPase by ouabain or activating Na+-K+-ATPase by DRm217 has functions on high glucose (HG) -induced myocardial injury was investigated. Here we found that addition of DRm217 or ouabain to HG-treated cells had opposite effects. DRm217 decreased but ouabain increased HG-induced cell injury and apoptosis. This was mediated by changing Na+-K+-ATPase activity and Na+-K+-ATPase cell surface expression. The inhibition of Na+-K+-ATPase endocytosis alleviated HG-induced ROS accumulation. Na+-K+-ATPase·c-Src dependent NADPH oxidase/ROS pathway was also involved in the effects of ouabain and DRm217 on HG-induced cell injury. These novel results may help us to understand the important role of the Na+-K+-ATPase in diabetic cardiovascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Metal fluoride complexes of Na,K-ATPase: characterization of fluoride-stabilized phosphoenzyme analogues and their interaction with cardiotonic steroids

    National Research Council Canada - National Science Library

    Cornelius, Flemming; Mahmmoud, Yasser A; Toyoshima, Chikashi

    2011-01-01

    .... Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations...

  18. Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens.

    Science.gov (United States)

    Hendrick, William A; Orr, Mona W; Murray, Samantha R; Lee, Vincent T; Melville, Stephen B

    2017-05-15

    The Gram-positive pathogen Clostridium perfringens possesses type IV pili (TFP), which are extracellular fibers that are polymerized from a pool of pilin monomers in the cytoplasmic membrane. Two proteins that are essential for pilus functions are an assembly ATPase (PilB) and an inner membrane core protein (PilC). Two homologues each of PilB and PilC are present in C. perfringens, called PilB1/PilB2 and PilC1/PilC2, respectively, along with four pilin proteins, PilA1 to PilA4. The gene encoding PilA2, which is considered the major pilin based on previous studies, is immediately downstream of the pilB2 and pilC2 genes. Purified PilB2 had ATPase activity, bound zinc, formed hexamers even in the absence of ATP, and bound the second messenger molecule cyclic di-GMP (c-di-GMP). Circular dichroism spectroscopy of purified PilC2 indicated that it retained its predicted degree of alpha-helical secondary structure. Even though no direct interactions between PilB2 and PilC2 could be detected in vivo or in vitro even in the presence of c-di-GMP, high levels of expression of a diguanylate cyclase from C. perfringens (CPE1788) stimulated polymerization of PilA2 in a PilB2- and PilC2-dependent manner. These results suggest that PilB2 activity is controlled by c-di-GMP levels in vivo but that PilB2-PilC2 interactions are either transitory or of low affinity, in contrast to results reported previously from in vivo studies of the PilB1/PilC1 pair in which PilC1 was needed for polar localization of PilB1. This is the first biochemical characterization of a c-di-GMP-dependent assembly ATPase from a Gram-positive bacterium.IMPORTANCE Type IV pili (TFP) are protein fibers involved in important bacterial functions, including motility, adherence to surfaces and host cells, and natural transformation. All clostridia whose genomes have been sequenced show evidence of the presence of TFP. The genetically tractable species Clostridium perfringens was used to study proteins involved in

  19. Na+/K+-ATPase and vacuolar-type H+-ATPase in the gills of the aquatic air-breathing fish Trichogaster microlepis in response to salinity variation.

    Science.gov (United States)

    Huang, Chun-Yen; Chao, Pei-Lin; Lin, Hui-Chen

    2010-03-01

    The aquatic air-breathing fish, Trichogaster microlepis, can be found in fresh water and estuaries. We further evaluated the changes in two important osmoregulatory enzymes, Na(+)/K(+)-ATPase (NKA) and vacuolar-type H(+)-ATPase (VHA), in the gills when fish were subjected to deionized water (DW), fresh water (FW), and salinated brackish water (salinity of 10 g/L). Fish were sampled only 4 days after experimental transfer. The mortality, plasma osmolality, and Na(+) concentration were higher in 10 g/L acclimated fish, while their muscle water content decreased with elevated external salinity. The highest NKA protein abundance was found in the fish gills in 10 g/L, and NKA activity was highest in the DW and 10 g/L acclimated fish. The VHA protein levels were highest in 10 g/L, and VHA activity was highest in the DW treatment. From immunohistochemical results, we found three different cell populations: (1) NKA-immunoreactive (NKA-IR) cells, (2) both NKA-IR and HA-IR cells, and (3) HA-IR cells. NKA-IR cells in the lamellar and interlamellar regions significantly increased in DW and 10 g/L treatments. Only HA-IR cells in the lamellar region were significantly increased in DW. In the interlamellar region, there was no difference in the number of HA-IR cells among the three treated. From these results, T. microlepis exhibited osmoregulatory ability in DW and 10 g/L treatments. The cell types involved in ionic regulation were also examined with immunofluorescence staining; three ionocyte types were found which were similar to the zebrafish model. Copyright 2009 Elsevier B.V. All rights reserved.

  20. Structures and immunolocalization of Na+, K+ -ATPase, Na+ /H+ exchanger 3 and vacuolar-type H+ -ATPase in the gills of blennies (Teleostei: Blenniidae) inhabiting rocky intertidal areas.

    Science.gov (United States)

    Uchiyama, M; Komiyama, M; Yoshizawa, H; Shimizu, N; Konno, N; Matsuda, K

    2012-05-01

    The structure and immunolocalization of the ion transporters Na(+) ,K(+) -ATPase (NKA), Na(+) /H(+) exchanger (NHE3) and vacuolar-type H(+) -ATPase (VHA) were examined in the gills of teleosts of the family Blenniidae, which inhabit rocky shores with vertical zonation in subtropical seas. These features were compared among the following species with different ecologies: the amphibious rockskipper blenny Andamia tetradactylus, the intertidal white-finned blenny Praealticus tanegasimae and the purely marine yaeyama blenny Ecsenius yaeyamaensis. Light and electron microscopic observations indicated that thick gill filaments were arranged close to each other and alternately on two hemibranches of a gill arch in the opercular space of A. tetradactylus. Many mucous cells (MC) and mitochondrion-rich cells (MRC) were present in the interlamellar regions of the gill filament. An immunohistochemical study demonstrated that numerous NKA, NHE3 and some VHA were located predominantly on presumed MRCs of gill filaments and at the base of the lamellae. Analyses using serial (mirror image) sections of the gills indicated that only a few NKA immunoreactive cells (IRC) were colocalized with VHA on some MRCs in the filaments. In the gills of P. tanegasimae, NKA- and NHE3-IRCs were observed in the interlamellar region of the filaments and at the base of the lamellae. VHA-IRCs were located sparsely on the lamellae and filaments. In the gills of E. yaeyamaensis, the lamellae and filaments were thin and straight, respectively. MCs were located at the tip as well as found scattered in the interlamellar region of gill filaments. NKA-, NHE3- and VHA-IRCs were moderately frequently observed in the filaments and rarely on the lamellae. This study shows that the structure and distribution of ion transporters in the gills differ among the three blennid species, presumably reflecting their different ecologies. © 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the

  1. ALT1, a Snf2 family chromatin remodeling ATPase, negatively regulates alkaline tolerance through enhanced defense against oxidative stress in rice.

    Directory of Open Access Journals (Sweden)

    Mingxin Guo

    Full Text Available Alkaline salt stress adversely affects rice growth, productivity and grain quality. However, the mechanism underlying this process remains elusive. We characterized here an alkaline tolerant mutant, alt1 in rice. Map-based cloning revealed that alt1 harbors a mutation in a chromatin remodeling ATPase gene. ALT1-RNAi transgenic plants under different genetic background mimicked the alt1 phenotype, exhibiting tolerance to alkaline stress in a transcript dosage-dependent manner. The predicted ALT1 protein belonged to the Ris1 subgroup of the Snf2 family and was localized in the nucleus, and transcription of ALT1 was transiently suppressed after alkaline treatment. Although the absorption of several metal ions maintained well in the mutant under alkaline stress, expression level of the genes involved in metal ions homeostasis was not altered in the alt1 mutant. Classification of differentially expressed abiotic stress related genes, as revealed by microarray analysis, found that the majority (50/78 were involved in ROS production, ROS scavenging, and DNA repair. This finding was further confirmed by that alt1 exhibited lower levels of H2O2 under alkaline stress and tolerance to methyl viologen treatment. Taken together, these results suggest that ALT1 negatively functions in alkaline tolerance mainly through the defense against oxidative damage, and provide a potential two-step strategy for improving the tolerance of rice plants to alkaline stress.

  2. Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase.

    Directory of Open Access Journals (Sweden)

    Anthonius A Eze

    2016-08-01

    Full Text Available Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine.Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15, being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance.Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F1 ATPase γ subunit, which

  3. Glutamate dehydrogenase and Na+-K+ ATPase expression and growth response of Litopenaeus vannamei to different salinities and dietary protein levels

    Science.gov (United States)

    Li, Erchao; Arena, Leticia; Lizama, Gabriel; Gaxiola, Gabriela; Cuzon, Gerard; Rosas, Carlos; Chen, Liqiao; van Wormhoudt, Alain

    2011-03-01

    Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture. The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp ( L. vannamei) were investigated. This involved an examination of growth performance, glutamate dehydrogenase (GDH) and Na+-K+ ATPase mRNA expression,, and GDH activity in muscles and gills. Three experimental diets were formulated, containing 25%, 40%, and 50% dietary protein, and fed to the shrimp at a salinity of 25. After 20 days, no significant difference was observed in weight gain, though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels. Subsequently, shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5, respectively, and sampled at weeks 1 and 2. Shrimp fed with 40% protein at 25 in salinity (optimal conditions) were used as a control. Regardless of the salinities, shrimp fed with 50% dietary protein had significantly higher growth performance than other diets; no significant differences were found in comparison with the control. Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks. Ambient salinity change also stimulated the hepatosomatic index, which increased in the first week and then recovered to a relatively normal level, as in the control, after 2 weeks. These findings indicate that in white shrimp, the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism. Increased dietary protein level could improve the osmoregulation capacity of L. vannamei with more energy resources allocated to GDH activity and expression.

  4. Directed mutagenesis of the strongly conserved aspartate 242 in the beta-subunit of Escherichia coli proton-ATPase.

    Science.gov (United States)

    Al-Shawi, M K; Parsonage, D; Senior, A E

    1988-12-25

    Oligonucleotide-directed mutagenesis was used to substitute Asn or Val for residue Asp-242 in the beta-subunit of Escherichia coli F1-ATPase. Asp-242 is strongly conserved in beta-subunits of F1-ATPase enzymes, in a region of sequence which shows homology with numerous nucleotide-binding proteins. By analogy with adenylate kinase (Fry, D.C., Kuby, S.A., and Mildvan, A.S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 907-911), beta-Asp-242 of F1-ATPase might participate in catalysis through electrostatic effects on the substrate Mg2+ or through hydrogen bonding to the substrate(s); an acid-base catalytic role is also plausible. The substitutions Asn and Val were chosen to affect the charge, hydrogen-bonding ability, and hydrophobicity of residue beta-Asp-242. Both mutations significantly impaired oxidative phosphorylation rates in vivo and membrane ATPase and ATP-driven proton-pumping activities in vitro. Asn-242 was more detrimental than Val-242. Purified soluble mutant F1-ATPases had normal molecular size and subunit composition, and displayed 7% (beta-Asn-242) and 17% (beta-Val-242) of normal specific Mg-ATPase activity. The relative MgATPase activities of both mutant enzymes showed similar pH dependence to normal. Relative MgATPase and CaATPase activities of normal and mutant enzymes were compared at widely varied pMg and pCa. The mutations had little effect on KM MgATP, but KM CaATP was reduced. The data showed that the carboxyl side-chain of beta-Asp-242 is not involved in catalysis either as a general acid-base catalyst or through direct involvement in any protonation/deprotonation-linked mechanism, nor is it likely to be directly involved in liganding to substrate Mg2+ during the reaction. Specificity constants (kcat/KM) for MgATP and CaATP were reduced in both mutant enzymes, showing that the mutations destabilized interactions between the catalytic nucleotide-binding domain and the transition state.

  5. BRAHMA ATPase of the SWI/SNF chromatin remodeling complex acts as a positive regulator of gibberellin-mediated responses in arabidopsis.

    Directory of Open Access Journals (Sweden)

    Rafal Archacki

    Full Text Available SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways.

  6. BRAHMA ATPase of the SWI/SNF Chromatin Remodeling Complex Acts as a Positive Regulator of Gibberellin-Mediated Responses in Arabidopsis

    Science.gov (United States)

    Archacki, Rafal; Buszewicz, Daniel; Sarnowski, Tomasz J.; Sarnowska, Elzbieta; Rolicka, Anna T.; Tohge, Takayuki; Fernie, Alisdair R.; Jikumaru, Yusuke; Kotlinski, Maciej; Iwanicka-Nowicka, Roksana; Kalisiak, Katarzyna; Patryn, Jacek; Halibart-Puzio, Joanna; Kamiya, Yuji; Davis, Seth J.; Koblowska, Marta K.; Jerzmanowski, Andrzej

    2013-01-01

    SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana) mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA) are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM) ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways. PMID:23536800

  7. Cloning and characterization of the heart muscle isoform of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) from crayfish.

    Science.gov (United States)

    Chen, Dongdong; Zhang, Zhiping; Wheatly, Michele G; Gao, Yongping

    2002-09-01

    This paper describes the cloning and functional characterization of the heart muscle isoform of Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish Procambarus clarkii. The complete crayfish heart SERCA, identified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), consists of 4495 bp with a 3060 bp open reading frame, coding for 1020 amino acids. This isoform differs from the previously identified axial abdominal (tail) muscle SERCA solely in its C-terminal amino acids. The last nine amino acids of the tail muscle isoform are replaced by 27 hydrophobic amino acids in the heart isoform that have the potential to form an additional transmembrane domain. Consistent with other invertebrate studies, Southern blot analysis suggested that the heart and tail muscle isoforms are encoded from the same gene that is equally related to SERCA-1, -2 and -3 of vertebrates. The tissue distributions of these two isoforms have been assessed using isoform-specific probes and northern analysis. A cardiac-specific probe bound only to a 5.8 kb species in heart and had minimal cross-hybridization with 7.6 and 5.8 kb species in eggs and no hybridization with tail muscle. A tail-isoform-specific probe hybridized with a 4.5 kb species in tail muscle and cross-hybridized with a 4.5 kb species in eggs and 8.8 kb in heart muscle. Both isoforms are expressed in eggs suggesting that transcripts are formed early in development and are subsequently broadly expressed in all tissue types. Expression of the cardiac muscle SERCA isoform varied with the stage of moulting. Expression was high in intermoult and decreased in premoult. However, expression was restored rapidly in postmoult (within 2 days) unlike expression of tail muscle SERCA, which remained downregulated for weeks. Differences in contractility between the two muscle types in the postmoult period may explain these expression patterns.

  8. Ablation of plasma membrane Ca(2+)-ATPase isoform 4 prevents development of hypertrophy in a model of hypertrophic cardiomyopathy.

    Science.gov (United States)

    Prasad, Vikram; Lorenz, John N; Lasko, Valerie M; Nieman, Michelle L; Jiang, Min; Gao, Xu; Rubinstein, Jack; Wieczorek, David F; Shull, Gary E

    2014-12-01

    The mechanisms linking the expression of sarcomeric mutant proteins to the development of pathological hypertrophy in hypertrophic cardiomyopathy (HCM) remain poorly understood. We investigated the role of the plasma membrane Ca(2+)-ATPase PMCA4 in the HCM phenotype using a transgenic model that expresses mutant (Glu180Gly) α-tropomyosin (Tm180) in heart. Immunoblot analysis revealed that cardiac PMCA4 expression was upregulated early in Tm180 disease pathogenesis. This was accompanied by an increase in levels of the L-type Ca(2+)-channel, which is implicated in pathological hypertrophy. When Tm180 mice were crossed with a PMCA4-null line, loss of PMCA4 caused the abrogation of hypertrophy in Tm180/PMCA4-null double mutant mice. RT-PCR analysis of Tm180/PMCA4-null hearts revealed blunting of the fetal program and reversion of pro-fibrotic Col1a1 and Col3a1 gene expression to wild-type levels. This was accompanied by evidence of reduced L-type Ca(2+)-channel expression, and diminished calcineurin activity. Expression of the metabolic substrate transporters glucose transporter 4 and carnitine palmitoyltransferase 1b was preserved and Tm180-related changes in mRNA levels of various contractile stress-related proteins including the cardiac ankyrin protein CARP and the N2B isoform of titin were reversed in Tm180/PMCA4-null hearts. cGMP levels were increased and phosphorylation of vasodilator-stimulated phosphoprotein was elevated in Tm180/PMCA4-null hearts. These changes were associated with a sharp reduction in left ventricular end-diastolic pressure in Tm180/PMCA4-null hearts, which occurred despite persistence of Tm180-related impairment of relaxation dynamics. These results reveal a novel and specific role for PMCA4 in the Tm180 hypertrophic phenotype, with the "protective" effects of PMCA4 deficiency encompassing multiple determinants of HCM-related hypertrophy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Different Modulatory Mechanisms of Renal FXYD12 for Na(+)-K(+)-ATPase between Two Closely Related Medakas upon Salinity Challenge.

    Science.gov (United States)

    Yang, Wen-Kai; Kang, Chao-Kai; Hsu, An-Di; Lin, Chia-Hao; Lee, Tsung-Han

    2016-01-01

    Upon salinity challenge, the Na(+)-K(+)-ATPase (NKA) of fish kidney plays a crucial role in maintaining ion and water balance. Moreover, the FXYD protein family was found to be a regulator of NKA. Our preliminary results revealed that fxyd12 was highly expressed in the kidneys of the two closely related euryhaline medaka species (Oryzias dancena and O. latipes) from different natural habitats (brackish water and fresh water). In this study, we investigated the expression and association of renal FXYD12 and NKA α-subunit as well as potential functions of FXYD12 in the two medakas. These findings illustrated and compared the regulatory roles of FXYD12 for NKA in kidneys of the two medakas in response to salinity changes. In this study, at the mRNA and/or protein level, the expression patterns were similar for renal FXYD12 and NKA in the two medakas. However, different patterns of NKA activities and different interaction levels between FXYD12 and NKA were found in the kidneys of these two medakas. The results revealed that different strategies were used in the kidneys of the two medaka species upon salinity challenge. On the other hand, gene knockdown experiments demonstrated that the function of O. dancena FXYD12 allowed maintenance of a high level of NKA activity. The results of the present study indicated that the kidneys of the examined euryhaline medakas originating from brackish water and fresh water exhibited different modulatory mechanisms through which renal FXYD12 enhanced NKA activity to maintain internal homeostasis. Our findings broadened the knowledge of expression and functions of FXYD proteins, the modulators of NKA, in vertebrates.

  10. [Preparation and application of monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit].

    Science.gov (United States)

    Yan, Xiaofei; Wu, Litao; DU, Xiaojuan; Li, Jing; Zhang, Fujun; Han, Yan; Lyu, Shemin; Li, Dongmin

    2016-12-01

    Objective To prepare monoclonal antibodies against DR region (897DVEDSYGQQWTYEQR911) of Na+-K+-ATPase α1 subunit and identify their properties. Methods BALB/c mice were immunized with DR-keyholelimpet hemocyanin (KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2/0 mouse myeloma cells. Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting, Western blotting and immunofluorescence assay. Na+-K+-ATPase activity was detected by SensoLyteR FDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line, named DRm217, was selected to prepare ascites. Dot blotting, Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na+-K+-ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na+-K+-ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit is prepared.

  11. Alternating Hemiplegia of Childhood-Related Neural and Behavioural Phenotypes in Na+,K+-ATPase α3 Missense Mutant Mice

    Science.gov (United States)

    Kirshenbaum, Greer S.; Dawson, Neil; Mullins, Jonathan G. L.; Johnston, Tom H.; Drinkhill, Mark J.; Edwards, Ian J.; Fox, Susan H.; Pratt, Judith A.; Brotchie, Jonathan M.; Roder, John C.; Clapcote, Steven J.

    2013-01-01

    Missense mutations in ATP1A3 encoding Na+,K+-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC), a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na+,K+-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N) that affects the same position in Na+,K+-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na+,K+-ATPase α3, including upon the K+ pore and predicted K+ binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality), directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na+,K+-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC. PMID:23527305

  12. Alternating hemiplegia of childhood-related neural and behavioural phenotypes in Na+,K+-ATPase α3 missense mutant mice.

    Directory of Open Access Journals (Sweden)

    Greer S Kirshenbaum

    Full Text Available Missense mutations in ATP1A3 encoding Na(+,K(+-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC, a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na(+,K(+-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N that affects the same position in Na(+,K(+-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na(+,K(+-ATPase α3, including upon the K(+ pore and predicted K(+ binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality, directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na(+,K(+-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC.

  13. Salinity fluctuation influencing biological adaptation: growth dynamics and Na+/K+-ATPase activity in a euryhaline bacterium.

    Science.gov (United States)

    Yang, Hao; Meng, Yang; Song, Youxin; Tan, Yalin; Warren, Alan; Li, Jiqiu; Lin, Xiaofeng

    2017-07-01

    Although salinity fluctuation is a prominent characteristic of many coastal ecosystems, its effects on biological adaptation have not yet been fully recognized. To test the salinity fluctuations on biological adaptation, population growth dynamics and Na + /K + -ATPase activity were investigated in the euryhaline bacterium Idiomarina sp. DYB, which was acclimated at different salinity exposure levels, exposure times, and shifts in direction of salinity. Results showed: (1) bacterial population growth dynamics and Na + /K + -ATPase activity changed significantly in response to salinity fluctuation; (2) patterns of variation in bacterial growth dynamics were related to exposure times, levels of salinity, and shifts in direction of salinity change; (3) significant tradeoffs were detected between growth rate (r) and carrying capacity (K) on the one hand, and Na + /K + -ATPase activity on the other; and (4) beneficial acclimation was confirmed in Idiomarina sp. DYB. In brief, this study demonstrated that salinity fluctuation can change the population growth dynamics, Na + /K + -ATPase activity, and tradeoffs between r, K, and Na + /K + -ATPase activity, thus facilitating bacterial adaption in a changing environment. These findings provide constructive information for determining biological response patterns to environmental change. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. In situ assembly states of (Na+,K+)-pump ATPase in human erythrocytes. Radiation target size analyses.

    Science.gov (United States)

    Hah, J; Goldinger, J M; Jung, C Y

    1985-11-15

    The in situ assembly state of the (Na+,K+)-pump ATPase of human erythrocytes was studied by applying the classical target theory to radiation inactivation data of the ouabain-sensitive sodium efflux and ATP hydrolysis. Erythrocytes and their extensively washed white ghosts were irradiated at -45 to -50 degrees C with an increasing dose of 1.5-MeV electron beam, and after thawing, the Na+-pump flux and/or enzyme activities were assayed. Each activity measured was reduced as a simple exponential function of radiation dose, from which a radiation sensitive mass (target size) was calculated. When intact cells were used, the target sizes for the pump and for the ATPase activities were equal and approximately 620,000 daltons. The target size for the ATPase activity was reduced to approximately 320,000 daltons if the cells were pretreated with digitoxigenin. When ghosts were used, the target size for the ATPase activity was again approximately 320,000 daltons. Our target size measurements together with other information available in literature suggest that (Na+,K+)-pump ATPase may exist in human erythrocytes either as a tetramer of alpha beta or as a dimer of alpha beta in tight association with other protein mass, probably certain glycolytic enzymes, and that this tetrameric or heterocomplex association is dissociable by digitoxigenin treatment or by extensive wash during ghost preparation.

  15. Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

    Directory of Open Access Journals (Sweden)

    Flora H.F. D'Angelis

    2014-09-01

    Full Text Available This study aims at standardizing the pre-incubation and incubation pH and temperature used in the metachromatic staining method of myofibrillar ATPase activity of myosin (mATPase used for asses and mules. Twenty four donkeys and 10 mules, seven females and three males, were used in the study. From each animal, fragments from the Gluteus medius muscle were collected and percutaneous muscle biopsy was performed using a 6.0-mm Bergström-type needle. In addition to the metachromatic staining method of mATPase, the technique of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR was also performed to confirm the histochemical data. The histochemical result of mATPase for acidic pre-incubation (pH=4.50 and alkaline incubation (pH=10.50, at a temperature of 37ºC, yielded the best differentiation of fibers stained with toluidine blue. Muscle fibers were identified according to the following colors: type I (oxidative, light blue, type IIA (oxidative-glycolytic, intermediate blue and type IIX (glycolytic, dark blue. There are no reports in the literature regarding the characterization and distribution of different types of muscle fibers used by donkeys and mules when performing traction work, cargo transportation, endurance sports (horseback riding and marching competitions. Therefore, this study is the first report on the standardization of the mATPase technique for donkeys and mules.

  16. Molecular and functional characterization of seven Na+/K+-ATPase β subunit paralogs in Senegalese sole (Solea senegalensis Kaup, 1858).

    Science.gov (United States)

    Armesto, Paula; Infante, Carlos; Cousin, Xavier; Ponce, Marian; Manchado, Manuel

    2015-04-01

    In the present work, seven genes encoding Na(+),K(+)-ATPase (NKA) β-subunits in the teleost Solea senegalensis are described for the first time. Sequence analysis of the predicted polypeptides revealed a high degree of conservation with those of other vertebrate species and maintenance of important motifs involved in structure and function. Phylogenetic analysis clustered the seven genes into four main clades: β1 (atp1b1a and atp1b1b), β2 (atp1b2a and atp1b2b), β3 (atp1b3a and atp1b3b) and β4 (atp1b4). In juveniles, all paralogous transcripts were detected in the nine tissues examined albeit with different expression patterns. The most ubiquitous expressed gene was atp1b1a whereas atp1b1b was mainly detected in osmoregulatory organs (gill, kidney and intestine), and atp1b2a, atp1b2b, atp1b3a, atp1b3b and atp1b4 in brain. An expression analysis in three brain regions and pituitary revealed that β1-type transcripts were more abundant in pituitary than the other β paralogs with slight differences between brain regions. Quantification of mRNA abundance in gills after a salinity challenge showed an activation of atp1b1a and atp1b1b at high salinity water (60 ppt) and atp1b3a and atp1b3b in response to low salinity (5 ppt). Transcriptional analysis during larval development showed specific expression patterns for each paralog. Moreover, no differences in the expression profiles between larvae cultivated at 10 and 35 ppt were observed except for atp1b4 with higher mRNA levels at 10 than 35 ppt at 18 days post hatch. Whole-mount in situ hybridization analysis revealed that atp1b1b was mainly localized in gut, pronephric tubule, gill, otic vesicle, and chordacentrum of newly hatched larvae. All these data suggest distinct roles of NKA β subunits in tissues, during development and osmoregulation with β1 subunits involved in the adaptation to hyperosmotic conditions and β3 subunits to hypoosmotic environments. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Cardiac function improved by sarcoplasmic reticulum Ca2+-ATPase overexpression in a heart failure model induced by chronic myocardial ischemia

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    Wei XIN

    2011-04-01

    Full Text Available Objective Chronic myocardial ischemia(CMI has become an important cause of heart failure(HF.The aim of present study was to examine the effects of Sarco-endoplasmic reticulum calcium ATPase(SERCA2a gene transfer in HF model in large animal induced by CMI.Methods HF was reproduced in minipigs by ligating the initial segment of proximal left anterior descending(LAD coronary artery with an ameroid constrictor to produce progressive vessel occlusion and ischemia.After confirmation of myocardial perfusion defect and cardiac function impairment by SPECT and echocardiography in the model,animals were divided into 4 groups: HF group;HF+enhanced green fluorescent protein(EGFP group;HF+SERCA2a group;and sham operation group as control.rAAV1-EGFP and rAAV1-SERCA2a(1×1012 vg for each animal were directly and intramyocardially injected to the animals of HF+EGFP and HF+SERCA2a groups.Sixty days after the gene transfer,the expression of SERCA2a at the protein level was examined by Western blotting and immunohistochemistry,the changes in cardiac function were determined by echocardiographic and hemodynamic analysis,and the changes in serum inflammatory and neuro-hormonal factors(including BNP,TNF-a,IL-6,ET-1 and Ang II were determined by radioimmunoassay.Results Sixty days after gene transfer,LVEF,Ev/Av and ±dp/dtmax increased significantly(P < 0.05,along with an increase of SERCA2a protein expression in the ischemic myocardium(PP < 0.05,accompanied by a significant decrease of inflammatory and neural-hormonal factors(PP < 0.05 in HF+SERCA2a group as compared with HF/HF+EGFP group.Conclusions Overexpression of SERCA2a may significantly improve the cardiac function of the ischemic myocardium of HF model induced by CMI and reverse the activation of neural-hormonal factors,implying that it has a potential therapeutic significance in CMI related heart failure.

  18. Genetic diversity and lack of artemisinin selection signature on the Plasmodium falciparum ATP6 in the Greater Mekong Subregion.

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    Miao Miao

    Full Text Available The recent detection of clinical Artemisinin (ART resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca(2+-ATPase (PfSERCA or PfATP6 has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (~3.6 Kb in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs, including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average d(N/d(S ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance.

  19. Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron

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    Cheval, L.; Doucet, A. (Centre National de la Recherche Scientifique, Paris (France))

    1990-07-01

    To determine the functioning rate of Na-K-ATPase in the rat nephron, a micromethod was developed to measure the rate of rubidium uptake in single nephron segments microdissected from collagenase-treated kidneys. Because the hydrolytic activity of Na-K-ATPase displayed the same apparent affinity for K and Rb ions, whereas the Vmax elicited by K was higher than that in the presence of Rb, experiments were performed in the presence of cold Rb plus 86Rb. Before the assay, tubules were preincubated for 10 min at 37 degrees C to restore the normal transmembrane cation gradients. 86Rb uptake was measured after washing out extracellular cations by rinsing the tubules in ice-cold choline chloride solution containing Ba2+. Rb uptake increased quasi-linearly as a function of incubation time up to 30 s in the thick ascending limb, 1 min in the proximal convoluted tubule, and 5 min in the collecting tubule, and reached an equilibrium after 5-30 min. The initial rates of Rb uptake increased in a saturable fashion as Rb concentration in the medium rose from 0.25 to 5 mM. In medullary thick ascending limb, the initial rate of Rb uptake was inhibited by greater than 90% by 2.5 mM ouabain and by 10(-5) M of the metabolic inhibitor carbonyl cyanide trifluoromethoxyphenylhydrazone. Correlation of Na-K-ATPase hydrolytic activity at Vmax and initial rates of ouabain-sensitive Rb uptake in the successive segments of nephron indicates that in intact cells the pump works at approximately 20-30% of its Vmax. Increasing intracellular Na concentration by tubule preincubation in a Rb- and K-free medium increased the initial rates of Rb intake up to the Vmax of the hydrolytic activity of the pump.

  20. Expression of sarcoplasmic-endoplasmic reticulum Ca-ATPase isoforms in masticatory muscles.

    Science.gov (United States)

    Sánchez, Gabriel A; Trinks, Pablo W; Richard, Susana B; Di Croce, Daniel E; Takara, Delia

    2014-02-01

    The aim of this study was to characterize the sarcoplasmic-endoplasmic reticulum Ca-ATPase (SERCA) isoforms in rabbit masticatory muscles compared with those in fast-twitch muscle. It was hypothesized that combined expression of the SERCA isoforms in fast- and slow-twitch muscles accounts for lower Ca-ATPase activity. SERCA was isolated by differential centrifugation, the isoforms were determined by ELISA, and the activity of each isoform was measured using a colorimetric method. Activity was tested for significance by anova, and the distribution of isoforms was assessed using the chi-square test (P < 0.05) and correlated to SERCA activity using Spearman's rank correlation. SERCA1 was predominant (90.5%) in fast-twitch muscle, whereas a mixture of SERCA isoforms was found in masticatory muscles: 62-78% was SERCA2, 20-37% was SERCA1, and the SERCA3 content was negligible. Depressor muscles showed a significantly higher content (77.8%) of SERCA2, and elevator muscles showed a higher content (35.4%) of SERCA1. Elevator muscles showed higher expression of SERCA2a (58%), and depressor muscles showed higher expression of SERCA2b (20%). The SERCA1 content was mainly SERCA1a and significantly higher for elevator muscles (33%), whereas depressor muscles showed a higher content of SERCA1b (4%). The SERCA1 content of fast-twitch muscle was mainly SERCA1a (88.5%). It is concluded that the mixture of different SERCA isoforms, along with a substantial content of SERCA2b, in masticatory muscles would support lower Ca-ATPase activity and calcium transport. © 2013 Eur J Oral Sci.

  1. Ataxia telangiectasia mutated (ATM) interacts with p400 ATPase for an efficient DNA damage response.

    Science.gov (United States)

    Smith, Rebecca J; Savoian, Matthew S; Weber, Lauren E; Park, Jeong Hyeon

    2016-11-04

    Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the ATM-p400 complex can be reconstituted without other mammalian bridging proteins. Overexpression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.

  2. Snf2 family gene distribution in higher plant genomes reveals DRD1 expansion and diversification in the tomato genome.

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    Joachim W Bargsten

    Full Text Available As part of large protein complexes, Snf2 family ATPases are responsible for energy supply during chromatin remodeling, but the precise mechanism of action of many of these proteins is largely unknown. They influence many processes in plants, such as the response to environmental stress. This analysis is the first comprehensive study of Snf2 family ATPases in plants. We here present a comparative analysis of 1159 candidate plant Snf2 genes in 33 complete and annotated plant genomes, including two green algae. The number of Snf2 ATPases shows considerable variation across plant genomes (17-63 genes. The DRD1, Rad5/16 and Snf2 subfamily members occur most often. Detailed analysis of the plant-specific DRD1 subfamily in related plant genomes shows the occurrence of a complex series of evolutionary events. Notably tomato carries unexpected gene expansions of DRD1 gene members. Most of these genes are expressed in tomato, although at low levels and with distinct tissue or organ specificity. In contrast, the Snf2 subfamily genes tend to be expressed constitutively in tomato. The results underpin and extend the Snf2 subfamily classification, which could help to determine the various functional roles of Snf2 ATPases and to target environmental stress tolerance and yield in future breeding.

  3. Identification of four genes involved in suppression of the pre-mRNA ...

    Indian Academy of Sciences (India)

    ... contrast to about > 50% level of unspliced rhp6 pre-mRNA in the sng1-1/rhp6- mutant, there is a restoration of normal splicing to varying degrees in the suppressors. The sur2 gene belongs to the AAA-ATPase family of proteins, with maximum homology to the SIN1-associated protein SAP1 of Saccharomyces cerevisia