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Sample records for calcium atpase pmca

  1. Plasma membrane calcium ATPase 4 (PMCA4) co-ordinates calcium and nitric oxide signaling in regulating murine sperm functional activity.

    Science.gov (United States)

    Olli, Kristine E; Li, Kun; Galileo, Deni S; Martin-DeLeon, Patricia A

    2018-01-01

    Reduced sperm motility (asthenospermia) and resulting infertility arise from deletion of the Plasma Membrane Ca 2+ -ATPase 4 (Pmca4) gene which encodes the highly conserved Ca 2+ efflux pump, PMCA4. This is the major Ca 2+ clearance protein in murine sperm. Since the mechanism underlying asthenospermia in PMCA4's absence or reduced activity is unknown, we investigated if sperm PMCA4 negatively regulates nitric oxide synthases (NOSs) and when absent NO, peroxynitrite, and oxidative stress levels are increased. Using co-immunoprecipitation (Co-IP) and Fluorescence Resonance Energy Transfer (FRET), we show an association of PMCA4 with the NOSs in elevated cytosolic [Ca 2+ ] in capacitated and Ca 2+ ionophore-treated sperm and with neuronal (nNOS) at basal [Ca 2+ ] (ucapacitated sperm). FRET efficiencies for PMCA4-eNOS were 35% and 23% in capacitated and uncapacitated sperm, significantly (p < 0.01) different, with the molecules being <10 nm apart. For PMCA4-nNOS, this interaction was seen only for capacitated sperm where FRET efficiency was 24%, significantly (p < 0.05) higher than in uncapacitated sperm (6%). PMCA4 and the NOSs were identified as interacting partners in a quaternary complex that includes Caveolin1, which co-immunoprecipitated with eNOS in a Ca 2+ -dependent manner. In Pmca4 -/- sperm NOS activity was elevated twofold in capacitated/uncapacitated sperm (vs. wild-type), accompanied by a twofold increase in peroxynitrite levels and significantly (p < 0.001) increased numbers of apoptotic germ cells. The data support a quaternary complex model in which PMCA4 co-ordinates Ca 2+ and NO signaling to maintain motility, with increased NO levels resulting in asthenospermia in Pmca4 -/- males. They suggest the involvement of PMCA4 mutations in human asthenospermia, with diagnostic relevance. © 2017 Wiley Periodicals, Inc.

  2. Plasma membrane calcium ATPase 4b inhibits nitric oxide generation through calcium-induced dynamic interaction with neuronal nitric oxide synthase.

    Science.gov (United States)

    Duan, Wenjuan; Zhou, Juefei; Li, Wei; Zhou, Teng; Chen, Qianqian; Yang, Fuyu; Wei, Taotao

    2013-04-01

    The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.

  3. Sperm Na+, K+-ATPase α4 and plasma membrane Ca2+-ATPase (PMCA) 4 regulation in asthenozoospermia.

    Science.gov (United States)

    Lestari, Silvia W; Miati, Dessy Noor; Seoharso, P; Sugiyanto, R; Pujianto, Dwi A

    2017-10-01

    Asthenozoospermia, which is characterized by reduced motility, is one of the etiologies of male infertility. Its biochemical and functional consequences include altered ATPase activity. This study investigated the activities of Na + , K + -ATPase and Ca 2+ -ATPase and the expression of Na + , K + -ATPase α4 and PMCA4 isoforms in human sperm of asthenozoospermic infertile men. Nineteen samples from asthenozoospermic infertile couples were examined in this study. Computerized-assisted semen analysis (CASA) was performed, and the enzyme activity was measured based on the ability of ATPase to release organic phosphate from ATP as a substrate. The Na + , K + -ATPase α4 and PMCA4 isoform expression levels were measured by western immunoblotting, whereas the protein distribution was examined by immunocytochemistry. This showed that the Na + , K + -ATPase activity and the Na + , K + -ATPase α4 isoform expression were lower in the asthenozoospermia group than in the normozoospermia group (8.688±1.161 versus 13.851±1.884 µmol Pi/mg protein/h, respectively; p>0.05). In contrast, the Ca 2+ -ATPase activity was significantly higher in the asthenozoospermia group than in the normozoospermia group (11.154±1.186 versus 2.725±0.545 µmol Pi/mg protein/h, respectively; p0.05). The altered ATPase activity and isoform expression in asthenozoospermia may impair sperm structure and function.

  4. Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling.

    Science.gov (United States)

    Supper, Verena; Schiller, Herbert B; Paster, Wolfgang; Forster, Florian; Boulègue, Cyril; Mitulovic, Goran; Leksa, Vladimir; Ohradanova-Repic, Anna; Machacek, Christian; Schatzlmaier, Philipp; Zlabinger, Gerhard J; Stockinger, Hannes

    2016-02-01

    The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression. Copyright © 2016 by The American Association of Immunologists, Inc.

  5. Evaluation of plasma membrane calcium/calmodulin-dependent ATPase isoform 4 as a potential target for fertility control.

    Science.gov (United States)

    Cartwright, Elizabeth J; Neyses, Ludwig

    2010-01-01

    The array of contraceptives currently available is clearly inadequate and does not meet consumer demands since it is estimated that up to a quarter of all pregnancies worldwide are unintended. There is, therefore, an overwhelming global need to develop new effective, safe, ideally non-hormonal contraceptives for both male and female use. The contraceptive field, unlike other areas such as cancer, has a dearth of new targets. We have addressed this issue and propose that isoform 4 of the plasma membrane calcium ATPase is a potentially exciting novel target for fertility control. The plasma membrane calcium ATPase is a ubiquitously expressed calcium pump whose primary function in the majority of cells is to extrude calcium to the extracellular milieu. Two isoforms of this gene family, PMCA1 and PMCA4, are expressed in spermatozoa, with PMCA4 being the predominant isoform. Although this gene is ubiquitously expressed, its function is highly tissue-specific. Genetic deletion of PMCA4, in PMCA4 knockout mice, led to 100% infertility specifically in the male mutant mice due to a selective defect in sperm motility. It is important to note that the gene deletion did not affect normal mating characteristics in these mice. This phenotype was mimicked in wild-type sperm treated with the non-specific PMCA inhibitor 5-(and 6-) carboxyeosin diacetate succinimidyl ester; a proof-of-principle that inhibition of PMCA4 has potential importance in the control of fertility. This review outlines the potential for PMCA4 to be a novel target for fertility control by acting to inhibit sperm motility. It will outline the characteristics that make this target drugable and will describe methodologies to identify and validate novel inhibitors of this target.

  6. 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...

  7. The PMCA pumps in genetically determined neuronal pathologies.

    Science.gov (United States)

    Calì, Tito; Brini, Marisa; Carafoli, Ernesto

    2018-01-10

    Ca 2+ signals regulate most aspects of animal cell life. They are of particular importance to the nervous system, in which they regulate specific functions, from neuronal development to synaptic plasticity. The homeostasis of cell Ca 2+ must thus be very precisely regulated: in all cells Ca 2+ pumps transport it from the cytosol to the extracellular medium (the Plasma Membrane Ca 2+ ATPases, hereafter referred to as PMCA pumps) or to the lumen of intracellular organelles (the Sarco/Endoplasmatic Reticulum Ca 2+ ATPase and the Secretory Pathway Ca 2+ ATPase, hereafter referred to as SERCA and SPCA pumps, respectively). In neurons and other excitable cells a powerful plasma membrane Na + /Ca 2+ exchanger (NCX) also exports Ca 2+ from cells. Quantitatively, the PMCA pumps are of minor importance to the bulk regulation of neuronal Ca 2+ . However, they are important in the regulation of Ca 2+ in specific sub-plasma membrane microdomains which contain a number of enzymes that are relevant to neuronal function. The PMCA pumps (of which 4 basic isoforms are expressed in animal cells) are P-type ATPases that are characterized by a long C-terminal cytosolic tail which is the site of interaction with most of the regulatory factors of the pump, the most important being calmodulin. In resting neurons, at low intracellular Ca 2+ the C-terminal tail of the PMCA interacts with the main body of the protein keeping it in an autoinhibited state. Local Ca 2+ increase activates calmodulin that removes the C-terminal tail from the inhibitory sites. Dysregulation of the Ca 2+ signals are incompatible with healthy neuronal life. A number of genetic mutations of PMCA pumps are associated with pathological phenotypes, those of the neuron-specific PMCA 2 and PMCA 3 being the best characterized. PMCA 2 mutations are associated with deafness and PMCA 3 mutations are linked to cerebellar ataxias. Biochemical analysis of the mutated pumps overexpressed in model cells have revealed their

  8. Mammary gland involution is associated with rapid down regulation of major mammary Ca**2+-ATPases

    Science.gov (United States)

    Sixty percent of calcium in milk is transported across the mammary cells apical membrane by the plasma membrane Ca**2+-ATPase 2 (PMCA2). The effect of abrupt cessation of milk production on the Ca**2+-ATPases and mammary calcium transport is unknown. We found that 24 hours after stopping milk prod...

  9. Secretory pathway Ca2+/Mn2+-ATPase isoform 2 and lactation: specific localization of plasmalemmal and secretory pathway Ca2+ pump isoforms in the mammary gland

    Energy Technology Data Exchange (ETDEWEB)

    Faddy, Helen M.; Smart, Chanel E.; Xu, Ren; Lee, Genee Y.; Kenny, Paraic A.; Feng, Mingye; Rao, Rajini; Brown, Melissa A.; Bissell, Mina J.; Roberts-Thomson, Sarah J.; Monteith, Gregory R.

    2008-04-09

    The supply of calcium to the developing neonate via milk is an important physiological process. Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely mediated via the secretory pathway. However, recent studies suggest that a specific isoform of the plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during different stages of development. SPCA2 levels increased over 35 fold during lactation, while SPCA1 increased only a modest two fold. The potential importance of SPCA2 in lactation was also highlighted by its localization to luminal secretory cells of the mammary gland during lactation, while SPCA1 was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1 during lactation. Using the SCp2 mouse mammary epithelial cell 3D culture model, differences in the sub-cellular distribution of PMCA2 and PMCA1 were clear. These studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation, and link the recently characterized SPCA2 calcium pump to the supply of calcium into milk and the regulation of Golgi resident enzymes important in lactation. They also indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.

  10. The Ca2+/H+ antiporter TMEM165 expression, localization in the developing, lactating and involuting mammary gland parallels the secretory pathway Ca2+ATPase (SPCA1)

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    Plasma membrane Ca2+-ATPase 2 (PMCA2) knockout mice showed that ~ 60 % of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca2+-ATPase’s 1 and/or 2 (SP...

  11. Plasma membrane calcium ATPases and related disorders.

    Science.gov (United States)

    Giacomello, Marta; De Mario, Agnese; Scarlatti, Chiara; Primerano, Simona; Carafoli, Ernesto

    2013-03-01

    The plasma membrane Ca(2+) ATPases (PMCA pumps) cooperate with other transport systems in the plasma membrane and in the organelles in the regulation of cell Ca(2+). They have high Ca(2+) affinity and are thus the fine tuners of cytosolic Ca(2+). They belong to the superfamily of P-type ATPases: their four basic isoforms share the essential properties of the reaction cycle and the general membrane topography motif of 10 transmembrane domains and three large cytosolic units. However they also differ in other important properties, e.g., tissue distribution and regulatory mechanisms. Their chief regulator is calmodulin, that removes their C-terminal cytosolic tail from autoinhibitory binding sites next to the active site of the pump, restoring activity. The number of pump isoforms is increased to over 30 by alternative splicing of the transcripts at a N-terminal site (site A) and at site C within the C-terminal calmodulin binding domain: the splice variants are tissue specific and developmentally regulated. The importance of PMCAs in the maintenance of cellular Ca(2+) homeostasis is underlined by the disease phenotypes, genetic or acquired, caused by their malfunction. Non-genetic PMCA deficiencies have long been considered possible causative factors in disease conditions as important as cancer, hypertension, or neurodegeneration. Those of genetic origin are better characterized: some have now been discovered in humans as well. They concern all four PMCA isoforms, and range from cardiac dysfunctions, to deafness, to hypertension, to cerebellar ataxia. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase

    OpenAIRE

    Andrews, Rachel E.; Galileo, Deni S.; Martin-DeLeon, Patricia A.

    2015-01-01

    Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca2+ efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca2+, and that these fertility-modulating proteins are present...

  13. 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

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    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.

  14. A V1143F mutation in the neuronal-enriched isoform 2 of the PMCA pump is linked with ataxia.

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    Vicario, Mattia; Zanni, Ginevra; Vallese, Francesca; Santorelli, Filippo; Grinzato, Alessandro; Cieri, Domenico; Berto, Paola; Frizzarin, Martina; Lopreiato, Raffaele; Zonta, Francesco; Ferro, Stefania; Sandre, Michele; Marin, Oriano; Ruzzene, Maria; Bertini, Enrico; Zanotti, Giuseppe; Brini, Marisa; Calì, Tito; Carafoli, Ernesto

    2018-04-12

    The fine regulation of intracellular calcium is fundamental for all eukaryotic cells. In neurons, Ca 2+ oscillations govern the synaptic development, the release of neurotransmitters and the expression of several genes. Alterations of Ca 2+ homeostasis were found to play a pivotal role in neurodegenerative progression. The maintenance of proper Ca 2+ signaling in neurons demands the continuous activity of Ca 2+ pumps and exchangers to guarantee physiological cytosolic concentration of the cation. The plasma membrane Ca 2+ ATPases (PMCA pumps) play a key role in the regulation of Ca 2+ handling in selected sub-plasma membrane microdomains. Among the four basic PMCA pump isoforms existing in mammals, isoforms 2 and 3 are particularly enriched in the nervous system. In humans, genetic mutations in the PMCA2 gene in association with cadherin 23 mutations have been linked to hearing loss phenotypes, while those occurring in the PMCA3 gene were associated with X-linked congenital cerebellar ataxias. Here we describe a novel missense mutation (V1143F) in the calmodulin binding domain (CaM-BD) of the PMCA2 protein. The mutant pump was present in a patient showing congenital cerebellar ataxia but no overt signs of deafness, in line with the absence of mutations in the cadherin 23 gene. Biochemical and molecular dynamics studies on the mutated PMCA2 have revealed that the V1143F substitution alters the binding of calmodulin to the CaM-BD leading to impaired Ca 2+ ejection. Copyright © 2018. Published by Elsevier Inc.

  15. Multiple Calcium Export Exchangers and Pumps Are a Prominent Feature of Enamel Organ Cells

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    Robertson, Sarah Y. T.; Wen, Xin; Yin, Kaifeng; Chen, Junjun; Smith, Charles E.; Paine, Michael L.

    2017-01-01

    Calcium export is a key function for the enamel organ during all stages of amelogenesis. Expression of a number of ATPase calcium transporting, plasma membrane genes (ATP2B1-4/PMCA1-4), solute carrier SLC8A genes (sodium/calcium exchanger or NCX1-3), and SLC24A gene family members (sodium/potassium/calcium exchanger or NCKX1-6) have been investigated in the developing enamel organ in earlier studies. This paper reviews the calcium export pathways that have been described and adds novel insights to the spatiotemporal expression patterns of PMCA1, PMCA4, and NCKX3 during amelogenesis. New data are presented to show the mRNA expression profiles for the four Atp2b1-4 gene family members (PMCA1-4) in secretory-stage and maturation-stage rat enamel organs. These data are compared to expression profiles for all Slc8a and Slc24a gene family members. PMCA1, PMCA4, and NCKX3 immunolocalization data is also presented. Gene expression profiles quantitated by real time PCR show that: (1) PMCA1, 3, and 4, and NCKX3 are most highly expressed during secretory-stage amelogenesis; (2) NCX1 and 3, and NCKX6 are expressed during secretory and maturation stages; (3) NCKX4 is most highly expressed during maturation-stage amelogenesis; and (4) expression levels of PMCA2, NCX2, NCKX1, NCKX2, and NCKX5 are negligible throughout amelogenesis. In the enamel organ PMCA1 localizes to the basolateral membrane of both secretory and maturation ameloblasts; PMCA4 expression is seen in the basolateral membrane of secretory and maturation ameloblasts, and also cells of the stratum intermedium and papillary layer; while NCKX3 expression is limited to Tomes' processes, and the apical membrane of maturation-stage ameloblasts. These new findings are discussed in the perspective of data already present in the literature, and highlight the multiplicity of calcium export systems in the enamel organ needed to regulate biomineralization. PMID:28588505

  16. Multiple Calcium Export Exchangers and Pumps Are a Prominent Feature of Enamel Organ Cells

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    Sarah Y. T. Robertson

    2017-05-01

    Full Text Available Calcium export is a key function for the enamel organ during all stages of amelogenesis. Expression of a number of ATPase calcium transporting, plasma membrane genes (ATP2B1-4/PMCA1-4, solute carrier SLC8A genes (sodium/calcium exchanger or NCX1-3, and SLC24A gene family members (sodium/potassium/calcium exchanger or NCKX1-6 have been investigated in the developing enamel organ in earlier studies. This paper reviews the calcium export pathways that have been described and adds novel insights to the spatiotemporal expression patterns of PMCA1, PMCA4, and NCKX3 during amelogenesis. New data are presented to show the mRNA expression profiles for the four Atp2b1-4 gene family members (PMCA1-4 in secretory-stage and maturation-stage rat enamel organs. These data are compared to expression profiles for all Slc8a and Slc24a gene family members. PMCA1, PMCA4, and NCKX3 immunolocalization data is also presented. Gene expression profiles quantitated by real time PCR show that: (1 PMCA1, 3, and 4, and NCKX3 are most highly expressed during secretory-stage amelogenesis; (2 NCX1 and 3, and NCKX6 are expressed during secretory and maturation stages; (3 NCKX4 is most highly expressed during maturation-stage amelogenesis; and (4 expression levels of PMCA2, NCX2, NCKX1, NCKX2, and NCKX5 are negligible throughout amelogenesis. In the enamel organ PMCA1 localizes to the basolateral membrane of both secretory and maturation ameloblasts; PMCA4 expression is seen in the basolateral membrane of secretory and maturation ameloblasts, and also cells of the stratum intermedium and papillary layer; while NCKX3 expression is limited to Tomes' processes, and the apical membrane of maturation-stage ameloblasts. These new findings are discussed in the perspective of data already present in the literature, and highlight the multiplicity of calcium export systems in the enamel organ needed to regulate biomineralization.

  17. Calcium pumps of plasma membrane and cell interior

    DEFF Research Database (Denmark)

    Strehler, Emanuel E; Treiman, Marek

    2004-01-01

    Calcium entering the cell from the outside or from intracellular organelles eventually must be returned to the extracellular milieu or to intracellular storage organelles. The two major systems capable of pumping Ca2+ against its large concentration gradient out of the cell or into the sarco....../endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue......- and cell type-specific patterns of expression. Different PMCA and SERCA isoforms are characterized by different regulatory and kinetic properties that likely are optimized for the distinct functional tasks fulfilled by each pump in setting resting cytosolic or intra-organellar Ca2+ levels, and in shaping...

  18. Decreased calcium pump expression in human erythrocytes is connected to a minor haplotype in the ATP2B4 gene.

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    Zámbó, Boglárka; Várady, György; Padányi, Rita; Szabó, Edit; Németh, Adrienn; Langó, Tamás; Enyedi, Ágnes; Sarkadi, Balázs

    2017-07-01

    Plasma membrane Ca 2+ -ATPases are key calcium exporter proteins in most tissues, and PMCA4b is the main calcium transporter in the human red blood cells (RBCs). In order to assess the expression level of PMCA4b, we have developed a flow cytometry and specific antibody binding method to quantitatively detect this protein in the erythrocyte membrane. Interestingly, we found several healthy volunteers showing significantly reduced expression of RBC-PMCA4b. Western blot analysis of isolated RBC membranes confirmed this observation, and indicated that there are no compensatory alterations in other PMCA isoforms. In addition, reduced PMCA4b levels correlated with a lower calcium extrusion capacity in these erythrocytes. When exploring the potential genetic background of the reduced PMCA4b levels, we found no missense mutations in the ATP2B4 coding regions, while a formerly unrecognized minor haplotype in the predicted second promoter region closely correlated with lower erythrocyte PMCA4b protein levels. In recent GWA studies, SNPs in this ATP2B4 haplotype have been linked to reduced mean corpuscular hemoglobin concentrations (MCHC), and to protection against malaria infection. Our data suggest that an altered regulation of gene expression is responsible for the reduced RBC-PMCA4b levels that is probably linked to the development of human disease-related phenotypes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Genome-wide analysis of wheat calcium ATPases and potential role of selected ACAs and ECAs in calcium stress.

    Science.gov (United States)

    Aslam, Roohi; Williams, Lorraine E; Bhatti, Muhammad Faraz; Virk, Nasar

    2017-10-27

    P 2 - type calcium ATPases (ACAs-auto inhibited calcium ATPases and ECAs-endoplasmic reticulum calcium ATPases) belong to the P- type ATPase family of active membrane transporters and are significantly involved in maintaining accurate levels of Ca 2+ , Mn 2+ and Zn 2+ in the cytosol as well as playing a very important role in stress signaling, stomatal opening and closing and pollen tube growth. Here we report the identification and possible role of some of these ATPases from wheat. In this study, ACA and ECA sequences of six species (belonging to Poaceae) were retrieved from different databases and a phylogenetic tree was constructed. A high degree of evolutionary relatedness was observed among P 2 sequences characterized in this study. Members of the respective groups from different plant species were observed to fall under the same clade. This pattern highlights the common ancestry of P 2- type calcium ATPases. Furthermore, qRT-PCR was used to analyse the expression of selected ACAs and ECAs from Triticum aestivum (wheat) under calcium toxicity and calcium deficiency. The data indicated that expression of ECAs is enhanced under calcium stress, suggesting possible roles of these ATPases in calcium homeostasis in wheat. Similarly, the expression of ACAs was significantly different in plants grown under calcium stress as compared to plants grown under control conditions. This gives clues to the role of ACAs in signal transduction during calcium stress in wheat. Here we concluded that wheat genome consists of nine P 2B and three P 2A -type calcium ATPases. Moreover, gene loss events in wheat ancestors lead to the loss of a particular homoeolog of a gene in wheat. To elaborate the role of these wheat ATPases, qRT-PCR was performed. The results indicated that when plants are exposed to calcium stress, both P 2A and P 2B gene expression get enhanced. This further gives clues about the possible role of these ATPases in wheat in calcium management. These findings can be

  20. Excessive signal transduction of gain-of-function variants of the calcium-sensing receptor (CaSR are associated with increased ER to cytosol calcium gradient.

    Directory of Open Access Journals (Sweden)

    Marianna Ranieri

    Full Text Available In humans, gain-of-function mutations of the calcium-sensing receptor (CASR gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA and reducing expression of Plasma Membrane Calcium-ATPase (PMCA. Wild-type CaSR (hCaSR-wt and its gain-of-function (hCaSR-R990G; hCaSR-N124K variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate receptor inputs to cell function.

  1. Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.

    Science.gov (United States)

    Sriram, M; Osipiuk, J; Freeman, B; Morimoto, R; Joachimiak, A

    1997-03-15

    The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes

  2. SwissProt search result: AK100483 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100483 J023097B04 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 7e-23 ...

  3. SwissProt search result: AK120057 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120057 J013000P10 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 1e-88 ...

  4. SwissProt search result: AK120057 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120057 J013000P10 (Q16720) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_HUMAN 2e-89 ...

  5. SwissProt search result: AK105082 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105082 001-046-C08 (Q01814) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_HUMAN 6e-15 ...

  6. SwissProt search result: AK241795 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241795 J065208E19 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 5e-15 ...

  7. SwissProt search result: AK058787 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058787 001-002-E05 (P20020) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_HUMAN 1e-65 ...

  8. SwissProt search result: AK120495 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120495 J013120H18 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 1e-142 ...

  9. SwissProt search result: AK070260 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070260 J023045A10 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 2e-78 ...

  10. SwissProt search result: AK110494 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110494 002-167-C09 (P58165) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) (Fragment) AT2B2_OREMO 2e-24 ...

  11. SwissProt search result: AK100435 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100435 J023088J16 (P20020) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_HUMAN 2e-24 ...

  12. SwissProt search result: AK110177 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110177 002-161-G12 (P11505) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_RAT 7e-20 ...

  13. SwissProt search result: AK100436 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100436 J023088M17 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 1e-166 ...

  14. SwissProt search result: AK100435 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100435 J023088J16 (Q64568) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_RAT 3e-23 ...

  15. SwissProt search result: AK107078 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK107078 002-121-F06 (P20020) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_HUMAN 2e-96 ...

  16. SwissProt search result: AK121509 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121509 J033023K22 (P20020) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_HUMAN 1e-77 ...

  17. SwissProt search result: AK067259 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067259 J013095I07 (P11506) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_RAT 1e-120 ...

  18. SwissProt search result: AK066838 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066838 J013084L23 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 8e-14 ...

  19. SwissProt search result: AK121272 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121272 J023107A16 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 7e-29 ...

  20. SwissProt search result: AK109037 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109037 002-154-C10 (P11506) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_RAT 6e-81 ...

  1. SwissProt search result: AK243441 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243441 J100068F13 (P20020) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_HUMAN 6e-14 ...

  2. SwissProt search result: AK243441 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243441 J100068F13 (Q01814) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_HUMAN 4e-14 ...

  3. SwissProt search result: AK068950 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK068950 J023001P16 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 3e-25 ...

  4. SwissProt search result: AK066864 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066864 J013085P21 (Q16720) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_HUMAN 1e-13 ...

  5. SwissProt search result: AK105840 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105840 001-203-F07 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 2e-28 ...

  6. SwissProt search result: AK067259 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067259 J013095I07 (Q01814) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_HUMAN 1e-119 ...

  7. SwissProt search result: AK063540 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK063540 001-117-C05 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 4e-12 ...

  8. SwissProt search result: AK070260 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070260 J023045A10 (P11505) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_RAT 3e-69 ...

  9. SwissProt search result: AK100435 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100435 J023088J16 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 7e-23 ...

  10. SwissProt search result: AK121509 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121509 J033023K22 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 1e-77 ...

  11. SwissProt search result: AK100878 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100878 J023127C21 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 5e-25 ...

  12. SwissProt search result: AK105840 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105840 001-203-F07 (Q16720) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_HUMAN 2e-28 ...

  13. SwissProt search result: AK100436 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100436 J023088M17 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 1e-168 ...

  14. SwissProt search result: AK241795 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241795 J065208E19 (Q16720) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_HUMAN 6e-15 ...

  15. SwissProt search result: AK242630 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242630 J090022D11 (P20020) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_HUMAN 1e-143 ...

  16. SwissProt search result: AK121250 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121250 J023101G05 (Q64568) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_RAT 1e-179 ...

  17. SwissProt search result: AK121250 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121250 J023101G05 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 1e-178 ...

  18. SwissProt search result: AK100483 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100483 J023097B04 (P58165) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) (Fragment) AT2B2_OREMO 1e-20 ...

  19. SwissProt search result: AK070260 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070260 J023045A10 (Q64568) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_RAT 1e-70 ...

  20. SwissProt search result: AK100483 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100483 J023097B04 (Q01814) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_HUMAN 2e-23 ...

  1. SwissProt search result: AK068950 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK068950 J023001P16 (P11506) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_RAT 2e-24 ...

  2. SwissProt search result: AK241795 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241795 J065208E19 (P11505) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_RAT 3e-15 ...

  3. SwissProt search result: AK072909 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072909 J023148A19 (Q01814) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_HUMAN 6e-23 ...

  4. SwissProt search result: AK072909 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072909 J023148A19 (P58165) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) (Fragment) AT2B2_OREMO 3e-20 ...

  5. SwissProt search result: AK070064 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070064 J023041B03 (P20020) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_HUMAN 1e-135 ...

  6. SwissProt search result: AK100415 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100415 J023087M18 (Q64568) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_RAT 1e-179 ...

  7. SwissProt search result: AK100483 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100483 J023097B04 (Q16720) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_HUMAN 9e-23 ...

  8. SwissProt search result: AK072909 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072909 J023148A19 (Q16720) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_HUMAN 3e-24 ...

  9. SwissProt search result: AK121509 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121509 J033023K22 (P11505) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_RAT 4e-78 ...

  10. SwissProt search result: AK105082 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105082 001-046-C08 (P11505) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_RAT 3e-16 ...

  11. SwissProt search result: AK065088 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065088 J013001L20 (P11505) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_RAT 1e-166 ...

  12. SwissProt search result: AK100415 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100415 J023087M18 (Q01814) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_HUMAN 1e-146 ...

  13. SwissProt search result: AK066259 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066259 J013059H11 (P11506) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_RAT 1e-143 ...

  14. SwissProt search result: AK073066 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK073066 J033023L01 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 5e-22 ...

  15. SwissProt search result: AK068950 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK068950 J023001P16 (P20020) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_HUMAN 4e-25 ...

  16. SwissProt search result: AK110494 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110494 002-167-C09 (Q16720) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_HUMAN 6e-27 ...

  17. SwissProt search result: AK109037 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109037 002-154-C10 (P11505) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_RAT 3e-81 ...

  18. SwissProt search result: AK066864 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066864 J013085P21 (P58165) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) (Fragment) AT2B2_OREMO 4e-13 ...

  19. SwissProt search result: AK100415 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100415 J023087M18 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 1e-178 ...

  20. SwissProt search result: AK073220 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK073220 J033000K23 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 1e-34 ...

  1. SwissProt search result: AK067535 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK067535 J013109J17 (P23634) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_HUMAN 2e-11 ...

  2. SwissProt search result: AK121509 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121509 J033023K22 (Q16720) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_HUMAN 6e-78 ...

  3. SwissProt search result: AK070260 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070260 J023045A10 (P11506) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_RAT 5e-58 ...

  4. SwissProt search result: AK071938 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK071938 J013083I02 (Q64568) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_RAT 1e-100 ...

  5. SwissProt search result: AK100415 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100415 J023087M18 (P11505) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_RAT 1e-179 ...

  6. SwissProt search result: AK243441 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243441 J100068F13 (P11505) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_RAT 4e-14 ...

  7. SwissProt search result: AK107078 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK107078 002-121-F06 (P11505) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_RAT 1e-96 ...

  8. SwissProt search result: AK065088 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065088 J013001L20 (P20020) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_HUMAN 1e-166 ...

  9. SwissProt search result: AK068950 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK068950 J023001P16 (Q01814) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_HUMAN 6e-25 ...

  10. SwissProt search result: AK110089 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110089 002-160-G08 (P58165) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) (Fragment) AT2B2_OREMO 1e-60 ...

  11. SwissProt search result: AK100436 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100436 J023088M17 (P58165) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) (Fragment) AT2B2_OREMO 1e-144 ...

  12. SwissProt search result: AK243441 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243441 J100068F13 (P11506) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_RAT 4e-14 ...

  13. SwissProt search result: AK058787 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK058787 001-002-E05 (Q64568) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_RAT 9e-64 ...

  14. SwissProt search result: AK120057 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120057 J013000P10 (P58165) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) (Fragment) AT2B2_OREMO 4e-88 ...

  15. SwissProt search result: AK072909 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072909 J023148A19 (P11506) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_RAT 1e-22 ...

  16. SwissProt search result: AK110020 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110020 002-159-H09 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 9e-24 ...

  17. SwissProt search result: AK066259 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066259 J013059H11 (Q64568) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_RAT 1e-179 ...

  18. SwissProt search result: AK073066 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK073066 J033023L01 (P58165) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) (Fragment) AT2B2_OREMO 6e-20 ...

  19. SwissProt search result: AK100483 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100483 J023097B04 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 2e-22 ...

  20. SwissProt search result: AK242894 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242894 J090080L10 (Q64542) Plasma membrane calcium-transporting ATPase 4 (EC 3.6....3.8) (PMCA4) (Plasma membrane calcium pump isoform 4) (Plasma membrane calcium ATPase isoform 4) AT2B4_RAT 2e-20 ...

  1. SwissProt search result: AK100435 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100435 J023088J16 (P58165) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) (Fragment) AT2B2_OREMO 9e-20 ...

  2. SwissProt search result: AK120057 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120057 J013000P10 (Q64568) Plasma membrane calcium-transporting ATPase 3 (EC 3.6....3.8) (PMCA3) (Plasma membrane calcium pump isoform 3) (Plasma membrane calcium ATPase isoform 3) AT2B3_RAT 1e-90 ...

  3. SwissProt search result: AK071938 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK071938 J013083I02 (P58165) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) (Fragment) AT2B2_OREMO 2e-97 ...

  4. SwissProt search result: AK121250 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121250 J023101G05 (P20020) Plasma membrane calcium-transporting ATPase 1 (EC 3.6....3.8) (PMCA1) (Plasma membrane calcium pump isoform 1) (Plasma membrane calcium ATPase isoform 1) AT2B1_HUMAN 1e-178 ...

  5. SwissProt search result: AK242894 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK242894 J090080L10 (Q9R0K7) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_MOUSE 1e-18 ...

  6. SwissProt search result: AK243441 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243441 J100068F13 (Q9R0K7) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_MOUSE 4e-14 ...

  7. SwissProt search result: AK105082 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105082 001-046-C08 (Q9R0K7) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_MOUSE 5e-15 ...

  8. SwissProt search result: AK071938 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK071938 J013083I02 (Q9R0K7) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_MOUSE 1e-99 ...

  9. SwissProt search result: AK121509 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK121509 J033023K22 (Q9R0K7) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_MOUSE 3e-81 ...

  10. SwissProt search result: AK110494 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110494 002-167-C09 (Q9R0K7) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_MOUSE 2e-29 ...

  11. SwissProt search result: AK120495 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120495 J013120H18 (Q9R0K7) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_MOUSE 1e-161 ...

  12. SwissProt search result: AK109037 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109037 002-154-C10 (Q9R0K7) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_MOUSE 2e-81 ...

  13. SwissProt search result: AK065088 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065088 J013001L20 (Q9R0K7) Plasma membrane calcium-transporting ATPase 2 (EC 3.6....3.8) (PMCA2) (Plasma membrane calcium pump isoform 2) (Plasma membrane calcium ATPase isoform 2) AT2B2_MOUSE 1e-170 ...

  14. Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

    International Nuclear Information System (INIS)

    Rocafull, Miguel A.; Thomas, Luz E.; Barrera, Girolamo J.; Castillo, Jesus R. del

    2010-01-01

    P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca 2+ , Na + , K + and H + ), have been reported. They include reticulum and plasma-membrane Ca 2+ -ATPases, Na + /K + -ATPase and H + /K + -ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg 2+ ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na + /K + -ATPase α1-isoform, H + /K + -ATPase α2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H + /K + -ATPase α2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

  15. Letrozole induced low estrogen levels affected the expressions of duodenal and renal calcium-processing gene in laying hens.

    Science.gov (United States)

    Li, Qiao; Zhao, Xingkai; Wang, Shujie; Zhou, Zhenlei

    2018-01-01

    Estrogen regulates the calcium homeostasis in hens, but the mechanisms involved are still unclear fully. In this study, we investigated whether letrozole (LZ) induced low estrogen levels affected the calcium absorption and transport in layers. In the duodenum, we observed a significant decrease of mRNA expressions of Calbindin-28k (CaBP-28k) and plasma membrane Ca 2+ -ATPase (PMCA 1b) while CaBP-28k protein expression was declined in birds with LZ treatment, and the mRNA levels of duodenal transient receptor potential vanilloid 6 (TRPV6) and Na + /Ca 2+ exchanger 1 (NCX1) were not affected. Interestingly, we observed the different changes in the kidney. The renal mRNA expressions of TRPV6 and NCX1 were unregulated while the PMCA1b was down-regulated in low estrogen layers, however, the CaBP-28k gene and protein expressions were no changed in the kidney. Furthermore, it showed that the duodenal estradiol receptor 2 (ESR2) transcripts rather than parathyroid hormone 1 receptor (PTH1R) and calcitonin receptor (CALCR) played key roles to down-regulate calcium transport in LZ-treated birds. In conclusion, CaBP-28k, PMCA 1b and ESR2 genes in the duodenum may be primary targets for estrogen regulation in order to control calcium homeostasis in hens. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

    International Nuclear Information System (INIS)

    Thatte, H.S.; Mickelson, J.R.; Addis, P.B.; Louis, C.F.

    1987-01-01

    To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45 Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration

  17. Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

    Energy Technology Data Exchange (ETDEWEB)

    Rocafull, Miguel A., E-mail: mrocaful@ivic.ve [Lab. Fisiologia Molecular, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of); Thomas, Luz E.; Barrera, Girolamo J.; Castillo, Jesus R. del [Lab. Fisiologia Molecular, Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (IVIC), Apartado 20632, Caracas 1020-A (Venezuela, Bolivarian Republic of)

    2010-01-01

    P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca{sup 2+}, Na{sup +}, K{sup +} and H{sup +}), have been reported. They include reticulum and plasma-membrane Ca{sup 2+}-ATPases, Na{sup +}/K{sup +}-ATPase and H{sup +}/K{sup +}-ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg{sup 2+}ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na{sup +}/K{sup +}-ATPase {alpha}1-isoform, H{sup +}/K{sup +}-ATPase {alpha}2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H{sup +}/K{sup +}-ATPase {alpha}2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

  18. A New Baltic Population-Specific Human Genetic Marker in the PMCA4 Gene.

    Science.gov (United States)

    Stavusis, Janis; Inashkina, Inna; Lace, Baiba; Pelnena, Dita; Limborska, Svetlana; Khrunin, Andrey; Kucinskas, Vaidutis; Krumina, Astrida; Piekuse, Linda; Zorn, Branko; Fodina, Violeta; Punab, Margus; Erenpreiss, Juris

    2016-01-01

    The PMCA gene family consists of 4 genes and at least 21 splice variants; among these, the Ca2+ ATPase 4 (PMCA4) gene encodes a plasma membrane protein abundantly expressed in several tissues, including the kidney, heart, and sperm. Knockout of PMCA4 causes infertility due to immotile sperm in mouse models. We therefore investigated variants in this gene for potential association with infertility in groups of Estonian (n = 191) and Latvian (n = 92) men with reduced sperm motility. All exons, exon-intron boundaries, 5' and 3' untranslated regions, and the promoter region of the PMCA4 gene were analysed by direct sequencing for a group of Estonian infertile men. Genotyping of guanine and adenine alleles of rs147729934 was performed, using a custom-designed TaqMan® probe for a group of Latvian infertile men as well as additional groups from Latvia and several groups of people with proven ethnicity from the Baltic region. Although we did not identify any significant associations between variants in the gene and infertility, our results indicated that in all studied Latvian and Estonian groups the adenine allele of the variant rs147729934 was present at a higher frequency than expected. Analysis of additional samples indicated that the adenine allele of rs147729934 likely originated once in the modern-day Baltic or western Russia area, as the frequency of the minor adenine allele observed in this region is remarkably higher than that in the general European population. Our results revealed no significant difference in frequencies of genetic variants in PMCA4 gene between men with normal and those with reduced sperm motility. The adenine allele of the variant rs147729934 is potentially an informative tool for future population studies concerning ancient Baltic and Finno-Ugric history. © 2017 S. Karger AG, Basel.

  19. Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a during murine estrus: association with oviductal exosomes and uptake in sperm.

    Directory of Open Access Journals (Sweden)

    Amal A Al-Dossary

    Full Text Available PMCA4, a membrane protein, is the major Ca(2+ efflux pump in murine sperm where its deletion leads to a severe loss of hyperactivated motility and to male infertility. We have previously shown that the PMCA4b splice variant interacts with CASK (Ca(2+/CaM-dependent serine kinase in regulating sperm Ca(2+. More recently we detected that PMCA4a isoform, in addition to its presence in testis, is secreted in the epididymal luminal fluid and transferred to sperm. Here we show that Pmca4 mRNA is expressed in both the 4a and 4b variants in the vagina, uterus, and oviduct. Immunofluorescence reveals that PMCA4a is similarly expressed and is elevated during estrus, appearing in the glandular and luminal epithelia. Western analysis detected PMCA4a in all tissues and in the luminal fluids (LF of the vagina (VLF, uterus (ULF, and the oviduct (OLF collected during estrus. It was ~9- and 4-fold higher in OLF than in VLF and ULF, and only marginally present in LF collected at metestrus/diestrus. Fractionation of the LF collected at estrus, via ultracentrifugation, revealed that 100% of the PMCA4a resides in the vesicular fraction of the ULF and OLF. Transmission electron microscopy (TEM revealed that OLF vesicles have an exosomal orientation (with the cytoplasmic-side inward, a size range of 25-100 nm, with the characteristic CD9 biomarker. Thus, we dubbed these vesicles "oviductosomes", to which PMCA4a was immunolocalized. Incubation of caudal sperm in the combined LF or exosomes resulted in up to a ~3-fold increase of sperm PMCA4a, as detected by flow cytometry, indicating in vitro uptake. Our results are consistent with the increased requirement of Ca(2+ efflux in the oviduct. They show for the first time the presence of oviductal exosomes and highlight their role, along with uterosomes and vaginal exosomes, in post-testicular sperm acquisition of PMCA4a which is essential for hyperactivated motility and fertility.

  20. Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm.

    Science.gov (United States)

    Al-Dossary, Amal A; Strehler, Emanuel E; Martin-Deleon, Patricia A

    2013-01-01

    PMCA4, a membrane protein, is the major Ca(2+) efflux pump in murine sperm where its deletion leads to a severe loss of hyperactivated motility and to male infertility. We have previously shown that the PMCA4b splice variant interacts with CASK (Ca(2+/)CaM-dependent serine kinase) in regulating sperm Ca(2+). More recently we detected that PMCA4a isoform, in addition to its presence in testis, is secreted in the epididymal luminal fluid and transferred to sperm. Here we show that Pmca4 mRNA is expressed in both the 4a and 4b variants in the vagina, uterus, and oviduct. Immunofluorescence reveals that PMCA4a is similarly expressed and is elevated during estrus, appearing in the glandular and luminal epithelia. Western analysis detected PMCA4a in all tissues and in the luminal fluids (LF) of the vagina (VLF), uterus (ULF), and the oviduct (OLF) collected during estrus. It was ~9- and 4-fold higher in OLF than in VLF and ULF, and only marginally present in LF collected at metestrus/diestrus. Fractionation of the LF collected at estrus, via ultracentrifugation, revealed that 100% of the PMCA4a resides in the vesicular fraction of the ULF and OLF. Transmission electron microscopy (TEM) revealed that OLF vesicles have an exosomal orientation (with the cytoplasmic-side inward), a size range of 25-100 nm, with the characteristic CD9 biomarker. Thus, we dubbed these vesicles "oviductosomes", to which PMCA4a was immunolocalized. Incubation of caudal sperm in the combined LF or exosomes resulted in up to a ~3-fold increase of sperm PMCA4a, as detected by flow cytometry, indicating in vitro uptake. Our results are consistent with the increased requirement of Ca(2+) efflux in the oviduct. They show for the first time the presence of oviductal exosomes and highlight their role, along with uterosomes and vaginal exosomes, in post-testicular sperm acquisition of PMCA4a which is essential for hyperactivated motility and fertility.

  1. In vitro effects of toxaphene on mitochondrial calcium ATPase and calcium uptake in selected rat tissues

    International Nuclear Information System (INIS)

    Trottman, C.H.; Rao, K.S.P.; Morrow, W.; Uzodinma, J.E.; Desaiah, D.

    1985-01-01

    In vitro effects of toxaphene on Ca 2+ -ATPase activity and 45 Ca 2+ -uptake were studied in mitochondrial fractions of heart, kidney and liver tissues of rat. Mitochondrial fractions were prepared by the conventional centrifugation method. Ca 2+ -ATPase activity was determined by measuring the inorganic phosphate liberated during ATP hydrolysis. Toxaphene inhibited Ca 2+ -ATPase in a concentration dependent manner in all the three tissues. Substrate activation kinetics, with heart, kidney and liver tissue fractions, revealed that toxaphene inhibited Ca 2+ -ATPase activity non-competetively by decreasing the maximum velocity of the enzyme without affecting the enzyme-substrate affinity. Toxaphene also inhibited mitochondrial 45 Ca 2+ -uptake in the three selected tissues in a concentration dependent manner. These results indicate that toxaphene is an inhibitor of mitochondrial Ca 2+ -ATPase and calcium transport in heart, kidney and liver tissues of rat. 19 references, 5 figures

  2. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    DEFF Research Database (Denmark)

    Tidow, Henning; Hein, Kim Langmach; Palmgren, Michael Broberg

    2010-01-01

    Plasma-membrane Ca2+-ATPases (PMCAs) are calcium pumps that expel Ca2+ from eukaryotic cells to maintain overall Ca2+ homoeostasis and to provide local control of intracellular Ca2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for p...... group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 Å, = 113.2°. A complete data set was collected to 3.0 Å resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin...

  3. Plasma Membrane Ca2+-ATPase 4 in Murine Epididymis: Secretion of Splice Variants in the Luminal Fluid and a Role in Sperm Maturation1

    OpenAIRE

    Patel, Ramkrishna; Al-Dossary, Amal A.; Stabley, Deborah L.; Barone, Carol; Galileo, Deni S.; Strehler, Emanuel E.; Martin-DeLeon, Patricia A.

    2013-01-01

    Plasma membrane Ca2+-ATPase isoform 4 (PMCA4) is the primary Ca2+ efflux pump in murine sperm, where it regulates motility. In Pmca4 null sperm, motility loss results in infertility. We have shown that murine sperm PMCA4b interacts with Ca2+/CaM-dependent serine kinase (CASK) in regulating Ca2+ homeostasis and motility. However, recent work indicated that the bovine PMCA4a splice variant (missing in testis) is epididymally expressed, along with 4b, and may be transferred to sperm. Here we sho...

  4. Zinc oxide nanoparticles decrease the expression and activity of plasma membrane calcium ATPase, disrupt the intracellular calcium homeostasis in rat retinal ganglion cells.

    Science.gov (United States)

    Guo, Dadong; Bi, Hongsheng; Wang, Daoguang; Wu, Qiuxin

    2013-08-01

    Zinc oxide nanoparticle is one of the most important materials with diverse applications. However, it has been reported that zinc oxide nanoparticles are toxic to organisms, and that oxidative stress is often hypothesized to be an important factor in cytotoxicity mediated by zinc oxide nanoparticles. Nevertheless, the mechanism of toxicity of zinc oxide nanoparticles has not been completely understood. In this study, we investigated the cytotoxic effect of zinc oxide nanoparticles and the possible molecular mechanism involved in calcium homeostasis mediated by plasma membrane calcium ATPase in rat retinal ganglion cells. Real-time cell electronic sensing assay showed that zinc oxide nanoparticles could exert cytotoxic effect on rat retinal ganglion cells in a concentration-dependent manner; flow cytometric analysis indicated that zinc oxide nanoparticles could lead to cell damage by inducing the overproduction of reactive oxygen species. Furthermore, zinc oxide nanoparticles could also apparently decrease the expression level and their activity of plasma membrane calcium ATPase, which finally disrupt the intracellular calcium homeostasis and result in cell death. Taken together, zinc oxide nanoparticles could apparently decrease the plasma membrane calcium ATPase expression, inhibit their activity, cause the elevated intracellular calcium ion level and disrupt the intracellular calcium homeostasis. Further, the disrupted calcium homeostasis will trigger mitochondrial dysfunction, generate excessive reactive oxygen species, and finally initiate cell death. Thus, the disrupted calcium homeostasis is involved in the zinc oxide nanoparticle-induced rat retinal ganglion cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Unilateral vestibular deafferentation-induced changes in calcium signaling-related molecules in the rat vestibular nuclear complex.

    Science.gov (United States)

    Masumura, Chisako; Horii, Arata; Mitani, Kenji; Kitahara, Tadashi; Uno, Atsuhiko; Kubo, Takeshi

    2007-03-23

    Inquiries into the neurochemical mechanisms of vestibular compensation, a model of lesion-induced neuronal plasticity, reveal the involvement of both voltage-gated Ca(2+) channels (VGCC) and intracellular Ca(2+) signaling. Indeed, our previous microarray analysis showed an up-regulation of some calcium signaling-related genes such as the alpha2 subunit of L-type calcium channels, calcineurin, and plasma membrane Ca(2+) ATPase 1 (PMCA1) in the ipsilateral vestibular nuclear complex (VNC) following unilateral vestibular deafferentation (UVD). To further elucidate the role of calcium signaling-related molecules in vestibular compensation, we used a quantitative real-time polymerase chain reaction (PCR) method to confirm the microarray results and investigated changes in expression of these molecules at various stages of compensation (6 h to 2 weeks after UVD). We also investigated the changes in gene expression during Bechterew's phenomenon and the effects of a calcineurin inhibitor on vestibular compensation. Real-time PCR showed that genes for the alpha2 subunit of VGCC, PMCA2, and calcineurin were transiently up-regulated 6 h after UVD in ipsilateral VNC. A subsequent UVD, which induced Bechterew's phenomenon, reproduced a complete mirror image of the changes in gene expressions of PMCA2 and calcineurin seen in the initial UVD, while the alpha2 subunit of VGCC gene had a trend to increase in VNC ipsilateral to the second lesion. Pre-treatment by FK506, a calcineurin inhibitor, decelerated the vestibular compensation in a dose-dependent manner. Although it is still uncertain whether these changes in gene expression are causally related to the molecular mechanisms of vestibular compensation, this observation suggests that after increasing the Ca(2+) influx into the ipsilateral VNC neurons via up-regulated VGCC, calcineurin may be involved in their synaptic plasticity. Conversely, an up-regulation of PMCA2, a brain-specific Ca(2+) pump, would increase an efflux of Ca

  6. Function of endoplasmic reticulum calcium ATPase in innate immunity-mediated programmed cell death

    Science.gov (United States)

    Zhu, Xiaohong; Caplan, Jeffrey; Mamillapalli, Padmavathi; Czymmek, Kirk; Dinesh-Kumar, Savithramma P

    2010-01-01

    Programmed cell death (PCD) initiated at the pathogen-infected sites during the plant innate immune response is thought to prevent the development of disease. Here, we describe the identification and characterization of an ER-localized type IIB Ca2+-ATPase (NbCA1) that function as a regulator of PCD. Silencing of NbCA1 accelerates viral immune receptor N- and fungal-immune receptor Cf9-mediated PCD, as well as non-host pathogen Pseudomonas syringae pv. tomato DC3000 and the general elicitor cryptogein-induced cell death. The accelerated PCD rescues loss-of-resistance phenotype of Rar1, HSP90-silenced plants, but not SGT1-silenced plants. Using a genetically encoded calcium sensor, we show that downregulation of NbCA1 results in the modulation of intracellular calcium signalling in response to cryptogein elicitor. We further show that NbCAM1 and NbrbohB function as downstream calcium decoders in N-immune receptor-mediated PCD. Our results indicate that ER-Ca2+-ATPase is a component of the calcium efflux pathway that controls PCD during an innate immune response. PMID:20075858

  7. Homer2 protein regulates plasma membrane Ca²⁺-ATPase-mediated Ca²⁺ signaling in mouse parotid gland acinar cells.

    Science.gov (United States)

    Yang, Yu-Mi; Lee, Jiae; Jo, Hae; Park, Soonhong; Chang, Inik; Muallem, Shmuel; Shin, Dong Min

    2014-09-05

    Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca(2+)-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca(2+) signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca(2+) signaling in parotid gland acinar cells using Homer2-deficient (Homer2(-/-)) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca(2+)-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca(2+)-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca(2+) extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca(2+) clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca(2+) signaling in parotid acinar cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase.

    Science.gov (United States)

    Andrews, Rachel E; Galileo, Deni S; Martin-DeLeon, Patricia A

    2015-11-01

    Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca(2+) efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca(2+), and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca(2+) ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca(2+)-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca(2+) concentration ([Ca(2+)]c) in capacitated sperm than at low [Ca(2+)]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca(2+)/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at

  9. Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b.

    Science.gov (United States)

    Tran, Quang-Kim; VerMeer, Mark; Burgard, Michelle A; Hassan, Ali B; Giles, Jennifer

    2015-05-22

    The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca(2+)-ATPase (PMCA) is essential for removal of cytoplasmic Ca(2+) and for shaping the time courses of Ca(2+)-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca(2+) extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca(2+) signaling and GPER/GPR30-mediated activities. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Deletion of the Intestinal Plasma Membrane Calcium Pump, Isoform 1, Atp2b1, in Mice is Associated with Decreased Bone Mineral Density and Impaired Responsiveness to 1, 25-Dihydroxyvitamin D3

    Science.gov (United States)

    Ryan, Zachary C.; Craig, Theodore A.; Filoteo, Adelaida G.; Westendorf, Jennifer J.; Cartwright, Elizabeth J.; Neyses, Ludwig; Strehler, Emanuel E.; Kumar, Rajiv

    2016-01-01

    The physiological importance of the intestinal plasma membrane calcium pump, isoform 1, (Pmca1, Atp2b1), in calcium absorption and homeostasis has not been previously demonstrated in vivo. Since global germ-line deletion of the Pmca1 in mice is associated with embryonic lethality, we selectively deleted the Pmca1 in intestinal absorptive cells. Mice with loxP sites flanking exon 2 of the Pmca1 gene (Pmca1fl/fl) were crossed with mice expressing Cre recombinase in the intestine under control of the villin promoter to give mice in which the Pmca1 had been deleted in the intestine (Pmca1EKO mice). Pmca1EKO mice were born at a reduced frequency and were small at the time of birth when compared to wild-type (Wt) litter mates. At two months of age, Pmca1EKO mice fed a 0.81% calcium, 0.34% phosphorus, normal vitamin D diet had reduced whole body bone mineral density (P <0.037), and reduced femoral bone mineral density (P <0.015). There was a trend towards lower serum calcium and higher serum parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) concentrations in Pmca1EKO mice compared to Wt mice but the changes were not statistically significant. The urinary phosphorus/creatinine ratio was increased in Pmca1EKO mice (P <0.004). Following the administration of 200 ng of 1α,25(OH)2D3 intraperitoneally to Wt mice, active intestinal calcium transport increased ∼2-fold, whereas Pmca1EKO mice administered an equal amount of 1α,25(OH)2D3 failed to show an increase in active calcium transport. Deletion of the Pmca1 in the intestine is associated with reduced growth and bone mineralization, and a failure to up-regulate calcium absorption in response to 1α,25(OH)2D3. PMID:26392310

  11. Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts.

    Science.gov (United States)

    Reisner, P D; Brandt, P C; Vanaman, T C

    1997-01-01

    It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.

  12. Plasma membrane Ca2+-ATPase 4 in murine epididymis: secretion of splice variants in the luminal fluid and a role in sperm maturation.

    Science.gov (United States)

    Patel, Ramkrishna; Al-Dossary, Amal A; Stabley, Deborah L; Barone, Carol; Galileo, Deni S; Strehler, Emanuel E; Martin-DeLeon, Patricia A

    2013-07-01

    Plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4) is the primary Ca(2+) efflux pump in murine sperm, where it regulates motility. In Pmca4 null sperm, motility loss results in infertility. We have shown that murine sperm PMCA4b interacts with Ca(2+)/CaM-dependent serine kinase (CASK) in regulating Ca(2+) homeostasis and motility. However, recent work indicated that the bovine PMCA4a splice variant (missing in testis) is epididymally expressed, along with 4b, and may be transferred to sperm. Here we show, via conventional and in situ RT-PCR, that both the splice variants of Pmca4 mRNA are expressed in murine testis and throughout the epididymis. Immunofluorescence localized PMCA4a to the apical membrane of the epididymal epithelium, and Western analysis not only confirmed its presence but showed for the first time that PMCA4a and PMCA4b are secreted in the epididymal luminal fluid (ELF), from which epididymosomes containing PMCA4a were isolated. Flow cytometry indicated the presence of PMCA4a on mature caudal sperm where it was increased ~5-fold compared to caput sperm (detected by Western blotting) and ~2-fold after incubation in ELF, revealing in vitro uptake and implicating PMCA4a in epididymal sperm maturation. Coimmunoprecipitation using pan-PMCA4 antibodies, revealed that both variants associate with CASK, suggesting their presence in a complex. Because they have different kinetic properties for Ca(2+) transport and different abilities to bind to CASK, our study suggests a mechanism for combining the functional attributes of both PMCA4 variants, leading to heightened efficiency of the pump in the maintenance of Ca(2+) homeostasis, which is crucial for normal motility and male fertility.

  13. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    International Nuclear Information System (INIS)

    Tidow, Henning; Hein, Kim L.; Baekgaard, Lone; Palmgren, Michael G.; Nissen, Poul

    2010-01-01

    Plant plasma-membrane Ca 2+ -ATPase is regulated via binding of calmodulin to its autoinhibitory N-terminal domain. In this study, the expression, purification, crystallization and preliminary X-ray diffraction analysis of this protein complex from A. thaliana are reported. Plasma-membrane Ca 2+ -ATPases (PMCAs) are calcium pumps that expel Ca 2+ from eukaryotic cells to maintain overall Ca 2+ homoeostasis and to provide local control of intracellular Ca 2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for presynaptic and postsynaptic Ca 2+ regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium-bound calmodulin (Ca 2+ -CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca 2+ -ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 Å, β = 113.2°. A complete data set was collected to 3.0 Å resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin

  14. N-Acetylcysteine-induced vasodilatation is modulated by KATP channels, Na+/K+-ATPase activity and intracellular calcium concentration: An in vitro study.

    Science.gov (United States)

    Vezir, Özden; Çömelekoğlu, Ülkü; Sucu, Nehir; Yalın, Ali Erdinç; Yılmaz, Şakir Necat; Yalın, Serap; Söğüt, Fatma; Yaman, Selma; Kibar, Kezban; Akkapulu, Merih; Koç, Meryem İlkay; Seçer, Didem

    2017-08-01

    In this study, we aimed to investigate the role of ATP-sensitive potassium (K ATP ) channel, Na + /K + -ATPase activity, and intracellular calcium levels on the vasodilatory effect of N-acetylcysteine (NAC) in thoracic aorta by using electrophysiological and molecular techniques. Rat thoracic aorta ring preparations and cultured thoracic aorta cells were divided into four groups as control, 2mM NAC, 5mM NAC, and 10mM NAC. Thoracic aorta rings were isolated from rats for measurements of relaxation responses and Na + /K + -ATPase activity. In the cultured thoracic aorta cells, we measured the currents of K ATP channel, the concentration of intracellular calcium and mRNA expression level of K ATP channel subunits (KCNJ8, KCNJ11, ABCC8 and ABCC9). The relaxation rate significantly increased in all NAC groups compared to control. Similarly, Na + /K + - ATPase activity also significantly decreased in NAC groups. Outward K ATP channel current significantly increased in all NAC groups compared to the control group. Intracellular calcium concentration decreased significantly in all groups with compared control. mRNA expression level of ABCC8 subunit significantly increased in all NAC groups compared to the control group. Pearson correlation analysis showed that relaxation rate was significantly associated with K ATP current, intracellular calcium concentration, Na + /K + -ATPase activity and mRNA expression level of ABCC8 subunit. Our findings suggest that NAC relaxes vascular smooth muscle cells through a direct effect on K ATP channels, by increasing outward K+ flux, partly by increasing mRNA expression of K ATP subunit ABCC8, by decreasing in intracellular calcium and by decreasing in Na + /K + -ATPase activity. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  15. Development and Validation of the Pediatric Medical Complexity Algorithm (PMCA) Version 3.0.

    Science.gov (United States)

    Simon, Tamara D; Haaland, Wren; Hawley, Katherine; Lambka, Karen; Mangione-Smith, Rita

    2018-02-26

    To modify the Pediatric Medical Complexity Algorithm (PMCA) to include both International Classification of Diseases, Ninth and Tenth Revisions, Clinical Modification (ICD-9/10-CM) codes for classifying children with chronic disease (CD) by level of medical complexity and to assess the sensitivity and specificity of the new PMCA version 3.0 for correctly identifying level of medical complexity. To create version 3.0, PMCA version 2.0 was modified to include ICD-10-CM codes. We applied PMCA version 3.0 to Seattle Children's Hospital data for children with ≥1 emergency department (ED), day surgery, and/or inpatient encounter from January 1, 2016, to June 30, 2017. Starting with the encounter date, up to 3 years of retrospective discharge data were used to classify children as having complex chronic disease (C-CD), noncomplex chronic disease (NC-CD), and no CD. We then selected a random sample of 300 children (100 per CD group). Blinded medical record review was conducted to ascertain the levels of medical complexity for these 300 children. The sensitivity and specificity of PMCA version 3.0 was assessed. PMCA version 3.0 identified children with C-CD with 86% sensitivity and 86% specificity, children with NC-CD with 65% sensitivity and 84% specificity, and children without CD with 77% sensitivity and 93% specificity. PMCA version 3.0 is an updated publicly available algorithm that identifies children with C-CD, who have accessed tertiary hospital emergency department, day surgery, or inpatient care, with very good sensitivity and specificity when applied to hospital discharge data and with performance to earlier versions of PMCA. Copyright © 2018 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.

  16. [Effects of desulfurization waste on calcium distribution, Ca(2+)-ATPase activity, and antioxidant characteristics of rice leaf under alkali stress].

    Science.gov (United States)

    Mao, Gui-Lian; Xu, Xing; Zeng, Jin; Yue, Zi-Hui; Yang, Shu-Juan

    2012-02-01

    To approach the action mechanisms of desulfurization waste on alleviating alkali stress-induced injury of rice, a pot experiment was conducted to study the variations of leaf total calcium content, calcium distribution, plasma membrane Ca(2+)-ATPase activity, and reactive oxygen content of rice seedlings under alkali stress after the application of desulfurization waste. In the control, a few calcium particulates scattered in the cell wall and chloroplasts, while applying desulfurization waste or CaSO4 increased the calcium particulates in the plasma membrane, intercellular space, cell wall, and vacuole significantly. With the increasing application rate of desulfurization waste or CaSO4, the leaf total calcium content increased, Ca(2+)-ATPase activity in plasma membrane and tonoplast presented an increasing trend, plasma membrane relative permeability, MDA content, and O2 production rate decreased, and SOD and POD activities increased. The desulfurization waste could relieve the alkali stress to rice in some extent, and the main reactive compound in the waste could be CaSO4.

  17. Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer.

    Science.gov (United States)

    Blackwell, Daniel J; Zak, Taylor J; Robia, Seth L

    2016-09-20

    The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  18. Effects of benzo(a)pyrene exposure on the ATPase activity and calcium concentration in the hippocampus of neonatal rats.

    Science.gov (United States)

    Yang, Kai; Chen, Chengzhi; Cheng, Shuqun; Cao, Xianqing; Tu, Baijie

    2017-03-30

    To investigate whether postnatal benzo(a)pyrene (B(a)P) exposure caused the impairments on the process of neurodevelopment and the alteration in the calcium medium in the neonatal rats. Eighty neonatal Sprague Dawley (SD) rats were randomly divided into 5 groups (untreated control group, vehicle group, 0.02 mg/kg, 0.2 mg/kg and 2 mg/kg B(a)P-exposed group). Rats were treated with B(a)P by the intragastric administration from postnatal day (PND) 4 to 25. Morris water maze (MWM) was employed to observe the spatial memory of rats. The activity of calcium adenosine triphosphatase (Ca2+-ATPase), sodium-potassium adenosine triphosphatase (Na+-K+-ATPase) and calcium-magnesium adenosine triphosphatase (Ca2+-Mg2+-ATPase) in the hippocampus were detected by commercial kits. Fura-2 pentakis(acetoxymethyl) (Fura-2/AM) probe and reactive oxygen species (ROS) reagent kit were used for measuring the concentration of Ca2+ and ROS in the hippocampus synapse, respectively. Rats exposed to B(a)P resulted in the deficits in the spatial memory manifested by the increased escape latency and decreased number of crossing platform and time spent in target quadrant in comparison with the control groups. Benzo(a)pyrene exposure caused the significant decrease in the ATPase activity in the hippocampus and caused Ca2+ overload in the synaptic, besides, the ROS concentration increased significantly which may further induce neurobehavioral impairment of the neonatal rats. Our findings suggest that postnatal B(a)P exposure may cause the neurobehavioral impairments in the neonatal rats, which were mediated by the decreased ATPase activity and elevated Ca2+ concentration. Int J Occup Med Environ Health 2017;30(2):203-211. This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

  19. Cardiac-specific inducible overexpression of human plasma membrane Ca2+ ATPase 4b is cardioprotective and improves survival in mice following ischemic injury.

    Science.gov (United States)

    Sadi, Al Muktafi; Afroze, Talat; Siraj, M Ahsan; Momen, Abdul; White-Dzuro, Colin; Zarrin-Khat, Dorrin; Handa, Shivalika; Ban, Kiwon; Kabir, M Golam; Trivieri, Maria G; Gros, Robert; Backx, Peter; Husain, Mansoor

    2018-03-30

    Background: Heart failure (HF) is associated with reduced expression of plasma membrane Ca 2+ -ATPase 4 (PMCA4). Cardiac-specific overexpression of human PMCA4b in mice inhibited nNOS activity and reduced cardiac hypertrophy by inhibiting calcineurin. Here we examine temporally regulated cardiac-specific overexpression of hPMCA4b in mouse models of myocardial ischemia reperfusion injury (IRI) ex vivo , and HF following experimental myocardial infarction (MI) in vivo Methods and results: Doxycycline-regulated cardiomyocyte-specific overexpression and activity of hPMCA4b produced adaptive changes in expression levels of Ca 2+ -regulatory genes, and induced hypertrophy without significant differences in Ca 2+ transients or diastolic Ca 2+ concentrations. Total cardiac NOS and nNOS-specific activities were reduced in mice with cardiac overexpression of hPMCA4b while nNOS, eNOS and iNOS protein levels did not differ. hMPCA4b-overexpressing mice also exhibited elevated systolic blood pressure vs. controls, with increased contractility and lusitropy in vivo In isolated hearts undergoing IRI, hPMCA4b overexpression was cardioprotective. NO donor-treated hearts overexpressing hPMCA4b showed reduced LVDP and larger infarct size versus vehicle-treated hearts undergoing IRI, demonstrating that the cardioprotective benefits of hPMCA4b-repressed nNOS are lost by restoring NO availability. Finally, both pre-existing and post-MI induction of hPMCA4b overexpression reduced infarct expansion and improved survival from HF. Conclusions: Cardiac PMCA4b regulates nNOS activity, cardiac mass and contractility, such that PMCA4b overexpression preserves cardiac function following IRI, heightens cardiac performance and limits infarct progression, cardiac hypertrophy and HF, even when induced late post-MI. These data identify PMCA4b as a novel therapeutic target for IRI and HF. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  20. Endoplasmic reticulum calcium transport ATPase expression during differentiation of colon cancer and leukaemia cells

    International Nuclear Information System (INIS)

    Papp, Bela; Brouland, Jean-Philippe; Gelebart, Pascal; Kovacs, Tuende; Chomienne, Christine

    2004-01-01

    The calcium homeostasis of the endoplasmic reticulum (ER) is connected to a multitude of cell functions involved in intracellular signal transduction, control of proliferation, programmed cell death, or the synthesis of mature proteins. Calcium is accumulated in the ER by various biochemically distinct sarco/endoplasmic reticulum calcium transport ATPase isoenzymes (SERCA isoforms). Experimental data indicate that the SERCA composition of some carcinoma and leukaemia cell types undergoes significant changes during differentiation, and that this is accompanied by modifications of SERCA-dependent calcium accumulation in the ER. Because ER calcium homeostasis can also influence cell differentiation, we propose that the modulation of the expression of various SERCA isoforms, and in particular, the induction of the expression of SERCA3-type proteins, is an integral part of the differentiation program of some cancer and leukaemia cell types. The SERCA content of the ER may constitute a new parameter by which the calcium homeostatic characteristics of the organelle are adjusted. The cross-talk between ER calcium homeostasis and cell differentiation may have some implications for the better understanding of the signalling defects involved in the acquisition and maintenance of the malignant phenotype

  1. Methods for Creating and Animating a Computer Model Depicting the Structure and Function of the Sarcoplasmic Reticulum Calcium ATPase Enzyme.

    Science.gov (United States)

    Chen, Alice Y.; McKee, Nancy

    1999-01-01

    Describes the developmental process used to visualize the calcium ATPase enzyme of the sarcoplasmic reticulum which involves evaluating scientific information, consulting scientists, model making, storyboarding, and creating and editing in a computer medium. (Author/CCM)

  2. Cavitation during the protein misfolding cyclic amplification (PMCA) method – The trigger for de novo prion generation?

    International Nuclear Information System (INIS)

    Haigh, Cathryn L.; Drew, Simon C.

    2015-01-01

    The protein misfolding cyclic amplification (PMCA) technique has become a widely-adopted method for amplifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo. - Highlights: • Sonication during PMCA generates free radicals at the surface of cavitation bubbles. • PrP-centered and RNA-centered radicals are formed in addition to PrP-RNA adducts. • De novo prions may result from ROS and structural constraints during cavitation

  3. Cavitation during the protein misfolding cyclic amplification (PMCA) method – The trigger for de novo prion generation?

    Energy Technology Data Exchange (ETDEWEB)

    Haigh, Cathryn L., E-mail: chaigh@unimelb.edu.au [Department of Pathology, The University of Melbourne, Victoria 3010 (Australia); Drew, Simon C., E-mail: sdrew@unimelb.edu.au [Florey Department of Neuroscience and Mental Health, The University of Melbourne, Victoria 3010 (Australia)

    2015-06-05

    The protein misfolding cyclic amplification (PMCA) technique has become a widely-adopted method for amplifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo. - Highlights: • Sonication during PMCA generates free radicals at the surface of cavitation bubbles. • PrP-centered and RNA-centered radicals are formed in addition to PrP-RNA adducts. • De novo prions may result from ROS and structural constraints during cavitation.

  4. The pmr gene, encoding a Ca2+-ATPase, is required for calcium and manganese homeostasis and normal development of hyphae and conidia in Neurospora crassa.

    Science.gov (United States)

    Bowman, Barry J; Abreu, Stephen; Johl, Jessica K; Bowman, Emma Jean

    2012-11-01

    The pmr gene is predicted to encode a Ca(2+)-ATPase in the secretory pathway. We examined two strains of Neurospora crassa that lacked PMR: the Δpmr strain, in which pmr was completely deleted, and pmr(RIP), in which the gene was extensively mutated. Both strains had identical, complex phenotypes. Compared to the wild type, these strains required high concentrations of calcium or manganese for optimal growth and had highly branched, slow-growing hyphae. They conidiated poorly, and the shape and size of the conidia were abnormal. Calcium accumulated in the Δpmr strains to only 20% of the wild-type level. High concentrations of MnCl(2) (1 to 5 mM) in growth medium partially suppressed the morphological defects but did not alter the defect in calcium accumulation. The Δpmr Δnca-2 double mutant (nca-2 encodes a Ca(2+)-ATPase in the plasma membrane) accumulated 8-fold more calcium than the wild type, and the morphology of the hyphae was more similar to that of wild-type hyphae. Previous experiments failed to show a function for nca-1, which encodes a SERCA-type Ca(2+)-ATPase in the endoplasmic reticulum (B. J. Bowman, S. Abreu, E. Margolles-Clark, M. Draskovic, and E. J. Bowman, Eukaryot. Cell 10:654-661, 2011). The pmr(RIP) Δnca-1 double mutant accumulated small amounts of calcium, like the Δpmr strain, but exhibited even more extreme morphological defects. Thus, PMR can apparently replace NCA-1 in the endoplasmic reticulum, but NCA-1 cannot replace PMR. The morphological defects in the Δpmr strain are likely caused, in part, by insufficient concentrations of calcium and manganese in the Golgi compartment; however, PMR is also needed to accumulate normal levels of calcium in the whole cell.

  5. Subcellular distribution of calcium-binding proteins and a calcium-ATPase in canine pancreas

    International Nuclear Information System (INIS)

    Nigam, S.K.; Towers, T.

    1990-01-01

    Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with Stains-All. Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca-ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca-ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER

  6. The parietal cell gastric H, K-ATPase also functions as the Na, K-ATPase and Ca-ATPase in altered states.

    Science.gov (United States)

    Ray, Tushar

    2013-01-01

    This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump) seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump) and/or Ca-ATPase (Ca-pump) depending on cellular needs.  This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM) fraction exhibits a (Ca or Mg)-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF), the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg)-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM) shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the  proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM) and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump.

  7. 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.

  8. The gastric H, K-ATPase system also functions as the Na, K-ATPase and Ca-ATPase in altered states [v1; ref status: indexed, http://f1000r.es/1eo

    Directory of Open Access Journals (Sweden)

    Tushar Ray

    2013-07-01

    Full Text Available This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump and/or Ca-ATPase (Ca-pump depending on cellular needs.  This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM fraction exhibits a (Ca or Mg-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF, the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the  proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump.

  9. The parietal cell gastric H, K-ATPase also functions as the Na, K-ATPase and Ca-ATPase in altered states [v2; ref status: indexed, http://f1000r.es/1tc

    Directory of Open Access Journals (Sweden)

    Tushar Ray

    2013-09-01

    Full Text Available This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump and/or Ca-ATPase (Ca-pump depending on cellular needs.  This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM fraction exhibits a (Ca or Mg-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF, the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the  proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump.

  10. Intracellular free calcium concentration and calcium transport in human erythrocytes of lead-exposed workers

    International Nuclear Information System (INIS)

    Quintanar-Escorza, M.A.; Gonzalez-Martinez, M.T.; Navarro, L.; Maldonado, M.; Arevalo, B.; Calderon-Salinas, J.V.

    2007-01-01

    Erythrocytes are the route of lead distribution to organs and tissues. The effect of lead on calcium homeostasis in human erythrocytes and other excitable cells is not known. In the present work we studied the effect of lead intoxication on the uptake and efflux (measured as (Ca 2+ -Mg 2+ )-ATPase activity) of calcium were studied in erythrocytes obtained from lead-exposed workers. Blood samples were taken from 15 workers exposed to lead (blood lead concentration 74.4 ± 21.9 μg/dl) and 15 non-exposed workers (9.9 ± 2 μg/dl). In erythrocytes of lead-exposed workers, the intracellular free calcium was 79 ± 13 nM, a significantly higher concentration (ANOVA, P 2+ -Mg 2+ )-ATPase activity. Lipid peroxidation was 1.7-fold higher in erythrocytes of lead-exposed workers as compared with control. The alteration on calcium equilibrium in erythrocytes is discussed in light of the toxicological effects in lead-exposed workers

  11. Inositol phosphates influence the membrane bound Ca2+/Mg2+ stimulated ATPase from human erythrocyte membranes

    International Nuclear Information System (INIS)

    Kester, M.; Ekholm, J.; Kumar, R.; Hanahan, D.J.

    1986-01-01

    The modulation by exogenous inositol phosphates of the membrane Ca 2+ /Mg 2+ ATPase from saponin/EGTA lysed human erythrocytes was determined in a buffer (pH 7.6) containing histidine, 80 mM, MgCl 2 , 3.3 mM, NaCl, 74 mM, KCl, 30 mM, Na 2 ATP, 2.3 mM, ouabain, 0.83 mM, with variable amounts of CaCl 2 and EGTA. The ATPase assay was linear with time at 44 0 C. The inositol phosphates were commercially obtained and were also prepared from 32 P labeled rabbit platelet inositol phospholipids. Inositol triphosphate (IP 3 ) elevated the Ca 2+ /Mg 2+ ATPase activity over basal levels in a dose, time, and calcium dependent manner and were increased up to 85% of control values. Activities for the Na + /K + -ATPase and a Mg 2+ ATPase were not effected by IP 3 . Ca 2+ /Mg 2+ APTase activity with IP 2 or IP 3 could be synergistically elevated with calmodulin addition. The activation of the ATPase with IP 3 was calcium dependent in a range from .001 to .02 mM. The apparent Km and Vmax values were determined for IP 3 stimulated Ca 2+ /Mg 2+ ATPase

  12. Purification and functional motifs of the recombinant ATPase of orf virus.

    Science.gov (United States)

    Lin, Fong-Yuan; Chan, Kun-Wei; Wang, Chi-Young; Wong, Min-Liang; Hsu, Wei-Li

    2011-10-01

    Our previous study showed that the recombinant ATPase encoded by the A32L gene of orf virus displayed ATP hydrolysis activity as predicted from its amino acids sequence. This viral ATPase contains four known functional motifs (motifs I-IV) and a novel AYDG motif; they are essential for ATP hydrolysis reaction by binding ATP and magnesium ions. The motifs I and II correspond with the Walker A and B motifs of the typical ATPase, respectively. To examine the biochemical roles of these five conserved motifs, recombinant ATPases of five deletion mutants derived from the Taiping strain were expressed and purified. Their ATPase functions were assayed and compared with those of two wild type strains, Taiping and Nantou isolated in Taiwan. Our results showed that deletions at motifs I-III or IV exhibited lower activity than that of the wild type. Interestingly, deletion of AYDG motif decreased the ATPase activity more significantly than those of motifs I-IV deletions. Divalent ions such as magnesium and calcium were essential for ATPase activity. Moreover, our recombinant proteins of orf virus also demonstrated GTPase activity, though weaker than the original ATPase activity. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Effect of inhibition of microsomal Ca(2+)-ATPase on cytoplasmic calcium and enzyme secretion in pancreatic acini.

    Science.gov (United States)

    Metz, D C; Pradhan, T K; Mrozinski, J E; Jensen, R T; Turner, R J; Patto, R J; Gardner, J D

    1994-01-13

    We used thapsigargin (TG), 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ) and cyclopiazonic acid (CPA), each of which inhibits microsomal Ca(2+)-ATPase, to evaluate the effects of this inhibition on cytoplasmic free calcium ([Ca2+]i) and secretagogue-stimulated enzyme secretion in rat pancreatic acini. Using single-cell microspectrofluorimetry of fura-2-loaded acini we found that all three agents caused a sustained increase in [Ca2+]i by mobilizing calcium from inositol-(1,4,5)-trisphosphate-sensitive intracellular calcium stores and by promoting influx of extracellular calcium. Concentrations of all three agents that increased [Ca2+]i potentiated the stimulation of enzyme secretion caused by secretagogues that activate adenylate cyclase but inhibited the stimulation of enzyme secretion caused by secretagogues that activate phospholipase C. With BHQ, potentiation of adenylate cyclase-mediated enzyme secretion occurred immediately whereas inhibition of phospholipase C-mediated enzyme secretion occurred only after several min of incubation. In addition, the effects of BHQ and CPA on both [Ca2+]i and secretagogue-stimulated enzyme secretion were reversed completely by washing whereas the actions of TG could not be reversed by washing. Concentrations of BHQ in excess of those that caused maximal changes in [Ca2+]i inhibited all modes of stimulated enzyme secretion by a mechanism that was apparently unrelated to changes in [Ca2+]i. Finally, in contrast to the findings with TG and BHQ, CPA inhibited bombesin-stimulated enzyme secretion over a range of concentrations that was at least 10-fold lower than the range of concentrations over which CPA potentiated VIP-stimulated enzyme secretion.

  14. Calcium transport in turtle bladder

    International Nuclear Information System (INIS)

    Sabatini, S.; Kurtzman, N.A.

    1987-01-01

    Unidirectional 45 Ca fluxes were measured in the turtle bladder under open-circuit and short-circuit conditions. In the open-circuited state net calcium flux (J net Ca ) was secretory (serosa to mucosa). Ouabain reversed J net Ca to an absorptive flux. Amiloride reduced both fluxes such that J net Ca was not significantly different from zero. Removal of mucosal sodium caused net calcium absorption; removal of serosal sodium caused calcium secretion. When bladders were short circuited, J net Ca decreased to approximately one-third of control value but remained secretory. When ouabain was added under short-circuit conditions, J net Ca was similar in magnitude and direction to ouabain under open-circuited conditions (i.e., absorptive). Tissue 45 Ca content was ≅30-fold lower when the isotope was placed in the mucosal bath, suggesting that the apical membrane is the resistance barrier to calcium transport. The results obtained in this study are best explained by postulating a Ca 2+ -ATPase on the serosa of the turtle bladder epithelium and a sodium-calcium antiporter on the mucosa. In this model, the energy for calcium movement would be supplied, in large part, by the Na + -K + -ATPase. By increasing cell sodium, ouabain would decrease the activity of the mucosal sodium-calcium exchanger (or reverse it), uncovering active calcium transport across the serosa

  15. HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes.

    Science.gov (United States)

    Meraviglia, Viviana; Bocchi, Leonardo; Sacchetto, Roberta; Florio, Maria Cristina; Motta, Benedetta M; Corti, Corrado; Weichenberger, Christian X; Savi, Monia; D'Elia, Yuri; Rosato-Siri, Marcelo D; Suffredini, Silvia; Piubelli, Chiara; Pompilio, Giulio; Pramstaller, Peter P; Domingues, Francisco S; Stilli, Donatella; Rossini, Alessandra

    2018-01-31

    SERCA2a is the Ca 2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.

  16. Ultrastructural and immunohistochemical localization of plasma membrane Ca2+-ATPase 4 in Ca2+-transporting epithelia

    DEFF Research Database (Denmark)

    Alexander, R Todd; Beggs, Megan R; Zamani, Reza

    2015-01-01

    role in transcellular Ca(2+) flux and investigated the localization and regulation of Pmca4 in Ca(2+)-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestine, while pan-specific Pmca antibodies...... the cortical thick ascending limbs, macula densa, and early distal tubules as well as smooth muscle layers surrounding renal vessels. In human kidney, a similar pattern of distribution was observed, with highest PMCA4 expression in NCC positive tubules. Electron microscopy demonstrated Pmca4 localization...... in distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments, but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca(2+) balance, pointing...

  17. 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.

  18. The role of calcium ions in cytological effects of hypogravity

    Science.gov (United States)

    Kordyum, E. L.; Belyavskaya, N. A.; Nedukha, E. M.; Palladina, T. A.; Tarasenko, V. A.

    Electron-cytochemical and biochemical methods made it possible to reveal certain differences in ATPase activity stimulation by calcium ions in root apex cells of pea seedlings and moss protonema Funaria hygrometrica grown under stationary and slow clinostatic (2 rev/min) conditions. It was showed that under clinostatic conditions in comparison with the control variant the ATPase activity decreases in plasmalemma. The protein content in the plasmalemma fraction was also twice as low under these conditions. The root apex cells of the pea seedlings grown under spaceflight conditions were found to contain high concentrations of membrane-bound calcium. The data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance and the system of active calcium ion transport through plasmalemma under hypogravity.

  19. A novel fluorescence imaging approach to monitor salt stress-induced modulation of ouabain-sensitive ATPase activity in sunflower seedling roots.

    Science.gov (United States)

    Mukherjee, Soumya; Bhatla, Satish Chander

    2014-04-01

    Seedlings exposed to salt stress are expected to show modulation of intracellular accumulation of sodium ions through a variety of mechanisms. Using a new methodology, this work demonstrates ouabain (OU)-sensitive ATPase activity in the roots of sunflower seedlings subjected to salt stress (120 mM NaCl). 9-Anthroylouabain (a derivative of ouabain known to inhibit Na(+), K(+) -ATPase activity in animal systems, EC 3.6.3.9) has been used as a probe to analyze OU-sensitive ATPase activity in sunflower (Helianthus annuus) seedling roots by spectrofluorometric estimation and localization of its spatial distribution using confocal laser scanning microscopy. Salt stress for 48 h leads to a significant induction of OU-sensitive ATPase activity in the meristematic region of the seedling roots. Calcium ions (10 mM) significantly inhibit enzyme activity and a parallel accumulation of sodium ions in the cytosol of the columella cells, epidermis and in the cells of the meristematic region of the roots is evident. As a rapid response to NaCl stress, the activity of OU-sensitive ATPase gets localized in the nuclear membrane of root protoplasts and it gets inhibited after treatment with calcium ions. Nuclear membrane localization of the OU-sensitive ATPase activity highlights a possible mechanism to efflux sodium ions from the nucleus. Thus, a correlation between OU-sensitive ATPase activity, its modulation by calcium ions and accumulation of sodium ions in various regions of the seedling roots, has been demonstrated using a novel approach in a plant system. © 2013 Scandinavian Plant Physiology Society.

  20. Inhibition of calmodulin - regulated calcium pump activity in rat brain by toxaphene

    International Nuclear Information System (INIS)

    Trottman, C.H.; Moorthy, K.S.

    1986-01-01

    In vivo effects of toxaphene on calcium pump activity in rat brain synaptosomes was studied. Male Sprague-Dawley rats were dosed with toxaphene at 0,25,50, and 100 mg/kg/day for 3 days and sacrificed 24 h after last dose. Ca 2+ -ATPase activity and 45 Ca uptake were determined in brain P 2 fraction. Toxaphene inhibited both Ca 2+ -ATPase activity and 45 Ca 2+ uptake and the inhibition was dose dependent. Both substrate and Ca 2+ activation kinetics of Ca 2+ -ATPase indicated non-competitive type of inhibition as evidenced by decreased catalytic velocity but not enzyme-substrate affinity. The inhibited Ca 2+ -ATPase activity and Ca 2+ uptake were restored to normal level by exogenously added calmodulin which increased both velocity and affinity. The inhibition of Ca 2+ -ATPase activity and Ca 2+ uptake and restoration by calmodulin suggests that toxaphene may impair active calcium transport mechanisms by decreasing regulator protein calmodulin levels

  1. Investigation of function similarities between the sarcoplasmic reticulum and platelet calcium-dependent adenosinetriphosphatases with the inhibitors quercetin and calmidazolium

    International Nuclear Information System (INIS)

    Fischer, T.H.; Campbell, K.P.; White, G.C. II

    1987-01-01

    The platelet and skeletal sarcoplasmic reticulum calcium-dependent adenosinetriphosphatases (Ca 2+ -ATPases) were functionally compared with respect to substrate activation by steady-state kinetic methods using the inhibitors quercetin and calmidazolium. Quercetin inhibited platelet and sarcoplasmic reticulum Ca 2+ -ATPase activities in a dose-dependent manner with IC 50 values of 25 and 10 μM, respectively. Calmidazolium also inhibited platelet and sarcoplasmic reticulum Ca 2+ -ATPase activities, with half-maximal inhibition measured at 5 and 4 μM, respectively. Both inhibitors also affected the [ 45 Ca] calcium transport activity of intact platelet microsomes at concentrations similar to those which reduced Ca 2+ -ATPase activity. These inhibitors were then used to examine substrate ligation by the platelet and sarcoplasmic reticulum calcium pump proteins. For both Ca 2+ -ATPase proteins, quercetin has an affinity for the E-Ca 2 (fully ligated with respect to calcium at the exterior high-affinity calcium binding sites, unligated with respect to ATP) conformational state of the protein that is approximately 10-fold grater than for other conformational states in the hydrolytic cycle. Quercetin can thus be considered a competitive inhibitor of the calcium pump proteins with respect to ATP. In contrast to the effect of quercetin, calmidazolium interacts with the platelet and sarcoplasmic reticulum Ca 2+ -ATPases in an uncompetitive manner. The dissociation constants for this inhibitor for the different conformational states of the calcium pump proteins were similar, indicating that calmidazolium has equal affinity for all of the reaction intermediates probed. These observations indicate that the substrate ligation processes are similar for the two pump proteins. This supports the concept that the hydrolytic cycles of the two proteins are comparable

  2. The ataxia related G1107D mutation of the plasma membrane Ca2+ ATPase isoform 3 affects its interplay with calmodulin and the autoinhibition process.

    Science.gov (United States)

    Calì, Tito; Frizzarin, Martina; Luoni, Laura; Zonta, Francesco; Pantano, Sergio; Cruz, Carlos; Bonza, Maria Cristina; Bertipaglia, Ilenia; Ruzzene, Maria; De Michelis, Maria Ida; Damiano, Nunzio; Marin, Oriano; Zanni, Ginevra; Zanotti, Giuseppe; Brini, Marisa; Lopreiato, Raffaele; Carafoli, Ernesto

    2017-01-01

    The plasma membrane Ca 2+ ATPases (PMCA pumps) have a long, cytosolic C-terminal regulatory region where a calmodulin-binding domain (CaM-BD) is located. Under basal conditions (low Ca 2+ ), the C-terminal tail of the pump interacts with autoinhibitory sites proximal to the active center of the enzyme. In activating conditions (i.e., high Ca 2+ ), Ca 2+ -bound CaM displaces the C-terminal tail from the autoinhibitory sites, restoring activity. We have recently identified a G1107D replacement within the CaM-BD of isoform 3 of the PMCA pump in a family affected by X-linked congenital cerebellar ataxia. Here, we investigate the effects of the G1107D replacement on the interplay of the mutated CaM-BD with both CaM and the pump core, by combining computational, biochemical and functional approaches. We provide evidence that the affinity of the isolated mutated CaM-BD for CaM is significantly reduced with respect to the wild type (wt) counterpart, and that the ability of CaM to activate the pump in vitro is thus decreased. Multiscale simulations support the conclusions on the detrimental effect of the mutation, indicating reduced stability of the CaM binding. We further show that the G1107D replacement impairs the autoinhibition mechanism of the PMCA3 pump as well, as the introduction of a negative charge perturbs the contacts between the CaM-BD and the pump core. Thus, the mutation affects both the ability of the pump to optimally transport Ca 2+ in the activated state, and the autoinhibition mechanism in its resting state. Copyright © 2016. Published by Elsevier B.V.

  3. Comparative immunolocalization of the plasma membrane calcium pump and calbindin D28K in chicken retina during embryonic development

    Directory of Open Access Journals (Sweden)

    N. Tolosa de Talamoni

    2010-05-01

    Full Text Available The immunolocalization of the plasma membrane calcium pump (PMCA was studied in 4-week-old chick retina in comparison with calbindin D28K (CaBP immunostaining. We have demonstrated that the monoclonal anti-PMCA antibody 5F10 from human erythrocyte plasma membrane crossreacts with a Ca2+ pump epitope of the cells from the neural retina. The immunolocalization of both proteins was also studied during the embryonic development of the chicken retina. At age 4.5 days, the cells of the retina were faintly immunoreactive to PMCA and CaBP antibodies, but the lack of cellular aggregation and differentiation did not allow discrimination between the two proteins. A clear difference in the localization was seen from the tenth day of development through post-hatching with slight variation. PMCA localized mainly in the outer and inner plexiform layers, in some cells in the ganglion layer, in the nerve fiber layer and slightly in the photoreceptor cells. CaBP was intensely stained in cones, cone pedicles and some amacrine cells. The number of CaBP positive amacrine cells declined after hatching. A few ganglion cells and several nerve fibers were CaBP 333 immunoreactive. The role of these proteins in the early stages of retinal development is unknown, but the results suggest that Ca2+ homeostasis in the retina is well regulated, probably to avoid excessive accumulation of Ca2+, which often leads to neurodegeneration.

  4. Cell cycle-related fluctuations in transcellular ionic currents and plasma membrane Ca2+/Mg2+ ATPase activity during early cleavages of Lymnaea stagnalis embryos.

    Science.gov (United States)

    Zivkovic, Danica; Créton, Robbert; Dohmen, René

    1991-08-01

    During the first four mitotic division cycles of Lymnaea stagnalis embryos, we have detected cell cycle-dependent changes in the pattern of transcellular ionic currents and membrane-bound Ca 2+ -stimulated ATPase activity. Ionic currents ranging from 0.05 to 2.50 μA/cm 2 have been measured using the vibrating probe technique. Enzyme activity was detected using Ando's cytochemical method (Ando et al. 1981) which reveals Ca 2+ /Mg 2+ ATPase localization at the ultrastructural level, and under high-stringency conditions with respect to calcium availability, it reveals Ca 2+ -stimulated ATPase. The ionic currents and Ca 2+ -stimulated ATPase localization have in common that important changes occur during the M-phase of the cell cycles. Minimal outward current at the vegetal pole coincides with metaphase/anaphase. Maximal inward current at the animal pole coincides with the onset of cytokinesis at that pole. Ca 2+ -stimulated ATPase is absent from one half of the embryo at metaphase/anaphase of the two- and four-cell stage, whereas it is present in all cells during the remaining part of the cell cycle. Since fluctuations of cytosolic free calcium concentrations appear to correlate with both karyokinesis and cytokinesis, we speculate that part of the cyclic pattern of Ca 2+ -stimulated ATPase localization and of the transcellular ionic currents reflects the elevation of cytosolic free calcium concentration during the M-phase.

  5. Testis-specific isoform of Na/K-ATPase (ATP1A4) regulates sperm function and fertility in dairy bulls through potential mechanisms involving reactive oxygen species, calcium and actin polymerization.

    Science.gov (United States)

    Rajamanickam, G D; Kroetsch, T; Kastelic, J P; Thundathil, J C

    2017-07-01

    Traditional bull breeding soundness evaluation (BBSE) eliminates bulls that are grossly abnormal; however, bulls classified as satisfactory potential breeders still vary in field fertility, implying submicroscopic differences in sperm characteristics. The testis-specific isoform of Na/K-ATPase (ATP1A4) is involved in regulation of sperm motility and capacitation in bulls through well-established enzyme activity and signaling functions. The objective was to determine ATP1A4 content, activity and their relationship to post-thaw sperm function and field fertility, using semen samples from low-fertility (LF) and high-fertility (HF) Holstein bulls (n = 20 each) with known FERTSOL rates (measure of field fertility, based on non-return rate). Frozen-thawed sperm from HF bulls had increased ATP1A4 content and activity compared to LF bulls. Furthermore, post-thaw sperm from HF bulls had increased tyrosine phosphorylation, ROS, F-actin content, and low intracellular calcium compared to LF bulls. Subsequent incubation of HF bull sperm with ouabain (a specific ligand of Na/K-ATPase) further augmented the post-thaw increase in tyrosine phosphorylation, ROS production, and F-actin content, whereas the increase in intracellular calcium was still low compared to LF bull sperm. ATP1A4 content and activity, ROS, F-actin and calcium were significantly correlated with fertility. In conclusion, we inferred that ATP1A4 content and activity differed among dairy bulls with satisfactory semen characteristics and that ATP1A4 may regulate sperm function through mechanisms involving ROS, F-actin and calcium in frozen-thawed sperm of HF and LF dairy bulls. © 2017 American Society of Andrology and European Academy of Andrology.

  6. Calcium movements and the cellular basis of gravitropism

    Science.gov (United States)

    Roux, S. J.; Biro, R. L.; Hale, C. C.

    An early gravity-transduction event in oat coleoptiles which precedes any noticeable bending is the accumulation of calcium on their prospective slower-growing side. Sub-cellular calcium localization studies indicate that the gravity-stimulated redistribution of calcium results in an increased concentration of calcium in the walls of responding cells. Since calcium can inhibit the extension growth of plant cell walls, this selective accumulation of calcium in walls may play a role in inducing the asymmetry of growth which characterizes gravitropism. The active transport of calcium from cells into walls is performed by a calcium-dependent ATPase localized in the plasma membrane. Evidence is presented in support of the hypothesis that this calcium pump is regulated by a feed-back mechanism which includes the participation of calmodulin.

  7. Myocardial Na,K-ATPase: Clinical aspects

    Science.gov (United States)

    Kjeldsen, Keld

    2003-01-01

    The specific binding of digitalis glycosides to Na,K-ATPase is used as a tool for Na,K-ATPase quantification with high accuracy and precision. In myocardial biopsies from patients with heart failure, total Na,K-ATPase concentration is decreased by around 40%; a correlation exists between a decrease in heart function and a decrease in Na,K-ATPase concentration. During digitalization, around 30% of remaining pumps are occupied by digoxin. Myocardial Na,K-ATPase is also influenced by other drugs used for the treatment of heart failure. Thus, potassium loss during diuretic therapy has been found to reduce myocardial Na,K-ATPase, whereas angiotensin-converting enzyme inhibitors may stimulate Na,K pump activity. Furthermore, hyperaldosteronism induced by heart failure has been found to decrease Na,K-ATPase activity. Accordingly, treatment with the aldosterone antagonist, spironolactone, may also influence Na,K-ATPase activity. The importance of Na,K pump modulation with heart disease, inhibition in digitalization and other effects of medication should be considered in the context of sodium, potassium and calcium regulation. It is recommended that digoxin be administered to heart failure patients who, after institution of mortality-reducing therapy, still have heart failure symptoms, and that the therapy be continued if symptoms are revealed or reduced. Digitalis glycosides are the only safe inotropic drugs for oral use that improve hemodynamics in heart failure. An important aspect of myocardial Na,K pump affection in heart disease is its influence on extracellular potassium (Ke) homeostasis. Two important aspects should be considered: potassium handling among myocytes, and effects of potassium entering the extracellular space of the heart via the bloodstream. It should be noted that both of these aspects of Ke homeostasis are affected by regulatory aspects, eg, regulation of the Na,K pump by physiological and pathophysiological conditions, as well as by medical

  8. Plasma membrane Ca2+-ATPase isoforms composition regulates cellular pH homeostasis in differentiating PC12 cells in a manner dependent on cytosolic Ca2+ elevations

    DEFF Research Database (Denmark)

    Boczek, Tomasz; Lisek, Malwina; Ferenc, Bozena

    2014-01-01

    isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca2+ overload evoked by 59 m....... In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca2+ overload. Cyclosporin and bongkrekic acid prevented Ψm loss suggesting the involvement of Ca2......+-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient...

  9. Lens ion transport: from basic concepts to regulation of Na,K-ATPase activity

    Science.gov (United States)

    Delamere, Nicholas A.; Tamiya, Shigeo

    2009-01-01

    In the late 1960s, studies by George Duncan explained many of the basic principles that underlie lens ion homeostasis. The experiments pointed to a permeability barrier close to the surface of the lens and illustrated the requirement for continuous Na,K-ATPase-mediated active sodium extrusion. Without active sodium extrusion, lens sodium and calcium content increases resulting in lens swelling and deterioration of transparency. Later, Duncan's laboratory discovered functional muscarinic and purinergic receptors at the surface of the lens. Recent studies using intact lens suggest purinergic receptors might be involved in short-term regulation of Na,K-ATPase in the epithelium. Purinergic receptor agonists ATP and UTP selectively activate certain Src family tyrosine kinases and stimulate Na,K-ATPase activity. This might represent part of a control mechanism capable of adjusting, perhaps fine tuning, lens ion transport machinery. PMID:18614168

  10. Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.).

    Science.gov (United States)

    Mirza, Neelofar; Taj, Gohar; Arora, Sandeep; Kumar, Anil

    2014-10-25

    Finger millet (Eleusine coracana) variably accumulates calcium in different tissues, due to differential expression of genes involved in uptake, translocation and accumulation of calcium. Ca(2+)/H(+) antiporter (CAX1), two pore channel (TPC1), CaM-stimulated type IIB Ca(2+) ATPase and two CaM dependent protein kinase (CaMK1 and 2) homologs were studied in finger millet. Two genotypes GP-45 and GP-1 (high and low calcium accumulating, respectively) were used to understand the role of these genes in differential calcium accumulation. For most of the genes higher expression was found in the high calcium accumulating genotype. CAX1 was strongly expressed in the late stages of spike development and could be responsible for accumulating high concentrations of calcium in seeds. TPC1 and Ca(2+) ATPase homologs recorded strong expression in the root, stem and developing spike and signify their role in calcium uptake and translocation, respectively. Calmodulin showed strong expression and a similar expression pattern to the type IIB ATPase in the developing spike only and indicating developing spike or even seed specific isoform of CaM affecting the activity of downstream target of calcium transportation. Interestingly, CaMK1 and CaMK2 had expression patterns similar to ATPase and TPC1 in various tissues raising a possibility of their respective regulation via CaM kinase. Expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf and developing spike inferring a surprising possibility of CAX1 regulation through 14-3-3 protein. Our results provide a molecular insight for explaining the mechanism of calcium accumulation in finger millet. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Movement of calcium signals and calcium-binding proteins: firewalls, traps and tunnels.

    Science.gov (United States)

    Barrow, S L; Sherwood, M W; Dolman, N J; Gerasimenko, O V; Voronina, S G; Tepikin, A V

    2006-06-01

    In the board game 'Snakes and Ladders', placed on the image of a pancreatic acinar cell, calcium ions have to move from release sites in the secretory region to the nucleus. There is another important contraflow - from calcium entry channels in the basal part of the cell to ER (endoplasmic reticulum) terminals in the secretory granule region. Both transport routes are perilous as the messenger can disappear in any place on the game board. It can be grabbed by calcium ATPases of the ER (masquerading as a snake but functioning like a ladder) and tunnelled through its low buffering environment, it can be lured into the whirlpools of mitochondria uniporters and forced to regulate the tricarboxylic acid cycle, and it can be permanently placed inside the matrix of secretory granules and released only outside the cell. The organelles could trade calcium (e.g. from the ER to mitochondria and vice versa) almost depriving this ion the light of the cytosol and noble company of cytosolic calcium buffers. Altogether it is a rich and colourful story.

  12. The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

    International Nuclear Information System (INIS)

    King, Taj D.; Gandy, Johanna C.; Bijur, Gautam N.

    2006-01-01

    The ubiquitously expressed protein glycogen synthase kinase-3 (GSK3) is constitutively active, however its activity is markedly diminished following phosphorylation of Ser21 of GSK3α and Ser9 of GSK3β. Although several kinases are known to phosphorylate Ser21/9 of GSK3, for example Akt, relatively much less is known about the mechanisms that cause the dephosphorylation of GSK3 at Ser21/9. In the present study KCl-induced plasma membrane depolarization of SH-SY5Y cells, which increases intracellular calcium concentrations caused a transient decrease in the phosphorylation of Akt at Thr308 and Ser473, and GSK3 at Ser21/9. Overexpression of the selective protein phosphatase-1 inhibitor protein, inhibitor-2, increased basal GSK3 phosphorylation at Ser21/9 and significantly blocked the KCl-induced dephosphorylation of GSK3β, but not GSK3α. The phosphorylation of Akt was not affected by the overexpression of inhibitor-2. GSK3 activity is known to affect sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) levels. Overexpression of inhibitor-2 or treatment of cells with the GSK3 inhibitors lithium and SB216763 increased the levels of SERCA2. These results indicate that the protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation induced by KCl and that GSK3 activity regulates SERCA2 levels

  13. A non-equilibrium thermodynamics model of reconstituted Ca(2+)-ATPase.

    Science.gov (United States)

    Waldeck, A R; van Dam, K; Berden, J; Kuchel, P W

    1998-01-01

    A non-equilibrium thermodynamics (NET) model describing the action of completely coupled or 'slipping' reconstituted Ca(2+)-ATPase is presented. Variation of the coupling stoichiometries with the magnitude of the electrochemical gradients, as the ATPase hydrolyzes ATP, is an indication of molecular slip. However, the Ca2+ and H+ membrane-leak conductances may also be a function of their respective gradients. Such non-ohmic leak typically yields 'flow-force' relationships that are similar to those that are obtained when the pump slips; hence, caution needs to be exercised when interpreting data of Ca(2+)-ATPase-mediated fluxes that display a non-linear dependence on the electrochemical proton (delta mu H) and/or calcium gradients (delta mu Ca). To address this issue, three experimentally verifiable relationships differentiating between membrane leak and enzymic slip were derived. First, by measuring delta mu H as a function of the rate of ATP hydrolysis by the enzyme. Second, by measuring the overall 'efficiency' of the pump as a function of delta mu H. Third, by measuring the proton ejection rate by the pump as a function of its ATP hydrolysis rate.

  14. Milrinone and thyroid hormone stimulate myocardial membrane Ca2+-ATPase activity and share structural homologies.

    Science.gov (United States)

    Mylotte, K M; Cody, V; Davis, P J; Davis, F B; Blas, S D; Schoenl, M

    1985-01-01

    We have recently shown that thyroid hormone in physiological concentrations stimulates sarcolemma-enriched rabbit-myocardial-membrane Ca2+-ATPase in vitro. In this study, milrinone [2-methyl-5-cyano-(3,4'-bipyridin)-6(1H)-one], a cardiac inotropic agent, was thyromimetic in the same system. At clinically achievable concentrations (50-500 nM), milrinone significantly stimulated membrane Ca2+-ATPase in vitro. This action was antagonized by W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an agent that also blocks thyroid hormone action on the Ca2+-ATPase, at concentrations as low as 5 microM. Progressive additions of milrinone to membranes incubated with a fixed concentration of thyroxine (0.10 nM) or triiodothyronine resulted in a progressive obliteration of the thyroid hormone effect on Ca2+-ATPase. Amrinone [5-amino-(3,4'-bipyridin)-6(1H)-one], the parent bipyridine of milrinone, had no effect on myocardial Ca2+-ATPase activity. X-ray crystallographic analysis of milrinone and amrinone revealed structural homologies between the phenolic ring of thyroxine and the substituted ring of milrinone, whereas amrinone did not share these homologies. The mechanism(s) of the inotropic actions of thyroxine and of milrinone is not clearly understood, but these observations implicate Ca2+-ATPase, a calcium pump-associated enzyme, as one mediator of the effects on the heart of these two compounds. PMID:2933747

  15. Hydrogen peroxide-mediated oxidative stress disrupts calcium binding on calmodulin: More evidence for oxidative stress in vitiligo

    International Nuclear Information System (INIS)

    Schallreuter, K.U.; Gibbons, N.C.J.; Zothner, C.; Abou Elloof, M.M.; Wood, J.M.

    2007-01-01

    Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10 -3 M H 2 O 2 . One consequence of this severe oxidative stress is an altered calcium homeostasis in epidermal keratinocytes and melanocytes. Here, we show decreased epidermal calmodulin expression in acute vitiligo. Since 10 -3 M H 2 O 2 oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H 2 O 2 utilising 45 calcium. The results showed that all four calcium atoms exchanged per molecule of calmodulin. Since oxidised calmodulin looses its ability to activate calcium ATPase, enzyme activities were followed in full skin biopsies from lesional skin of patients with acute vitiligo (n = 6) and healthy controls (n = 6). The results yielded a 4-fold decrease of ATPase activities in the patients. Computer simulation of native and oxidised calmodulin confirmed the loss of all four calcium ions from their specific EF-hand domains. Taken together H 2 O 2 -mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed L-phenylalanine-uptake in the epidermis of acute vitiligo

  16. Novel Sulfur Metabolites of Garlic Attenuate Cardiac Hypertrophy and Remodeling through Induction of Na+/K+-ATPase Expression.

    Science.gov (United States)

    Khatua, Tarak N; Borkar, Roshan M; Mohammed, Soheb A; Dinda, Amit K; Srinivas, R; Banerjee, Sanjay K

    2017-01-01

    Epidemiologic studies show an inverse correlation between garlic consumption and progression of cardiovascular disease. However, the molecular basis for the beneficial effect of garlic on the heart is not known. Therefore, the objective of the present study was to (1) investigate the effect of raw garlic on isoproterenol (Iso) induced cardiac hypertrophy (2) find the active metabolites of garlic responsible for the beneficial effect. Cardiac hypertrophy was induced in rats by subcutaneous single injection of Iso 5 mg kg -1 day -1 for 15 days and the effect of garlic (250 mg/kg/day orally) was evaluated. Garlic metabolites in in vivo were identified by LC/MS study. The effect of garlic and its metabolites were evaluated against hypertrophy in H9C2 cells. Garlic normalized cardiac oxidative stress after Iso administration. Cardiac pathology and mitochondrial enzyme activities were improved in hypertrophy heart after garlic administration. Decreased Na + /K + -ATPase protein level that observed in hypertrophy heart was increased after garlic administration. We identified three garlic metabolites in rat serum. To confirm the role of garlic metabolites on cardiac hypertrophy, Na + /K + -ATPase expression and intracellular calcium levels were measured after treating H9C2 cells with raw garlic and two of its active metabolites, allyl methyl sulfide and allyl methyl sulfoxide. Raw garlic and both metabolites increased Na + /K + -ATPase protein level and decreased intracellular calcium levels and cell size in Iso treated H9C2 cells. This antihypertrophic effect of garlic and its sulfur metabolites were lost in H9C2 cells in presence of Na + /K + -ATPase inhibitor. In conclusion, garlic and its active metabolites increased Na + /K + -ATPase in rat heart, and attenuated cardiac hypertrophy and associated remodeling. Our data suggest that identified new garlic metabolites may be useful for therapeutic intervention against cardiac hypertrophy.

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

    International Nuclear Information System (INIS)

    Harper, Jeffrey F.

    2004-01-01

    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

  18. Goatpoxvirus ATPase activity is increased by dsDNA and decreased by zinc ion.

    Science.gov (United States)

    Lee, Ming-Liang; Hsu, Wei-Li; Wang, Chi-Young; Chen, Hui-Yu; Lin, Fong-Yuan; Chang, Ming-Huang; Chang, Hong-You; Wong, Min-Liang; Chan, Kun-Wei

    2016-10-01

    Viral-encoded ATPase can act as a part of molecular motor in genome packaging of DNA viruses, such as vaccinia virus and adenovirus, by ATP hydrolysis and interaction with DNA. Poxviral ATPase (also called A32) is involved in genomic double-stranded DNA (dsDNA) encapsidation, and inhibition of the expression of A32 causes formation of immature virions lacking viral DNA. However, the role of A32 in goatpoxvirus genome packaging and its dsDNA binding property are not known. In this study, purified recombinant goatpoxvirus A32 protein (rA32) was examined for its dsDNA binding property as well as the effect of dsDNA on ATP hydrolysis. We found that rA32 could bind dsDNA, and its ATPase activity was significant increased with dsDNA binding. Effects of magnesium and calcium ions on ATP hydrolysis were investigated also. The ATPase activity was dramatically enhanced by dsDNA in the presence of Mg(2+); in contrast, ATPase function was not altered by Ca(2+). Furthermore, the enzyme activity of rA32 was completely blocked by Zn(2+). Regarding DNA-protein interaction, the rA32-ATP-Mg(2+) showed lower dsDNA binding affinity than that of rA32-ATP-Ca(2+). The DNA-protein binding was stronger in the presence of zinc ion. Our results implied that A32 may play a role in viral genome encapsidation and DNA condensation.

  19. Oviductal extracellular vesicles (oviductosomes, OVS) are conserved in humans: murine OVS play a pivotal role in sperm capacitation and fertility.

    Science.gov (United States)

    Bathala, Pradeepthi; Fereshteh, Zeinab; Li, Kun; Al-Dossary, Amal A; Galileo, Deni S; Martin-DeLeon, Patricia A

    2018-03-01

    Are extracellular vesicles (EVs) in the murine oviduct (oviductosomes, OVS) conserved in humans and do they play a role in the fertility of Pmca4-/- females? OVS and their fertility-modulating proteins are conserved in humans, arise via the apocrine pathway, and mediate a compensatory upregulation of PMCA1 (plasma membrane Ca2+-ATPase 1) in Pmca4-/- female mice during proestrus/estrus, to account for their fertility. Recently murine OVS were identified and shown during proestrus/estrus to express elevated levels of PMCA4 which they can deliver to sperm. PMCA4 is the major Ca2+ efflux pump in murine sperm and Pmca4 deletion leads to loss of sperm motility and male infertility as there is no compensatory upregulation of the remaining Ca2+ pump, PMCA1. Of the four family members of PMCAs (PMCA1-4), PMCA1 and PMCA4 are ubiquitous, and to date there have been no reports of one isoform being upregulated to compensate for another in any organ/tissue. Since Pmca4-/- females are fertile, despite the abundant expression of PMCA4 in wild-type (WT) OVS, we propose that OVS serve a role of packaging and delivering to sperm elevated levels of PMCA1 in Pmca4-/- during proestrus/estrus to compensate for PMCA4's absence. Fallopian tubes from pre-menopausal women undergoing hysterectomy were used to study EVs in the luminal fluid. Oviducts from sexually mature WT mice were sectioned after perfusion fixation to detect EVs in situ. Oviducts were recovered from WT and Pmca4-/- after hormonally induced estrus and sectioned for PMCA1 immunofluorescence (IF) (detected with confocal microscopy) and hematoxylin and eosin staining. Reproductive tissues, luminal fluids and EVs were recovered after induced estrus and after natural cycling for western blot analysis of PMCA1 and qRT-PCR of Pmca1 to compare expression levels in WT and Pmca4-/-. OVS, uterosomes, and epididymal luminal fluid were included in the comparisons. WT and Pmca4-/- OVS were analyzed for the presence of known PMCA4 partners

  20. Calcium uptake and proton transport by acidocalcisomes of Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Peter Rohloff

    Full Text Available Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+/H(+ countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.

  1. Calcium in pollen-pistil interaction in 'Petunia hybrida Hat'. Pt. 3. Localization of Ca2+ ions and Ca2+-ATPase in pollinated pistil

    International Nuclear Information System (INIS)

    Bednarska, E.; Butowt, R.

    1995-01-01

    Studies were carried out of Ca 2+ and Ca 2+ -ATPase localization in pollinated (6 and 48 h after pollination) pistils of 'Petunia hybrida'. The results were confronted with Ca 2+ localization in mature pollen grain and in unpollinated pistil. It has been found that after pollination the number of Ca 2+ sequestered in the stigmal exudate and in the sporoderm of the pollen grain gets lower. That phenomenon was associated with the appearance of a large number of Sb/Ca precipitates in the submembrane cytoplasm of the germinating pollen. In the vacuolized pollen grain, i.e. grown into a pollen tube, there were only a few precipitates. In the pollen tube, Ca 2+ were found in the organelles of the tip cytoplasm and in the external pectin cell wall. Studies with the use of 45 Ca 2+ have revealed that the source of calcium ions incorporated into the pollen tube tip and its pectin wall is the transmitting tract of the style. In the transmitting tract overgrown with pollen tubes, Ca 2+ were localized in the intercellular matrix and in the transmitting cell. Sb/Ca precipitates occurred in the nuclei, around the secretary vesicles and on the plasmalemma in the transverse walls region. Elevated Ca 2+ level was found in degenerating cells (inhibited pollen tubes, transmitting cells, nucellar cells). The progressing degeneration process of the cells of the transmitting tract of the pollinated pistil was associated with a decrease in the activity of plasmalemma Ca 2+ -ATPase. (author). 30 refs, 19 figs

  2. Hofmeister effect of anions on calcium translocation by sarcoplasmic reticulum Ca2+-ATPase

    Science.gov (United States)

    Tadini-Buoninsegni, Francesco; Moncelli, Maria Rosa; Peruzzi, Niccolò; Ninham, Barry W.; Dei, Luigi; Nostro, Pierandrea Lo

    2015-10-01

    The occurrence of Hofmeister (specific ion) effects in various membrane-related physiological processes is well documented. For example the effect of anions on the transport activity of the ion pump Na+, K+-ATPase has been investigated. Here we report on specific anion effects on the ATP-dependent Ca2+ translocation by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Current measurements following ATP concentration jumps on SERCA-containing vesicles adsorbed on solid supported membranes were carried out in the presence of different potassium salts. We found that monovalent anions strongly interfere with ATP-induced Ca2+ translocation by SERCA, according to their increasing chaotropicity in the Hofmeister series. On the contrary, a significant increase in Ca2+ translocation was observed in the presence of sulphate. We suggest that the anions can affect the conformational transition between the phosphorylated intermediates E1P and E2P of the SERCA cycle. In particular, the stabilization of the E1P conformation by chaotropic anions seems to be related to their adsorption at the enzyme/water and/or at the membrane/water interface, while the more kosmotropic species affect SERCA conformation and functionality by modifying the hydration layers of the enzyme.

  3. Lithium-induced inhibition of Na-K ATPase and Ca ATPase activities in rat brain synaptosome.

    Science.gov (United States)

    Cho, Y. W.

    1995-01-01

    To explore the action mechanism of lithium in the brain, the author investigated the effects of lithium on Na-K ATPase and Ca ATPase in rat brain synaptosomes prepared from forebrains by the method of Booth and Clark. The activities of Na-K ATPase and Ca ATPase were assayed by the level of inorganic phosphate liberated from the hydrolysis of ATP. Lithium at the optimum therapeutic concentration of 1 mM decreased the activity of Na-K ATPase from the control value of 19.08 +/- 0.29 to 18.27 +/- 0.10 micromoles Pi/mg protein/h and also reduced the activity of Ca ATPase from 6.38 +/- 0.12 to 5.64 +/- 0.12 micromoles Pi/mg protein/h. The decreased activity of Na-K ATPase will decrease the rate of Ca2+ efflux, probably via an Na-Ca exchange mechanism and will increase the rate of Ca2+ entry by the depolarization of nerve terminals. The reduced activity of Ca ATPase will result in the decreased efflux of Ca2+. As a Conclusion, it can be speculated that lithium elevates the intrasynaptosomal Ca2+ concentration via inhibition of the activities of Na-K ATPase and Ca ATPase, and this increased [Ca2+]i will cause the release of neurotransmitters and neurological effects of lithium. PMID:7598829

  4. Iron overload impact on P-ATPases.

    Science.gov (United States)

    Sousa, Leilismara; Pessoa, Marco Tulio C; Costa, Tamara G F; Cortes, Vanessa F; Santos, Herica L; Barbosa, Leandro Augusto

    2018-03-01

    Iron is a chemical element that is active in the fundamental physiological processes for human life, but its burden can be toxic to the body, mainly because of the stimulation of membrane lipid peroxidation. For this reason, the action of iron on many ATPases has been studied, especially on P-ATPases, such as the Na + ,K + -ATPase and the Ca 2+ -ATPase. On the Fe 2+ -ATPase activity, the free iron acts as an activator, decreasing the intracellular Fe 2+ and playing a protection role for the cell. On the Ca 2+ -ATPase activity, the iron overload decreases the enzyme activity, raising the cytoplasmic Ca 2+ and decreasing the sarco/endoplasmic reticulum and the Golgi apparatus Ca 2+ concentrations, which could promote an enzyme oxidation, nitration, and fragmentation. However, the iron overload effect on the Na + ,K + -ATPase may change according to the tissue expressions. On the renal cells, as well as on the brain and the heart, iron promotes an enzyme inactivation, whereas its effect on the erythrocytes seems to be the opposite, directly stimulating the ATPase activity, or stimulating it by signaling pathways involving ROS and PKC. Modulations in the ATPase activity may impair the ionic transportation, which is essential for cell viability maintenance, inducing irreversible damage to the cell homeostasis. Here, we will discuss about the iron overload effect on the P-ATPases, such as the Na + ,K + -ATPase, the Ca 2+ -ATPase, and the Fe 2+ -ATPase.

  5. 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...... to include the regulation of membrane traffic, cytoskeletal dynamics, cell division, lipid metabolism, and lipid signaling. In this review, we will summarize the basic features of P4-ATPases and the physiological implications of their lipid transport activity in the cell....

  6. Calcium in plant cells

    Directory of Open Access Journals (Sweden)

    V. V. Schwartau

    2014-04-01

    Full Text Available The paper gives the review on the role of calcium in many physiological processes of plant organisms, including growth and development, protection from pathogenic influences, response to changing environmental factors, and many other aspects of plant physiology. Initial intake of calcium ions is carried out by Ca2+-channels of plasma membrane and they are further transported by the xylem owing to auxins’ attractive ability. The level of intake and selectivity of calcium transport to ove-ground parts of the plant is controlled by a symplast. Ca2+enters to the cytoplasm of endoderm cells through calcium channels on the cortical side of Kaspary bands, and is redistributed inside the stele by the symplast, with the use of Ca2+-АТPases and Ca2+/Н+-antiports. Owing to regulated expression and activity of these calcium transporters, calclum can be selectively delivered to the xylem. Important role in supporting calcium homeostasis is given to the vacuole which is the largest depo of calcium. Regulated quantity of calcium movement through the tonoplast is provided by a number of potential-, ligand-gated active transporters and channels, like Ca2+-ATPase and Ca2+/H+ exchanger. They are actively involved in the inactivation of the calcium signal by pumping Ca2+ to the depo of cells. Calcium ATPases are high affinity pumps that efficiently transfer calcium ions against the concentration gradient in their presence in the solution in nanomolar concentrations. Calcium exchangers are low affinity, high capacity Ca2+ transporters that are effectively transporting calcium after raising its concentration in the cell cytosol through the use of protons gradients. Maintaining constant concentration and participation in the response to stimuli of different types also involves EPR, plastids, mitochondria, and cell wall. Calcium binding proteins contain several conserved sequences that provide sensitivity to changes in the concentration of Ca2+ and when you

  7. 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.

  8. Polar localization of plasma membrane Ca2+/Mg2+ ATPase correlates with the pattern of steady ionic currents in eggs ofLymnaea stagnalis andBithynia tentaculata (Mollusca).

    Science.gov (United States)

    Zivkovic, Danica; Créton, Robbert; Zwaan, Gideon; de Bruijn, Willem C; Dohmen, M René

    1990-11-01

    During extrusion of the first polar body in eggs ofLymnaea stagnalis andBithynia tentaculata a localized Ca 2+ /Mg 2+ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705-726]. The enzyme activity was distributed in a polar fashion, along the cytoplasmic face of the plasma membrane. In the eggs ofLymnaea it was found only in the vegetal hemisphere, whereas inBithynia eggs it was localized both in the vegetal hemisphere and at the animal pole. This pattern of enzyme activity corresponds to the polar pattern of transcellular ionic currents measured with the vibrating probe, which we showed to be partially carried or regulated by calcium [Zivkovic and Dohmen (1989) Biol Bull (Woods Hole) 176 (Suppl):103-109]. The characteristics of the ATPase were studied using a variety of approaches such as ion and substrate depletions and substitutions, addition of specific inhibitors of ATPase activity, treatment with EDTA/EGTA and electron energy-loss spectrometry. The results indicate that, inLymnaea, there are at least two enzymatic entities. The first one is a Ca 2+ /Mg 2+ ATPase localized along the membrane and in the cortex of the vegetal hemisphere. The second one is a Ca 2+ -stimulated ATPase (calcium pump of the plasma membrane) localized in a small region of the membrane at the vegetal pole. We speculate that in the eggs ofLymnaea andBithynia a functional relationship exists between the plasma-membrane-associated ATPase activity and the transcellular ionic currents measured in the same region.

  9. 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.

  10. A pivotal role of vacuolar H(+)-ATPase in regulation of lipid production in Phaeodactylum tricornutum.

    Science.gov (United States)

    Zhang, Huiying; Zeng, Rensen; Chen, Daoyi; Liu, Jian

    2016-08-08

    Microalgal lipids have been considered as a promising source for biodiesel production. Alkaline pH can induce neutral lipid accumulation in microalgae cells. However, whether and how proton pumps, especially vacuolar H(+)-ATPase (V-ATPase), function in these processes is not well known. In this study, we treated Phaeodactylum tricornutum with V-ATPase specific inhibitor bafilomycin A1 (BFA1) to determine its role in lipid production. Firstly, V-ATPase activity was increased in the latter phase of microalgae growth. BFA1 treatment decreased the cell density and lipid contents. Further analysis showed that BFA1 treatment reduced the number and size of oil bodies. GC-MS analysis showed that lipid components were not affected by BFA1 treatment. Intracellular pH was decreased and nitrogen depletion was delayed after BFA1 treatment. RNA-Seq analysis showed that expression of genes involved in calcium signaling, sulfur metabolism, cell cycle, glycolysis, pentose phosphate pathway, porphyrin, chlorophyll metabolism and lipid catabolic metabolism were upregulated, while expression of genes involved in ion transmembrane transport, ubiquitin mediated proteolysis, SNARE interactions in vesicular transport, fatty acid biosynthesis were downregulated under BFA1 treatment. Our findings provided insights into the molecular mechanisms underlying lipid accumulation and the key genes involved in lipid metabolism in Phaeodactylum tricornutum in response to BFA1.

  11. AAA-ATPases in Protein Degradation

    Directory of Open Access Journals (Sweden)

    Ravikiran S. Yedidi

    2017-06-01

    Full Text Available Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

  12. AAA-ATPases in Protein Degradation.

    Science.gov (United States)

    Yedidi, Ravikiran S; Wendler, Petra; Enenkel, Cordula

    2017-01-01

    Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

  13. 3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity.

    Science.gov (United States)

    Jardim-Messeder, Douglas; Camacho-Pereira, Juliana; Galina, Antonio

    2012-05-01

    3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Response of Ca2+-ATPase to clinorotaion of pea seedlings. O. M. Nedukha and E. L. Kordyum

    Science.gov (United States)

    Nedukha, Olena

    2016-07-01

    The present study was aimed to reveal of response of Ca2+-ATPase activity of cortex cells in distal elongation zone of Pisum sativum root to slow clinorotation. Pea seedlings were grown on a horizontal clinostat (2 rpm) and in the stationary control for 6 days. The electron-cytochemical method was used to examine the effects of imitated microgravity on the distribution of Ca2+-ATPase in outer layers of root cortex. The quantitative analysis of the density of cytochemical reaction products was measured using the Image J program. Electron microscopy showed the presence of electron-dense lead phosphate precipitated grains, the enzymatic activity reaction products on the plasma membrane, membranes of vesicular structures, endoplasmic reticulum (ER) and on organelles envelope in both of samples of the stationary control and clinorotated seedlings. We revealed the sensitivity of Ca2+-ATPase to clinorotation. The quantitative analysis of the area and density of enzymatic activity reaction products revealed that clinorotation led to the decrease of 3.4 times the density of reaction products on the plasma membrane and the increase of reaction products density on endomembranes and organelles membranes, in particular: in 2.2 times on mitochondria membranes; in 1.3 times - on membranes of ER; in 2.5 times - on tonoplast; by an order of magnitude greater - on contacting membranes of organelles with plasma membrane in comparison with such in cells of control samples. The data analysis can indicate an intensification of calcium pump on endomembranes, on envelopes of cytoplasmic organelles and nucleus. The obtained data suggest that the redistribution of Ca2+-ATPase activity in cells can be mediated by the activation of certain isoforms of enzyme or/and by an activation of Ca2+/H+ antiporter in plasma membrane that helps to maintain optimal calcium balance in plant cells under imitated microgravity.

  15. Calcium efflux systems in stress signalling and adaptation in plants

    Directory of Open Access Journals (Sweden)

    Jayakumar eBose

    2011-12-01

    Full Text Available Transient cytosolic calcium ([Ca2+]cyt elevation is an ubiquitous denominator of the signalling network when plants are exposed to literally every known abiotic and biotic stress. These stress-induced [Ca2+]cyt elevations vary in magnitude, frequency and shape, depending on the severity of the stress as well the type of stress experienced. This creates a unique stress-specific calcium signature that is then decoded by signal transduction networks. While most published papers have been focused predominantly on the role of Ca2+ influx mechanisms in shaping [Ca2+]cyt signatures, restoration of the basal [Ca2+]cyt levels is impossible without both cytosolic Ca2+ buffering and efficient Ca2+ efflux mechanisms removing excess Ca2+ from cytosol, to reload Ca2+ stores and to terminate Ca2+ signalling. This is the topic of the current review. The molecular identity of two major types of Ca2+ efflux systems, Ca2+-ATPase pumps and Ca2+/H+ exchangers, is described, and their regulatory modes are analysed in detail. The spatial and temporal organisation of calcium signalling networks is described, and the importance of existence of intracellular calcium microdomains is discussed. Experimental evidence for the role of Ca2+ efflux systems in plant responses to a range of abiotic and biotic factors is summarised. Contribution of Ca2+-ATPase pumps and Ca2+/H+ exchangers in shaping [Ca2+]cyt signatures is then modelled by using a four-component model (plasma- and endo- membrane-based Ca2+-permeable channels and efflux systems taking into account the cytosolic Ca2+ buffering. It is concluded that physiologically relevant variations in the activity of Ca2+-ATPase pumps and Ca2+/H+ exchangers are sufficient to fully describe all the reported experimental evidence and determine the shape of [Ca2+]cyt signatures in response to environmental stimuli, emphasising the crucial role these active efflux systems play in plant adaptive responses to environment.

  16. Neurotransmitter modulation of extracellular H+ fluxes from isolated retinal horizontal cells of the skate

    Science.gov (United States)

    Molina, Anthony J A; Verzi, Michael P; Birnbaum, Andrea D; Yamoah, Ebenezer N; Hammar, Katherine; Smith, Peter J S; Malchow, Robert Paul

    2004-01-01

    Self-referencing H+-selective microelectrodes were used to measure extracellular H+ fluxes from horizontal cells isolated from the skate retina. A standing H+ flux was detected from quiescent cells, indicating a higher concentration of free hydrogen ions near the extracellular surface of the cell as compared to the surrounding solution. The standing H+ flux was reduced by removal of extracellular sodium or application of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), suggesting activity of a Na+–H+ exchanger. Glutamate decreased H+ flux, lowering the concentration of free hydrogen ions around the cell. AMPA/kainate receptor agonists mimicked the response, and the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) eliminated the effects of glutamate and kainate. Metabotropic glutamate agonists were without effect. Glutamate-induced alterations in H+ flux required extracellular calcium, and were abolished when cells were bathed in an alkaline Ringer solution. Increasing intracellular calcium by photolysis of the caged calcium compound NP-EGTA also altered extracellular H+ flux. Immunocytochemical localization of the plasmalemma Ca2+–H+-ATPase (PMCA pump) revealed intense labelling within the outer plexiform layer and on isolated horizontal cells. Our results suggest that glutamate modulation of H+ flux arises from calcium entry into cells with subsequent activation of the plasmalemma Ca2+–H+-ATPase. These neurotransmitter-induced changes in extracellular pH have the potential to play a modulatory role in synaptic processing in the outer retina. However, our findings argue against the hypothesis that hydrogen ions released by horizontal cells normally act as the inhibitory feedback neurotransmitter onto photoreceptor synaptic terminals to create the surround portion of the centre-surround receptive fields of retinal neurones. PMID:15272044

  17. Na(+),K (+)-ATPase as a docking station: protein-protein complexes of the Na(+),K (+)-ATPase.

    Science.gov (United States)

    Reinhard, Linda; Tidow, Henning; Clausen, Michael J; Nissen, Poul

    2013-01-01

    The Na(+),K(+)-ATPase, or sodium pump, is well known for its role in ion transport across the plasma membrane of animal cells. It carries out the transport of Na(+) ions out of the cell and of K(+) ions into the cell and thus maintains electrolyte and fluid balance. In addition to the fundamental ion-pumping function of the Na(+),K(+)-ATPase, recent work has suggested additional roles for Na(+),K(+)-ATPase in signal transduction and biomembrane structure. Several signaling pathways have been found to involve Na(+),K(+)-ATPase, which serves as a docking station for a fast-growing number of protein interaction partners. In this review, we focus on Na(+),K(+)-ATPase as a signal transducer, but also briefly discuss other Na(+),K(+)-ATPase protein-protein interactions, providing a comprehensive overview of the diverse signaling functions ascribed to this well-known enzyme.

  18. Inhibition of calcium uptake during hypoxia in developing zebrafish is mediated by hypoxia-inducible factor.

    Science.gov (United States)

    Kwong, Raymond W M; Kumai, Yusuke; Tzaneva, Velislava; Azzi, Estelle; Hochhold, Nina; Robertson, Cayleih; Pelster, Bernd; Perry, Steve F

    2016-12-15

    The present study investigated the potential role of hypoxia-inducible factor (HIF) in calcium homeostasis in developing zebrafish (Danio rerio). It was demonstrated that zebrafish raised in hypoxic water (30 mmHg; control, 155 mmHg P O 2 ) until 4 days post-fertilization exhibited a substantial reduction in whole-body Ca 2+ levels and Ca 2+ uptake. Ca 2+ uptake in hypoxia-treated fish did not return to pre-hypoxia (control) levels within 2 h of transfer back to normoxic water. Results from real-time PCR showed that hypoxia decreased the whole-body mRNA expression levels of the epithelial Ca 2+ channel (ecac), but not plasma membrane Ca 2+ -ATPase (pmca2) or Na + /Ca 2+ -exchanger (ncx1b). Whole-mount in situ hybridization revealed that the number of ecac-expressing ionocytes was reduced in fish raised in hypoxic water. These findings suggested that hypoxic treatment suppressed the expression of ecac, thereby reducing Ca 2+ influx. To further evaluate the potential mechanisms for the effects of hypoxia on Ca 2+ regulation, a functional gene knockdown approach was employed to prevent the expression of HIF-1αb during hypoxic treatment. Consistent with a role for HIF-1αb in regulating Ca 2+ balance during hypoxia, the results demonstrated that the reduction of Ca 2+ uptake associated with hypoxic exposure was not observed in fish experiencing HIF-1αb knockdown. Additionally, the effects of hypoxia on reducing the number of ecac-expressing ionocytes was less pronounced in HIF-1αb-deficient fish. Overall, the current study revealed that hypoxic exposure inhibited Ca 2+ uptake in developing zebrafish, probably owing to HIF-1αb-mediated suppression of ecac expression. © 2016. Published by The Company of Biologists Ltd.

  19. Effect of ionizing radiation on Ca2+-ATPase and Mg2+-ATPase: the role of ligands

    International Nuclear Information System (INIS)

    Dreval', V.I.

    1994-01-01

    The change of Ca 2+ -ATPase and Mg 2+ -ATPase activity in plasma membranes of thymocytes irradiated with doses of 10 2 , 10 3 and 10 4 Gy in the presence of Ca 2+ , Mg 2+ and ATP was studied. Stabilizing effect of Ca 2+ and Mg 2+ on Ca 2+ -ATPase and ATP on Mg 2+ -ATPase under irradiation was established

  20. Myocardial Na,K-ATPase: Clinical aspects

    OpenAIRE

    Kjeldsen, Keld

    2003-01-01

    The specific binding of digitalis glycosides to Na,K-ATPase is used as a tool for Na,K-ATPase quantification with high accuracy and precision. In myocardial biopsies from patients with heart failure, total Na,K-ATPase concentration is decreased by around 40%; a correlation exists between a decrease in heart function and a decrease in Na,K-ATPase concentration. During digitalization, around 30% of remaining pumps are occupied by digoxin. Myocardial Na,K-ATPase is also influenced by other drugs...

  1. 3-Methylcholanthrene inhibits lymphocyte proliferation and increases intracellular calcium levels in common carp (Cyprinus carpio L)

    International Nuclear Information System (INIS)

    Reynaud, S.; Duchiron, C.; Deschaux, P.

    2003-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. Many authors have focused on macrophage activities in fish exposed to PAHs. However, fewer studies have reported decrease in specific immunity in such fish. We investigated the intracellular mechanisms by which the 3-methylcholanthrene (3-MC) decreased lymphocyte proliferation in carp. T- and B-lymphocyte proliferation induced by Concanavalin A (Con A) and lipopolysaccharide (LPS) were inhibited by 3-MC (0.5-50 μM). 3-MC also produced a rapid and a sustained increase in intracellular calcium concentration ([Ca 2+ ] i ) (2 h minimum). However, the cytochrome P450 1A and Ah receptor inhibitor, α-naphtoflavone (a-NF), also inhibited lymphocyte proliferation and did not reverse the effects of 3-MC. Moreover, since a-NF and 3-MC increased [Ca 2+ ] i and inhibited lymphocyte proliferation it was possible that calcium release played a role in 3-MC-inhibited lymphocyte proliferation. The rise in [Ca 2+ ] i induced by 3-MC was potentiated by the inhibitor of the endoplasmic reticulum calcium ATPases, thapsigargin. Treating cells with 3-MC decreased calcium mobilization caused by thapsigargin. These results suggest that 3-MC acts on the endoplasmic reticulum, perhaps directly on calcium ATPases, to increase intracellular calcium levels in carp leucocytes

  2. Calcium in pollen-pistil interaction in `Petunia hybrida Hat`. Pt. 3. Localization of Ca{sup 2+} ions and Ca{sup 2+}-ATPase in pollinated pistil

    Energy Technology Data Exchange (ETDEWEB)

    Bednarska, E.; Butowt, R. [Uniwersytet Mikolaja Kopernika, Torun (Poland)

    1995-12-31

    Studies were carried out of Ca{sup 2+} and Ca{sup 2+}-ATPase localization in pollinated (6 and 48 h after pollination) pistils of `Petunia hybrida`. The results were confronted with Ca{sup 2+} localization in mature pollen grain and in unpollinated pistil. It has been found that after pollination the number of Ca{sup 2+} sequestered in the stigmal exudate and in the sporoderm of the pollen grain gets lower. That phenomenon was associated with the appearance of a large number of Sb/Ca precipitates in the submembrane cytoplasm of the germinating pollen. In the vacuolized pollen grain, i.e. grown into a pollen tube, there were only a few precipitates. In the pollen tube, Ca{sup 2+} were found in the organelles of the tip cytoplasm and in the external pectin cell wall. Studies with the use of {sup 45}Ca{sup 2+} have revealed that the source of calcium ions incorporated into the pollen tube tip and its pectin wall is the transmitting tract of the style. In the transmitting tract overgrown with pollen tubes, Ca{sup 2+} were localized in the intercellular matrix and in the transmitting cell. Sb/Ca precipitates occurred in the nuclei, around the secretary vesicles and on the plasmalemma in the transverse walls region. Elevated Ca{sup 2+} level was found in degenerating cells (inhibited pollen tubes, transmitting cells, nucellar cells). The progressing degeneration process of the cells of the transmitting tract of the pollinated pistil was associated with a decrease in the activity of plasmalemma Ca{sup 2+}-ATPase. (author). 30 refs, 19 figs.

  3. Roles of transmembrane segment M1 of Na(+),K (+)-ATPase and Ca (2+)-ATPase, the gatekeeper and the pivot

    DEFF Research Database (Denmark)

    Einholm, Anja P.; Andersen, Jens Peter; Vilsen, Bente

    2007-01-01

    In this review we summarize mutagenesis work on the structure-function relationship of transmembrane segment M1 in the Na(+),K(+)-ATPase and the sarco(endo)plasmic reticulum Ca(2+)-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported...... cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca(2+) interaction/occlusion in Ca(2+)-ATPase and K(+) interaction/occlusion in Na(+),K(+)-ATPase. Leu(65) of the Ca(2+)-ATPase and Leu(99) of the Na(+),K(+)-ATPase, located...... of the extracytoplasmic gate in both the Ca(2+)-ATPase and the Na(+),K(+)-ATPase. Udgivelsesdato: 2007-Dec...

  4. On archaebacterial ATPase from Halobacterium saccharovorum

    Science.gov (United States)

    Kristjansson, H.; Ponnamperuma, C.; Hochstein, L.; Altekar, W.

    1984-01-01

    The energy transducing ATPase from Halobacterium saccharovorum was studied in order to define the origin of energy transducing systems. The ATPase required high salt concentration (4M NaCl) for activity; activity was rapidly lost when NaCl was below 1 Molar. At low salt concentration, the membrane bound ATPase activity could be stabilized in presence of spermine. However, following solubilization spermine was ineffective. Furthermore, F1 ATPase activity was stabilized by ammonium sulfate even when the NaCl concentration was less than 1 Molar. These studies suggest that stabilization by hydrophobic interactions preceded ionic ones in the evolution of the energy transducing ATPases.

  5. Transcriptional regulators of Na, K-ATPase subunits

    Directory of Open Access Journals (Sweden)

    Zhiqin eLi

    2015-10-01

    Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

  6. Best time window for the use of calcium-modulating agents to improve functional recovery in injured peripheral nerves-An experiment in rats.

    Science.gov (United States)

    Yan, Yuhui; Shen, Feng-Yi; Agresti, Michael; Zhang, Lin-Ling; Matloub, Hani S; LoGiudice, John A; Havlik, Robert; Li, Jifeng; Gu, Yu-Dong; Yan, Ji-Geng

    2017-09-01

    Peripheral nerve injury can have a devastating effect on daily life. Calcium concentrations in nerve fibers drastically increase after nerve injury, and this activates downstream processes leading to neuron death. Our previous studies showed that calcium-modulating agents decrease calcium accumulation, which aids in regeneration of injured peripheral nerves; however, the optimal therapeutic window for this application has not yet been identified. In this study, we show that calcium clearance after nerve injury is positively correlated with functional recovery in rats suffering from a crushed sciatic nerve injury. After the nerve injury, calcium accumulation increased. Peak volume is from 2 to 8 weeks post injury; calcium accumulation then gradually decreased over the following 24-week period. The compound muscle action potential (CMAP) measurement from the extensor digitorum longus muscle recovered to nearly normal levels in 24 weeks. Simultaneously, real-time polymerase chain reaction results showed that upregulation of calcium-ATPase (a membrane protein that transports calcium out of nerve fibers) mRNA peaked at 12 weeks. These results suggest that without intervention, the peak in calcium-ATPase mRNA expression in the injured nerve occurs after the peak in calcium accumulation, and CMAP recovery continues beyond 24 weeks. Immediately using calcium-modulating agents after crushed nerve injury improved functional recovery. These studies suggest that a crucial time frame in which to initiate effective clinical approaches to accelerate calcium clearance and nerve regeneration would be prior to 2 weeks post injury. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  7. Further characterization of the red beet plasma membrane Ca2+-ATPase using GTP as an alternative substrate

    International Nuclear Information System (INIS)

    Williams, L.E.; Schueler, S.B.; Briskin, D.P.

    1990-01-01

    The GTP-driven component of Ca 2+ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca 2+ -translocating ATPase and assess its utility as a probe for this transport system. Uptake of 45 Ca 2+ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca 2+ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca 2+ -ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of 45 Ca 2+ -uptake by exogeneous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven 45 Ca 2+ uptake represents the capacity of the plasma membrane Ca 2+ -translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ- 32 P]GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca 2+ -ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca 2+ -ATPases present in animal cells

  8. Comprehensive comparison of two protein family of P-ATPases (13A1 and 13A3) in insects.

    Science.gov (United States)

    Seddigh, Samin

    2017-06-01

    The P-type ATPases (P-ATPases) are present in all living cells where they mediate ion transport across membranes on the expense of ATP hydrolysis. Different ions which are transported by these pumps are protons like calcium, sodium, potassium, and heavy metals such as manganese, iron, copper, and zinc. Maintenance of the proper gradients for essential ions across cellular membranes makes P-ATPases crucial for cell survival. In this study, characterization of two families of P-ATPases including P-ATPase 13A1 and P-ATPase 13A3 protein was compared in two different insect species from different orders. According to the conserved motifs found with MEME, nine motifs were shared by insects of 13A1 family but eight in 13A3 family. Seven different insect species from 13A1 and five samples from 13A3 family were selected as the representative samples for functional and structural analyses. The structural and functional analyses were performed with ProtParam, SOPMA, SignalP 4.1, TMHMM 2.0, ProtScale and ProDom tools in the ExPASy database. The tertiary structure of Bombus terrestris as a sample of each family of insects were predicted by the Phyre2 and TM-score servers and their similarities were verified by SuperPose server. The tertiary structures were predicted via the "c3b9bA" model (PDB Accession Code: 3B9B) in P-ATPase 13A1 family and "c2zxeA" model (PDB Accession Code: 2ZXE) in P-ATPase 13A3 family. A phylogenetic tree was constructed with MEGA 6.06 software using the Neighbor-joining method. According to the results, there was a high identity of P-ATPase families so that they should be derived from a common ancestor however they belonged to separate groups. In protein-protein interaction analysis by STRING 10.0, six common enriched pathways of KEGG were identified in B. terrestris in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of other insects and organisms. Copyright © 2017 Elsevier Ltd. All rights

  9. 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

  10. Decoding P4-ATPase substrate interactions.

    Science.gov (United States)

    Roland, Bartholomew P; Graham, Todd R

    Cellular membranes display a diversity of functions that are conferred by the unique composition and organization of their proteins and lipids. One important aspect of lipid organization is the asymmetric distribution of phospholipids (PLs) across the plasma membrane. The unequal distribution of key PLs between the cytofacial and exofacial leaflets of the bilayer creates physical surface tension that can be used to bend the membrane; and like Ca 2+ , a chemical gradient that can be used to transduce biochemical signals. PL flippases in the type IV P-type ATPase (P4-ATPase) family are the principle transporters used to set and repair this PL gradient and the asymmetric organization of these membranes are encoded by the substrate specificity of these enzymes. Thus, understanding the mechanisms of P4-ATPase substrate specificity will help reveal their role in membrane organization and cell biology. Further, decoding the structural determinants of substrate specificity provides investigators the opportunity to mutationally tune this specificity to explore the role of particular PL substrates in P4-ATPase cellular functions. This work reviews the role of P4-ATPases in membrane biology, presents our current understanding of P4-ATPase substrate specificity, and discusses how these fundamental aspects of P4-ATPase enzymology may be used to enhance our knowledge of cellular membrane biology.

  11. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

    Science.gov (United States)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

    1999-07-01

    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  12. Partial purification of the ATP-driven calcium pump of Streptococcus sanguis

    International Nuclear Information System (INIS)

    Lynn, A.R.; Rosen, B.P.

    1986-01-01

    ATP-dependent transport of calcium has been observed in several species of streptococci as uptake of 45 Ca 2+ into everted membrane vesicles. Membranes from Streptococcus sanguis and Streptococcus faecalis were solubilized with octyl-β-D-glucoside or Triton X-100, and the extracts reconstituted into proteoliposomes containing Escherichia coli or soybean phospholipid. Calcium transport in reconstituted proteoliposomes was insensitive to the ionophores nigericin and valinomycin and was unaffected by the F 0 F 1 inhibitor N,N'-dicyclohexylcarbodiimide. Uptake was inhibited by ortho-vanadate with a K/sub i/ in the micromolar range. These results demonstrate that the reconstituted transport activities are not the result of ATP-driven proton pumping via the F 0 F 1 coupled to a calcium/proton antiporter and suggest that existence of a calcium translocating ATPase. Partial purification of the transport activity from Streptococcus sanguis has been achieved using density gradient centrifugation and FPLC

  13. Calcium homeostasis and vitamin D metabolism and expression in strongly calcifying laying birds.

    Science.gov (United States)

    Bar, Arie

    2008-12-01

    Egg laying and shell calcification impose severe extra demands on ionic calcium (Ca2+) homeostasis; especially in birds characterized by their long clutches (series of eggs laid sequentially before a "pause day"). These demands induce vitamin D metabolism and expression. The metabolism of vitamin D is also altered indirectly, by other processes associated with increased demands for calcium, such as growth, bone formation and egg production. A series of intestinal, renal or bone proteins are consequently expressed in the target organs via mechanisms involving a vitamin D receptor. Some of these proteins (carbonic anhydrase, calbindin and calcium-ATPase) are also found in the uterus (eggshell gland) or are believed to be involved in calcium transport in the intestine or kidney (calcium channels). The present review deals with vitamin D metabolism and the expression of the above-mentioned proteins in birds, with special attention to the strongly calcifying laying bird.

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

    International Nuclear Information System (INIS)

    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/ ≅ 70,000 and ≅ 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-[ 14 C]ethylmaleimide and 7-chloro-4-nitro[ 14 C]benzo-2-oxa-1,3-diazole, labeled the M/sub r/ ≅ 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-[ 14 C]dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ ≅ 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 10 5 , 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/ ≅ 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 F 0 F 1 ATPases

  15. Peripheral serotonin regulates maternal calcium trafficking in mammary epithelial cells during lactation in mice.

    Directory of Open Access Journals (Sweden)

    Jimena Laporta

    Full Text Available Lactation is characterized by massive transcellular flux of calcium, from the basolateral side of the mammary alveolar epithelium (blood into the ductal lumen (milk. Regulation of calcium transport during lactation is critical for maternal and neonatal health. The monoamine serotonin (5-HT is synthesized by the mammary gland and functions as a homeostatic regulation of lactation. Genetic ablation of tryptophan hydroxylase 1 (Tph1, which encodes the rate-limiting enzyme in non-neuronal serotonin synthesis, causes a deficiency in circulating serotonin. As a consequence maternal calcium concentrations decrease, mammary epithelial cell morphology is altered, and cell proliferation is decreased during lactation. Here we demonstrate that serotonin deficiency decreases the expression and disrupts the normal localization of calcium transporters located in the apical (PMCA2 and basolateral (CaSR, ORAI-1 membranes of the lactating mammary gland. In addition, serotonin deficiency decreases the mRNA expression of calcium transporters located in intracellular compartments (SERCA2, SPCA1 and 2. Mammary expression of serotonin receptor isoform 2b and its downstream pathways (PLCβ3, PKC and MAP-ERK1/2 are also decreased by serotonin deficiency, which might explain the numerous phenotypic alterations described above. In most cases, addition of exogenous 5-hydroxy-L-tryptophan to the Tph1 deficient mice rescued the phenotype. Our data supports the hypothesis that serotonin is necessary for proper mammary gland structure and function, to regulate blood and mammary epithelial cell transport of calcium during lactation. These findings can be applicable to the treatment of lactation-induced hypocalcemia in dairy cows and can have profound implications in humans, given the wide-spread use of selective serotonin reuptake inhibitors as antidepressants during pregnancy and lactation.

  16. 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.

  17. V-ATPase-mediated granular acidification is regulated by the V-ATPase accessory subunit Ac45 in POMC-producing cells.

    NARCIS (Netherlands)

    Jansen, E.J.S.; Hafmans, T.G.M.; Martens, G.J.

    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

  18. Calcium and cargoes as regulators of myosin 5a activity

    International Nuclear Information System (INIS)

    Sellers, James R.; Thirumurugan, Kavitha; Sakamoto, Takeshi; Hammer, John A.; Knight, Peter J.

    2008-01-01

    Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins

  19. Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

    Science.gov (United States)

    Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

    2017-07-17

    Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

  20. Novel aspects of Na+,K+-ATPase

    OpenAIRE

    Aizman, Oleg

    2002-01-01

    Na,K-ATPase, an integral membrane protein expressed in each eukaryotic cell, serves as the major determinant of intracellular ion composition. In the current study we investigated novel aspects of Na,K-ATPase function and regulation. It is well established that Na,K-ATPase activity is regulated by reversible phosphorylation. New findings in this study are: 1) the level of intracellular Ca 2. concentration determines the functional effects of PKA and PKC-mediated Na,K-ATP...

  1. The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase.

    NARCIS (Netherlands)

    Pont, J.J.H.H.M. de; Swarts, H.G.P.; Karawajczyk, A.; Schaftenaar, G.; Willems, P.H.G.M.; Koenderink, J.B.

    2009-01-01

    Based on studies with chimeras between (non-)gastric H,K-ATPase and Na,K-ATPase, a model for the ouabain binding site has recently been presented (Qiu et al. J.Biol.Chem. 280 (2005) 32349). In this model, hydrogen bonds between specific amino acid residues of Na,K-ATPase and hydroxyl groups of

  2. Mg,Ca-ATPase activity under irradiation

    International Nuclear Information System (INIS)

    Ladutin, V.V.; Orlova, V.V.; Lob, P.A.; Gerasiminko, I.V.; Mack, E.I.

    2003-01-01

    Full text: The influence of different doses irradiation at the Mg,Ca-ATPase activity at the rat brain has been investigated. The analyses were made at the apparatus of LKB and Carl-Ceis-Jena firm with help of reagents of Sigma and Boehringer firm. Rats decapitated after 1, 3, 6, 24 and 48 h after action of irradiation. Dose 0.206 C/kg. Erythrocytes. 1 and 3h after irradiation influence- decrease of Mg,Ca-ATPase activity to 86-87% relatively control level, 24 and 48 h - increase of activity to the control level. Dose 0.312 C/kg. Large hemispheres. 1h - decrease of ATPase activity to 90% relatively control, 3h - increase to control level, 24h - fall to 86%, after 48h small increase to 93% relatively control. Dose 9.287 C/kg. Large hemispheres. 1h - sharp fall of Mg, Ca-ATPase activity to 67 % relatively control, increase of activity to 96% after 3h and sharp fall of activity to 64% 6h after action of irradiation. Dose 9.287 C/kg. Cerebellum. 1h - sharp decrease of ATPase activity to 80%. After 3h -sharp increase to 160% relatively control level and sharp fall of ATPase activity to 47% relatively control after 6h. The mechanism of radiation pathology of active ion transport has been discussed

  3. Alterations in calcium metabolism during human monocyte activation

    International Nuclear Information System (INIS)

    Scully, S.P.

    1984-01-01

    Human peripheral blood monocytes have been prepared from plateletpheresis residues by counterflow centrifugal elutriation in sufficient quantities to enable quantitative studies of cell calcium. Kinetic analysis of 45 Ca exchange data in resting monocytes was compatible with a model of cellular calcium containing three exchangeable calcium pools. These pools are thought to represent a putative ectocellular pool, a putative cytoplasmic chelated pool, and a putative organelle sequestered pool. Exposure of monocytes to the plant lectin Con A at a concentration that maximally simulated superoxide production caused an increase in the size and a doubling in the exchange rate of the putative cytoplasmic pool without a change in the other cellular pools. The cytoplasmic ionized calcium, [Ca]/sub i/, measured with the fluorescent probe, Quin 2 rose from a resting level of 83 nM to 165 mN within 30 sec of exposure to Con A. This increase in cytoplasmic calcium preceded the release of superoxide radicals. Calcium transport and calcium ATPase activities were identified and characterized in plasma membrane vesicles prepared from monocytes. Both activities were strictly dependent on ATP and Mg, had a Km/sub Ca/ in the submicromolar range and were stimulated by calmodulin. Thus, it seems that monocyte calcium is in a dynamic steady state that is a balance between efflux and influx rates, and that the activation of these cells results in the transition to a new steady state. The alteration in [Ca]/sub i/ that accompany the new steady state are essential for superoxide production by human monocytes

  4. Identification of a receptor for extracellular renalase.

    Directory of Open Access Journals (Sweden)

    Ling Wang

    Full Text Available An increased risk for developing essential hypertension, stroke and diabetes is associated with single nucleotide gene polymorphisms in renalase, a newly described secreted flavoprotein with oxidoreductase activity. Gene deletion causes hypertension, and aggravates acute ischemic kidney (AKI and cardiac injury. Independent of its intrinsic enzymatic activities, extracellular renalase activates MAPK signaling and prevents acute kidney injury (AKI in wild type (WT mice. Therefore, we sought to identity the receptor for extracellular renalase.RP-220 is a previously identified, 20 amino acids long renalase peptide that is devoid of any intrinsic enzymatic activity, but it is equally effective as full-length recombinant renalase at protecting against toxic and ischemic injury. Using biotin transfer studies with RP-220 in the human proximal tubular cell line HK-2 and protein identification by mass spectrometry, we identified PMCA4b as a renalase binding protein. This previously characterized plasma membrane ATPase is involved in cell signaling and cardiac hypertrophy. Co-immunoprecipitation and co-immunolocalization confirmed protein-protein interaction between endogenous renalase and PMCA4b. Down-regulation of endogenous PMCA4b expression by siRNA transfection, or inhibition of its enzymatic activity by the specific peptide inhibitor caloxin1b each abrogated RP-220 dependent MAPK signaling and cytoprotection. In control studies, these maneuvers had no effect on epidermal growth factor mediated signaling, confirming specificity of the interaction between PMCA4b and renalase.PMCA4b functions as a renalase receptor, and a key mediator of renalase dependent MAPK signaling.

  5. Effect of ionizing radiation on catalytic properties of Ca2+-ATP-ase from sarcoplasmic reticulum of skeletal muscle

    International Nuclear Information System (INIS)

    Bagel', I.M.; Shafranovskaya, E.V.; Gorokh, G.A.; Markova, A.G.

    1999-01-01

    It was studied kinetic and thermodynamic characteristics of Ca 2+ -ATP-ase of rat skeletal muscle (membranes of sarcoplasmic reticulum) after irradiation in doses 0,5, 4,0 and 8,0 Gy. It was shown that external gamma-irradiation at different doses changed kinetic and thermodynamic characteristics of the enzyme of sarcoplasmic reticulum membranes of skeletal muscle. These alterations probably correlate with disbalance of hormonal regulation of intracellular calcium metabolism and changes in membrane structure and functions

  6. Isolation and characterization of DNA-dependent ATPases from the Novikoff Hepatoma

    International Nuclear Information System (INIS)

    Thomas, D.C.

    1984-01-01

    Four DNA-dependent ATPases have been purified to apparent homogeneity from extracts of the Novikoff Hepatoma, and named ATPases II, III, IV, and V. The physical and enzymological properties of ATPases II, III, and V are nearly identical, and from tryptic peptide mapping these proteins were determined to be related, though they are still chromatographically distinct; all appear to be dimers. ATPaseIV is unique among the ATPases, and is probably a monomer. ATPase V appears much more stable to thermal inactivation than the similar curves generated by ATPases II, and III. ATPase IV, however, projects of a heat-inactivation curve intermediate to these two types. ATPase II is labelled to a much higher degree than the others when treated with a heterologous protein kinase using gamma-[ 32 P]-ATP. When ATPase II was treated with this kinase, and subsequently run over a DNA-cellulose column, the profile of ATPase II was found to contain small peaks of activity in the positions where ATPases III and V normally elute, suggesting that ATPase II may be a dephosphorylated form of the other two. The ATPases have been extensively characterized with respect to reaction products and requirements, substrate utilization, DNA effector requirements, and effects of ATP analogs

  7. 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.

  8. 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

    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......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......(+),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....

  9. 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

    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(+),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.......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...

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu(+)-ATPase, ......Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu...

  11. The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.

    OpenAIRE

    Ishii, T; Takeyasu, K

    1993-01-01

    Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity,...

  12. Inactivation of mitochondrial ATPase by ultraviolet light

    International Nuclear Information System (INIS)

    Chavez, E.; Cuellar, A.

    1984-01-01

    The present work describes experiments that show that far-ultraviolet irradiation induce the inhibition of ATPase activity in both membrane-bound and soluble F1. It was also found that ultraviolet light promotes the release of tightly bound adenine nucleotides from F1-ATPase. Experiments carried out with submitochondrial particles indicate that succinate partially protects against these effects of ultraviolet light. Titration of sulfhydryl groups in both irradiated submitochondrial particles and soluble F1-ATPase indicates that a conformational change induced by photochemical modifications of amino acid residues appears involved in the inactivation of the enzyme. Finally, experiments are described which show that the tyrosine residue located in the active site of F1-ATPase is modified by ultraviolet irradiation

  13. 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.

  14. Transcriptional regulators of Na, K-ATPase subunits

    OpenAIRE

    Zhiqin eLi; Sigrid A Langhans

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during developme...

  15. The Role of the N-Domain in the ATPase Activity of the Mammalian AAA ATPase p97/VCP*

    Science.gov (United States)

    Niwa, Hajime; Ewens, Caroline A.; Tsang, Chun; Yeung, Heidi O.; Zhang, Xiaodong; Freemont, Paul S.

    2012-01-01

    p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase domains, D1 and D2; and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N-domain with D1. Here we have tested ten major inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia-linked mutants for ATPase activity and found that all have increased activity over the wild type, with one mutant, p97A232E, having three times higher activity. Further mutagenesis of p97A232E shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a coplanar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97A232E suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N-domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive. PMID:22270372

  16. The role of the N-domain in the ATPase activity of the mammalian AAA ATPase p97/VCP.

    Science.gov (United States)

    Niwa, Hajime; Ewens, Caroline A; Tsang, Chun; Yeung, Heidi O; Zhang, Xiaodong; Freemont, Paul S

    2012-03-09

    p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase domains, D1 and D2; and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N-domain with D1. Here we have tested ten major inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia-linked mutants for ATPase activity and found that all have increased activity over the wild type, with one mutant, p97(A232E), having three times higher activity. Further mutagenesis of p97(A232E) shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a coplanar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97(A232E) suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N-domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive.

  17. P4 ATPases: Flippases in Health and Disease

    Directory of Open Access Journals (Sweden)

    Coen C. Paulusma

    2013-04-01

    Full Text Available P4 ATPases catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes, a process termed “lipid flipping”. Accumulating evidence obtained in lower eukaryotes points to an important role for P4 ATPases in vesicular protein trafficking. The human genome encodes fourteen P4 ATPases (fifteen in mouse of which the cellular and physiological functions are slowly emerging. Thus far, deficiencies of at least two P4 ATPases, ATP8B1 and ATP8A2, are the cause of severe human disease. However, various mouse models and in vitro studies are contributing to our understanding of the cellular and physiological functions of P4-ATPases. This review summarizes current knowledge on the basic function of these phospholipid translocating proteins, their proposed action in intracellular vesicle transport and their physiological role.

  18. Regulation of cardiac remodeling by cardiac Na/K-ATPase isoforms

    Directory of Open Access Journals (Sweden)

    Lijun Catherine Liu

    2016-09-01

    Full Text Available Cardiac remodeling occurs after cardiac pressure/volume overload or myocardial injury during the development of heart failure and is a determinant of heart failure. Preventing or reversing remodeling is a goal of heart failure therapy. Human cardiomyocyte Na+/K+-ATPase has multiple α isoforms (1-3. The expression of the α subunit of the Na+/K+-ATPase is often altered in hypertrophic and failing hearts. The mechanisms are unclear. There are limited data from human cardiomyocytes. Abundant evidences from rodents show that Na+/K+-ATPase regulates cardiac contractility, cell signaling, hypertrophy and fibrosis. The α1 isoform of the Na+/K+-ATPase is the ubiquitous isoform and possesses both pumping and signaling functions. The α2 isoform of the Na+/K+-ATPase regulates intracellular Ca2+ signaling, contractility and pathological hypertrophy. The α3 isoform of the Na+/K+-ATPase may also be a target for cardiac hypertrophy. Restoration of cardiac Na+/K+-ATPase expression may be an effective approach for prevention of cardiac remodeling. In this article, we will overview: (1 the distribution and function of isoform specific Na+/K+-ATPase in the cardiomyocytes. (2 the role of cardiac Na+/K+-ATPase in the regulation of cell signaling, contractility, cardiac hypertrophy and fibrosis in vitro and in vivo. Selective targeting of cardiac Na+/K+-ATPase isoform may offer a new target for the prevention of cardiac remodeling.

  19. H+-ATPase activity from storage tissue of Beta vulgaris. IV. N,N'-dicyclohexylcarbodiimide binding and inhibition of the plasma membrane H+-ATPase

    International Nuclear Information System (INIS)

    Oleski, N.A.; Bennett, A.B.

    1987-01-01

    The molecular weight and isoelectric point of the plasma membrane H + -ATPase from red beet storage tissue were determined using N,N'-dicyclohexylcarbodiimide (DCCD) and a H + -ATPase antibody. When plasma membrane vesicles were incubated with 20 micromolar [ 14 C]-DCCD at 0 0 C, a single 97,000 dalton protein was visualized on a fluorography of a sodium dodecyl sulfate polyacrylamide gel. A close correlation between [ 14 C]DCCD labeling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentration suggests that this 97,000 dalton protein is a component of the plasma membrane H + -ATPase. An antibody raised against the plasma membrane H + -ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two-dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the H + -ATPase to be 6.5

  20. Characterization of the plasma membrane H+-ATPase in the liverwort Marchantia polymorpha.

    Science.gov (United States)

    Okumura, Masaki; Inoue, Shin-ichiro; Takahashi, Koji; Ishizaki, Kimitsune; Kohchi, Takayuki; Kinoshita, Toshinori

    2012-06-01

    The plasma membrane H(+)-ATPase generates an electrochemical gradient of H(+) across the plasma membrane that provides the driving force for solute transport and regulates pH homeostasis and membrane potential in plant cells. Recent studies have demonstrated that phosphorylation of the penultimate threonine in H(+)-ATPase and subsequent binding of a 14-3-3 protein is the major common activation mechanism for H(+)-ATPase in vascular plants. However, there is very little information on the plasma membrane H(+)-ATPase in nonvascular plant bryophytes. Here, we show that the liverwort Marchantia polymorpha, which is the most basal lineage of extant land plants, expresses both the penultimate threonine-containing H(+)-ATPase (pT H(+)-ATPase) and non-penultimate threonine-containing H(+)-ATPase (non-pT H(+)-ATPase) as in the green algae and that pT H(+)-ATPase is regulated by phosphorylation of its penultimate threonine. A search in the expressed sequence tag database of M. polymorpha revealed eight H(+)-ATPase genes, designated MpHA (for M. polymorpha H(+)-ATPase). Four isoforms are the pT H(+)-ATPase; the remaining isoforms are non-pT H(+)-ATPase. An apparent 95-kD protein was recognized by anti-H(+)-ATPase antibodies against an Arabidopsis (Arabidopsis thaliana) isoform and was phosphorylated on the penultimate threonine in response to the fungal toxin fusicoccin in thalli, indicating that the 95-kD protein contains pT H(+)-ATPase. Furthermore, we found that the pT H(+)-ATPase in thalli is phosphorylated in response to light, sucrose, and osmotic shock and that light-induced phosphorylation depends on photosynthesis. Our results define physiological signals for the regulation of pT H(+)-ATPase in the liverwort M. polymorpha, which is one of the earliest plants to acquire pT H(+)-ATPase.

  1. Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme

    International Nuclear Information System (INIS)

    Kasho, V.N.; Boyer, P.D.

    1989-01-01

    Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F 1 , F 0 -ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F 1 ,F 0 -ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and P i as medium ATP concentration was lowered was determined by 18 O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the P i formed as ATP concentration is decreased to the micromolar range. The F 1 ,F 0 -ATPase from neurospora mitochondria showed an event more pronounced modulation, similar to that of other F 1 -type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F 1 ,F 0 -ATPases

  2. Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

    Science.gov (United States)

    Schep, Daniel G.; Rubinstein, John L.

    2016-01-01

    Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases. PMID:26951669

  3. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

    Science.gov (United States)

    Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

    2016-05-01

    Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. © 2016 American Society of Plant Biologists. All Rights Reserved.

  4. 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)P 2 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 V o 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).

  5. Molecular aspects of calcium signalling at the crossroads of unikont and bikont eukaryote evolution--the ciliated protozoan Paramecium in focus.

    Science.gov (United States)

    Plattner, Helmut

    2015-03-01

    The ciliated protozoan, Paramecium tetraurelia has a high basic Ca(2+) leakage rate which is counteracted mainly by export through a contractile vacuole complex, based on its V-type H(+)-ATPase activity. In addition Paramecium cells dispose of P-type Ca(2+)-ATPases, i.e. a plasmamembrane and a sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (PMCA, SERCA). Antiporter systems are to be expected, as inferred from indirect evidence. Among the best known cytosolic Ca(2+)-binding proteins, calmodulin activates Ca(2+) influx channels in the somatic cell membrane, but inactivates Ca(2+) influx channels in cilia, where it, thus, ends ciliary reversal induced by depolarization via channels in the somatic cell membrane. Centrin inactivates Ca(2+) signals after stimulation by its high capacity/low affinity binding sites, whereas its high affinity sites regulate some other functions. Cortical Ca(2+) stores (alveolar sacs) are activated during stimulated trichocyst exocytosis and thereby mediate store-operated Ca(2+) entry (SOCE). Ca(2+) release channels (CRCs) localised to alveoli and underlying SOCE are considered as Ryanodine receptor-like proteins (RyR-LPs) which are members of a CRC family with 6 subfamilies. These also encompass genuine inositol 1,4,5-trisphosphate receptors (IP3Rs) and intermediates between the two channel types. All IP3R/RyR-type CRCs possess six carboxyterminal transmembrane domains (TMD), with a pore domain between TMD 5 and 6, endowed with a characteristic selectivity filter. There are reasons to assume a common ancestor molecule for such channels and diversification further on in evolution. The distinct distribution of specific CRCs in the different vesicles undergoing intracellular trafficking suggests constitutive formation of very locally restricted Ca(2+) signals during vesicle-vesicle interaction. In summary, essential steps of Ca(2+) signalling already occur at this level of evolution, including an unexpected multitude of CRCs. For dis

  6. Conditions of activation of yeast plasma membrane ATPase.

    Science.gov (United States)

    Sychrová, H; Kotyk, A

    1985-04-08

    The in vivo activation of the H+-ATPase of baker's yeast plasma membrane found by Serrano in 1983 was demonstrated with D-glucose aerobically and anaerobically (as well as in a respiration-deficient mutant) and, after suitable induction, with maltose, trehalose, and galactose. The activated but not the control ATPase was sensitive to oligomycin. No activation was possible in a cell-free extract with added glucose. The ATPase was not activated in yeast protoplasts which may account for the absence of glucose-stimulated secondary active transports in these wall-less cells and provide support for a microscopic coupling between ATPase activity and these transports in yeast cells.

  7. Arginine substitution of a cysteine in transmembrane helix M8 converts Na+,K+-ATPase to an electroneutral pump similar to H+,K+-ATPase.

    Science.gov (United States)

    Holm, Rikke; Khandelwal, Jaanki; Einholm, Anja P; Andersen, Jens P; Artigas, Pablo; Vilsen, Bente

    2017-01-10

    Na + ,K + -ATPase and H + ,K + -ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H + ,K + -ATPase avoids binding of Na + at the site corresponding to the Na + -specific site of the Na + ,K + -ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na + ,K + -ATPase with arginine, present in the H + ,K + -ATPase at the corresponding position, converted the normal 3Na + :2K + :1ATP stoichiometry of the Na + ,K + -ATPase to electroneutral 2Na + :2K + :1ATP stoichiometry similar to the electroneutral transport mode of the H + ,K + -ATPase. The electroneutral C932R mutant of the Na + ,K + -ATPase retained a wild-type-like enzyme turnover rate for ATP hydrolysis and rate of cellular K + uptake. Only a relatively minor reduction of apparent Na + affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na + affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine + group of the M8 arginine replaces Na + at the third site, thus preventing Na + binding there, although allowing Na + to bind at the two other sites and become transported. Hence, in the H + ,K + -ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

  8. The mechanism of Torsin ATPase activation.

    Science.gov (United States)

    Brown, Rebecca S H; Zhao, Chenguang; Chase, Anna R; Wang, Jimin; Schlieker, Christian

    2014-11-11

    Torsins are membrane-associated ATPases whose activity is dependent on two activating cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain-like LAP1 (LULL1). The mechanism by which these cofactors regulate Torsin activity has so far remained elusive. In this study, we identify a conserved domain in these activators that is predicted to adopt a fold resembling an AAA+ (ATPase associated with a variety of cellular activities) domain. Within these domains, a strictly conserved Arg residue present in both activating cofactors, but notably missing in Torsins, aligns with a key catalytic Arg found in AAA+ proteins. We demonstrate that cofactors and Torsins associate to form heterooligomeric assemblies with a defined Torsin-activator interface. In this arrangement, the highly conserved Arg residue present in either cofactor comes into close proximity with the nucleotide bound in the neighboring Torsin subunit. Because this invariant Arg is strictly required to stimulate Torsin ATPase activity but is dispensable for Torsin binding, we propose that LAP1 and LULL1 regulate Torsin ATPase activity through an active site complementation mechanism.

  9. The structural basis of calcium transport by the calcium pump

    DEFF Research Database (Denmark)

    Olesen, Claus; Picard, Martin; Winther, Anne-Marie Lund

    2007-01-01

    The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding,...

  10. Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca2+-ATPase

    International Nuclear Information System (INIS)

    Pestov, Nikolay B.; Dmitriev, Ruslan I.; Kostina, Maria B.; Korneenko, Tatyana V.; Shakhparonov, Mikhail I.; Modyanov, Nikolai N.

    2012-01-01

    Highlights: ► Full-length secretory pathway Ca-ATPase (SPCA2) cloned from rat duodenum. ► ATP2C2 gene (encoding SPCA2) exists only in genomes of Tetrapoda. ► Rat and pig SPCA2 are expressed in intestines, lung and some secretory glands. ► Subcellular localization of SPCA2 may depend on tissue type. ► 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 gene radiated from ATP2C1 (encoding SPCA1) during adaptation of tetrapod ancestors to terrestrial habitats.

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

    Directory of Open Access Journals (Sweden)

    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

  12. Regulation of vacuolar H+-ATPase in microglia by RANKL

    International Nuclear Information System (INIS)

    Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian; Ochotny, Noelle; Manolson, Morris F.; Holliday, L. Shannon

    2009-01-01

    Vacuolar H + -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 κB-ligand (RANKL). We found that Receptor Activator of Nuclear Factor κB (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.

  13. The non-gastric H,K-ATPase is oligomycin-sensitive and can function as an H+,NH4(+)-ATPase.

    NARCIS (Netherlands)

    Swarts, H.G.P.; Koenderink, J.B.; Willems, P.H.G.M.; Pont, J.J.H.H.M. de

    2005-01-01

    We used the baculovirus/Sf9 expression system to gain new information on the mechanistic properties of the rat non-gastric H,K-ATPase, an enzyme that is implicated in potassium homeostasis. The alpha2-subunit of this enzyme (HKalpha2) required a beta-subunit for ATPase activity thereby showing a

  14. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Science.gov (United States)

    Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

    2016-01-01

    Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase

  15. 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...

  16. Differential effects of inhibitors and detergents on the Ca2+-ATPase and Mg2+-ATPase activities of the plasma membrane of a human oat cell carcinoma

    International Nuclear Information System (INIS)

    Knowles, A.F.; Lawrence, C.M.

    1986-01-01

    Plasma membranes of human oat cell carcinoma possess Mg 2+ - and Ca 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 2+ -ATPase activity appears to be more sensitive to both DTT and FSBA. The Ca 2+ -ATPase activity is more easily inactivated by Triton X-100, while the Mg 2+ -ATPase is preferentially activated by digitonin. These differential effects of inhibitors and detergents suggest that the Ca 2+ -ATPase and Mg 2+ -ATPase are separate enzymes. Incubation of oat cell carcinoma plasma membrane with [ 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 2+ -ATPase previously purified in this laboratory. The identity of one or more of the other labelled proteins with the Mg 2+ -ATPase has not been demonstrated, but is presently under investigation

  17. P4 ATPases - lipid flippases and their role in disease

    NARCIS (Netherlands)

    Folmer, Dineke E.; Elferink, Ronald P. J. Oude; Paulusma, Coen C.

    2009-01-01

    P4 ATPases (type 4 P-type ATPases) are multispan transmembrane proteins that have been implicated in phospholipid translocation from the exoplasmic to the cytoplasmic leaflet of biological membranes. Studies in Saccharomyces cerevisiae have indicated that P4 ATPases are important in vesicle

  18. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls.

    Science.gov (United States)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T V; Alyethodi, Rafeeque R; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly (P ATPase Β1, ATPase B2, and ATPase B3 is highly correlated (P ATPase beta family genes for cellular thermotolerance in cattle.

  19. Sperm Na+, K+-ATPase and Ca2+-ATPase activity: A preliminary study of comparison of swim up and density gradient centrifugation methods for sperm preparation

    Science.gov (United States)

    Lestari, Silvia W.; Larasati, Manggiasih D.; Asmarinah, Mansur, Indra G.

    2018-02-01

    As one of the treatment for infertility, the success rate of Intrauterine Insemination (IUI) is still relatively low. Several sperm preparation methods, swim-up (SU) and the density-gradient centrifugation (DGC) are frequently used to select for better sperm quality which also contribute to IUI failure. Sperm selection methods mainly separate the motile from the immotile sperm, eliminating the seminal plasma. The sperm motility involves the structure and function of sperm membrane in maintaining the balance of ion transport system which is regulated by the Na+, K+-ATPase, and Ca2+-ATPase enzymes. This study aims to re-evaluate the efficiency of these methods in selecting for sperm before being used for IUI and based the evaluation on sperm Na+,K+-ATPase and Ca2+-ATPase activities. Fourteen infertile men from couples who underwent IUI were involved in this study. The SU and DGC methods were used for the sperm preparation. Semen analysis was performed based on the reference value of World Health Organization (WHO) 2010. After isolating the membrane fraction of sperms, the Na+, K+-ATPase activity was defined as the difference in the released inorganic phosphate (Pi) with and without the existence of 10 mM ouabain in the reaction, while the Ca2+-ATPase was determined as the difference in Pi contents with and without the existence of 55 µm CaCl2. The prepared sperm demonstrated a higher percentage of motile sperm compared to sperm from the whole semen. Additionally, the percentage of motile sperm of post-DGC showed higher result than the sperm from post-SU. The velocity of sperm showed similar pattern with the percentage of motile sperm, in which the velocity of prepared sperm was higher than the sperm from whole semen. Furthermore, the sperm velocity of post-DGC was higher compared to the sperm from post-SU. The Na+, K+-ATPase activity of prepared sperm was higher compared to whole semen, whereas Na+, K+-ATPase activity in the post DGC was higher than post SU. The Ca2

  20. 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

  1. 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

    group is propelled along against its concentration gradient with the hydrocarbon chains projecting out into the lipid phase by movement of an isoleucine located at the position corresponding to an ion binding glutamate in the Ca2+- and Na+/K+-ATPases. Hence, the P4-ATPase mechanism is quite similar...... 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....

  2. Sodium ions as substitutes for protons in the gastric H,K-ATPase

    International Nuclear Information System (INIS)

    Polvani, C.; Sachs, G.; Blostein, R.

    1989-01-01

    In view of the striking homology among various ion-translocating ATPases including Na,K-ATPase, Ca-ATPase, and H,K-ATPase, and the recent evidence that protons can replace cytoplasmic sodium as well as potassium in the reaction mechanism of the Na,K-ATPase (Polvani, C., and Blostein, R. (1988) J. Biol. Chem. 263, 16757-16763), we studied the role of sodium as a substitute for protons in the H,K-ATPase reaction. Using hog gastric H,K-ATPase-rich inside-out membrane vesicles we observed 22Na+ influx which was stimulated by intravesicular potassium ions (K+i) at pH 8.5 but not at pH 7.1. This sodium influx was observed in medium containing ATP and was inhibited by vanadate and SCH28080, a selective inhibitor of the gastric H,K-ATPase. At least 2-fold accumulation of sodium was observed at pH 8.5. Experiments aimed to determine the sidedness of the alkaline pH requirement for K+i-dependent sodium influx showed that K+i-activated sodium influx depends on pHout and is unaffected by changes in pHin. These results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na,K-ATPase and the gastric H,K-ATPase enzymes

  3. Kinase-Mediated Regulation of P4-ATPases

    DEFF Research Database (Denmark)

    Frøsig, Merethe Mørch

    designed a fast and efficient screening strategy to identify novel regulator proteins of P4-ATPases. The system is based on heterologous expression in a specially designed yeast strain, and regulatory proteins can be identified via change in activity of the P4-ATPase of interest. Hereby the first steps...

  4. Thermophilic P-loop transport ATPases : Enzyme function and energetics at high temperature

    NARCIS (Netherlands)

    Pretz, Monika Gyöngyi

    2007-01-01

    Primary transport ATPases are divided into several superfamilies; amongst others including ATPases of the ABC transporter superfamily, the F-ATPase superfamily or the motor ATPases of the General Secretory (Sec) pathway. Motor proteins from these superfamilies show a low sequence similarity, except

  5. Changes in the distribution of lens calcium during development of x-ray cataract

    International Nuclear Information System (INIS)

    Hightower, K.R.; Giblin, F.J.; Reddy, V.N.

    1983-01-01

    The present study was designed to examine the possible role of calcium in the opacification of x-ray-induced cataract in rabbit. The results demonstrate that the concentration of calcium in x-rayed lenses, just prior to lens hydration (7.5 weeks postirradiation), was twice that present in contralateral control lenses. At this stage of immature cataract, the lens nucleus remained transparent and maintained a normal level of calcium, but the lens cortex, containing regions of subcapsular opacification, accumulated a level of calcium that was twice that of the control. In the completely opaque mature cataract, (8-9 weeks post x-ray), both the cortex and nucleus had gained significant amounts of calcium. As the concentration of total calcium increased in the immature x-ray cataract, the amount of the cation bound to membranes and insoluble proteins of the cytosol also increased comparably. However, the relative proportion of calcium in the various fractions remained unaltered in the immature cataract; in both control lenses and immature cataracts, 20% of the total calcium remained in the membrane pellet and 70% was located in the soluble protein fraction. Only in the mature stage of cataract was a shift in the distribution of calcium apparent, as the proportion of calcium in the soluble protein fraction increased to 90%. Although only 7% of the total calcium in a mature cataract was bound to membrane, the amount represented a fivefold increase over the control. The results of this study demonstrate that an elevation in lens calcium accompanies the opacification process in x-ray cataract. The work also suggests that changes in calcium levels are not likely to result from inactivation of Ca-ATPase

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

    DEFF Research Database (Denmark)

    Mattle, Daniel; Sitsel, Oleg; Autzen, Henriette Elisabeth

    2013-01-01

    P-type ATPases perform active transport of various compounds across biological membranes and are crucial for ion homeostasis and the asymmetric composition of lipid bilayers. Although their functional cycle share principles of phosphoenzyme intermediates, P-type ATPases also show subclass...... 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...

  7. Anticancer ruthenium(III) complex KP1019 interferes with ATP-dependent Ca2+ translocation by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA).

    Science.gov (United States)

    Sadafi, Fabrizio-Zagros; Massai, Lara; Bartolommei, Gianluca; Moncelli, Maria Rosa; Messori, Luigi; Tadini-Buoninsegni, Francesco

    2014-08-01

    Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), a P-type ATPase that sustains Ca2+ transport and plays a major role in intracellular Ca2+ homeostasis, represents a therapeutic target for cancer therapy. Here, we investigated whether ruthenium-based anticancer drugs, namely KP1019 (indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)]), NAMI-A (imidazolium [trans-tetrachloro(1H-imidazole)(S-dimethylsulfoxide)ruthenate(III)]) and RAPTA-C ([Ru(η6-p-cymene)dichloro(1,3,5-triaza-7-phosphaadamantane)]), and cisplatin (cis-diammineplatinum(II) dichloride) might act as inhibitors of SERCA. Charge displacement by SERCA adsorbed on a solid-supported membrane was measured after ATP or Ca2+ concentration jumps. Our results show that KP1019, in contrast to the other metal compounds, is able to interfere with ATP-dependent translocation of Ca2+ ions. An IC50 value of 1 μM was determined for inhibition of calcium translocation by KP1019. Conversely, it appears that KP1019 does not significantly affect Ca2+ binding to the ATPase from the cytoplasmic side. Inhibition of SERCA at pharmacologically relevant concentrations may represent a crucial aspect in the overall pharmacological and toxicological profile of KP1019. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Characterization of sarcoplasmic reticulum Ca(2+) ATPase pumps in muscle of patients with myotonic dystrophy and with hypothyroid myopathy.

    Science.gov (United States)

    Guglielmi, V; Oosterhof, A; Voermans, N C; Cardani, R; Molenaar, J P; van Kuppevelt, T H; Meola, G; van Engelen, B G; Tomelleri, G; Vattemi, G

    2016-06-01

    Sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) pumps play the major role in lowering cytoplasmic calcium concentration in skeletal muscle by catalyzing the ATP-dependent transport of Ca(2+) from the cytosol to the lumen of the sarcoplasmic reticulum (SR). Although SERCA abnormalities have been hypothesized to contribute to the dysregulation of intracellular Ca(2+) homeostasis and signaling in muscle of patients with myotonic dystrophy (DM) and hypothyroid myopathy, the characterization of SERCA pumps remains elusive and their impairment is still unclear. We assessed the activity of SR Ca(2+)-ATPase, expression levels and fiber distribution of SERCA1 and SERCA2, and oligomerization of SERCA1 protein in muscle of patients with DM type 1 and 2, and with hypothyroid myopathy. Our data provide evidence that SR Ca(2+) ATPase activity, protein levels and muscle fiber distribution of total SERCA1 and SERCA2, and SERCA1 oligomerization pattern are similar in patients with both DM1 and DM2, hypothyroid myopathy and in control subjects. We prove that SERCA1b, the neonatal isoform of SERCA1, is expressed at protein level in muscle of patients with DM2 and, in lower amount, of patients with DM1. Our present study demonstrates that SERCA function is not altered in muscle of patients with DM and with hypothyroid myopathy. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Juel, C

    2016-04-01

    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. 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. Exposure to the NO donor spermine NONOate (10 μm) increased the maximal Na,K-ATPase activity by 27% in isolated glycolytic muscles, but had no effect in oxidative muscles. Spermine NONOate increased the maximal Na,K-ATPase activity by 58% (P Na,K-ATPase α-isoform. Incubation with cGMP (1 mm) increased the maximal Na,K-ATPase activity in homogenates from glycolytic muscle by 16% (P 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, the NO/cGMP/protein kinase G signalling pathway is involved. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  10. Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury 1

    Science.gov (United States)

    Iswari, S.; Palta, Jiwan P.

    1989-01-01

    Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a Km around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its Km was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in Km, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress. Images Figure 1 Figure 2 PMID:16666856

  11. 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

    International Nuclear Information System (INIS)

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

    1985-01-01

    Using radiation inactivation, the authors 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 60 Co. 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. They also used radiation inactivation to measure the size of the Ca 2+ -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, they directly compared the sizes of these two ATPases and found them to be essentially the same. The authors conclude that both H + -ATPase and Ca 2+ -ATPase are oligomeric enzymes, most likely composed of two approximately 100,000-dalton polypeptides

  12. Influence of hexavanadates on Na+/K+- ATPase activity

    Directory of Open Access Journals (Sweden)

    Zdravković Aleksandra

    2016-01-01

    Full Text Available Introduction: There is a great interest in use of polioximetalates in clinical medicine, primary as antibacterial, antiviral and antitumoral agents. Considering the key role of Na+/ K+- ATPase in normal functioning of most animal cells, as well as pivotal roles in cancer cell migration, the aim of this paper was to examine the influence of new synthesized hexavandates [V6-CH3][Na]2, [V6-NO2][TBA]2, [V6-C3][H]2, [V6-C5d][TBA]2 on Na+/K+- ATPase activity. Material and methods: The enzymatic activity of porcine cerebral cortex Na+/K+- ATPase was followed in both the absence and presence of increasing concentration of [V6-CH3] [Na]2, [V6-NO2][TBA]2, [V6-C3][H]2, [V6-C5d][TBA]2 (within the range 10-8 - 10-3 mol/L. The released Pi, liberated from the enzymatic hydrolysis of ATP, was determined by spectrophotometric method, using Perkin Elmer Lambda 35 UV-VIS spectrophotometer. Results: Investigated compounds inhibit the activity of Na+/K+ ATPase in dose-dependent manner within the investigated range. Obtained results indicate that all investigated compounds inhibit the Na+/K+ ATPase activity, but with different inhibiting power. [V6-NO2] [TBA]2 (IC50 = 1,87 × 10-5 mol/L was the most potent inhibitor of Na+/K+ ATPase, while [V6-C5d][TBA]2 showed the least potent inhibiting power (IC50 = 1,31 × 10-4 mol/L . The results are consistent with previously published concentration-dependent inhibitory effect of polyoxometalates (including polioxovandates on ATPase activity from different model syistems. Conclusion: Based on the results, we can conclude that the examined compounds inhibit Na+/K+- ATPase activity in a dose-dependent manner. Inhibiting power of tested hexavanadates are different, and weaker than inhibiting power of decavanadates (tested earlier on Na+/K+- ATPase activity, which is probably due to differences in charge, size and shape of these polioxometalates. Considering the role of this enzymes in the functioning of healthy cells and the

  13. Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines.

    Science.gov (United States)

    Monroe, Nicole; Hill, Christopher P

    2016-05-08

    Meiotic clade AAA ATPases (ATPases associated with diverse cellular activities), which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT (endosomal sorting complexes required for transport) pathway and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT (microtubule interacting and trafficking) domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and they appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops and that recruitment to the protein lattice via MIT domains also relieves autoinhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, as well as potentially katanin and fidgetin, are highly coordinated. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Fast calcium transients translate the distribution and conduction of neural activity in different regions of a single sensory neuron.

    Science.gov (United States)

    Purali, Nuhan

    2017-09-01

    In the present study, cytosolic calcium concentration changes were recorded in response to various forms of excitations, using the fluorescent calcium indicator dye OG-BAPTA1 together with the current or voltage clamp methods in stretch receptor neurons of crayfish. A single action potential evoked a rise in the resting calcium level in the axon and axonal hillock, whereas an impulse train or a large saturating current injection would be required to evoke an equivalent response in the dendrite region. Under voltage clamp conditions, amplitude differences between axon and dendrite responses vanished completely. The fast activation time and the modulation of the response by extracellular calcium concentration changes indicated that the evoked calcium transients might be mediated by calcium entry into the cytosol through a voltage-gated calcium channel. The decay of the responses was slow and sensitive to extracellular sodium and calcium concentrations as well as exposure to 1-10 mM NiCl 2 and 10-500 µM lanthanum. Thus, a sodium calcium exchanger and a calcium ATPase might be responsible for calcium extrusion from the cytosol. Present results indicate that the calcium indicator OG-BAPTA1 might be an efficient but indirect way of monitoring regional membrane potential differences in a single neuron.

  15. Functional Analysis of P4-ATPases

    DEFF Research Database (Denmark)

    Theorin, Lisa

    and mammalian P4-ATPases have been studied extensively and the physiological function is mostly known, while the exact biochemistry and specific activity is mostly unknown. Even though the plant Arabidopsis thaliana has 12 P4-ATPases, not much is known about their function. In this study, the biochemical...... for purification of the complex by one-step purification. The ATPase activity of the ALA2/ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine. Changes in the phosphatidylserine headgroup or alteration of the stereochemistry affected enzymatic activity. The results demonstrate that ALA2...... is specific for phosphatidylserine and that binding of the lipid to the substrate binding site requires a unique spatial configuration of the lipid head group. Detailed information on the substrate requirements lead the way towards the full function and transport pathway of lipid flippases in plants. Recent...

  16. Overexpression of Sarcoendoplasmic Reticulum Calcium ATPase 2a Promotes Cardiac Sympathetic Neurotransmission via Abnormal Endoplasmic Reticulum and Mitochondria Ca2+ Regulation

    Science.gov (United States)

    Shanks, Julia; Herring, Neil; Johnson, Errin; Liu, Kun; Li, Dan

    2017-01-01

    Reduced cardiomyocyte excitation–contraction coupling and downregulation of the SERCA2a (sarcoendoplasmic reticulum calcium ATPase 2a) is associated with heart failure. This has led to viral transgene upregulation of SERCA2a in cardiomyocytes as a treatment. We hypothesized that SERCA2a gene therapy expressed under a similar promiscuous cytomegalovirus promoter could also affect the cardiac sympathetic neural axis and promote sympathoexcitation. Stellate neurons were isolated from 90 to 120 g male, Sprague–Dawley, Wistar Kyoto, and spontaneously hypertensive rats. Neurons were infected with Ad-mCherry or Ad-mCherry-hATP2Aa (SERCA2a). Intracellular Ca2+ changes were measured using fura-2AM in response to KCl, caffeine, thapsigargin, and carbonylcyanide-p-trifluoromethoxyphenylhydrazine to mobilize intracellular Ca2+ stores. The effect of SERCA2a on neurotransmitter release was measured using [3H]-norepinephrine overflow from 340 to 360 g Sprague–Dawley rat atria in response to right stellate ganglia stimulation. Upregulation of SERCA2a resulted in greater neurotransmitter release in response to stellate stimulation compared with control (empty: 98.7±20.5 cpm, n=7; SERCA: 186.5±28.41 cpm, n=8; Pneurons, SERCA2a overexpression facilitated greater depolarization-induced Ca2+ transients (empty: 0.64±0.03 au, n=57; SERCA: 0.75±0.03 au, n=68; Pneurons resulted in increased neurotransmission and increased Ca2+ loading into intracellular stores. Whether the increased Ca2+ transient and neurotransmission after SERCA2A overexpression contributes to enhanced sympathoexcitation in heart failure patients remains to be determined. PMID:28223472

  17. Stochastic Four-State Mechanochemical Model of F1-ATPase

    International Nuclear Information System (INIS)

    Wu Weixia; Zhan Yong; Zhao Tongjun; Han Yingrong; Chen Yafei

    2010-01-01

    F 1 -ATPase, a part of ATP synthase, can synthesize and hydrolyze ATP moleculars in which the central γ-subunit rotates inside the α 3 β 3 cylinder. A stochastic four-state mechanochemical coupling model of F 1 -ATPase is studied with the aid of the master equation. In this model, the ATP hydrolysis and synthesis are dependent on ATP, ADP, and Pi concentrations. The effects of ATP concentration, ADP concentration, and the external torque on the occupation probability of binding-state, the rotation rate and the diffusion coefficient of F 1 -ATPase are investigated. Moreover, the results from this model are compared with experiments. The mechanochemical mechanism F 1 -ATPase is qualitatively explained by the model. (general)

  18. Regulation of alpha1 Na/K-ATPase expression by cholesterol.

    Science.gov (United States)

    Chen, Yiliang; Li, Xin; Ye, Qiqi; Tian, Jiang; Jing, Runming; Xie, Zijian

    2011-04-29

    We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol trafficking to the plasma membrane by compound U18666A had the same effect on α1 Na/K-ATPase. Similarly, the expression of α1, but not α2 and α3, Na/K-ATPase was significantly reduced in the target organs of Niemann-Pick type C mice where the intracellular cholesterol trafficking is blocked. Mechanistically, decreases in the plasma membrane cholesterol activated Src kinase and stimulated the endocytosis and degradation of α1 Na/K-ATPase through Src- and ubiquitination-dependent pathways. Thus, the new findings, taken together with what we have already reported, revealed a previously unrecognized feed-forward mechanism by which cells can utilize the Src-dependent interplay among Na/K-ATPase, caveolin-1, and cholesterol to effectively alter the structure and function of the plasma membrane.

  19. Specialized functional diversity and interactions of the Na,K-ATPase

    Directory of Open Access Journals (Sweden)

    Igor I. Krivoi

    2016-05-01

    Full Text Available 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 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.

  20. MicroRNAs and cardiac sarcoplasmic reticulum calcium ATPase-2 in human myocardial infarction: expression and bioinformatic analysis.

    Science.gov (United States)

    Boštjančič, Emanuela; Zidar, Nina; Glavač, Damjan

    2012-10-15

    Cardiac sarco(endo)plasmic reticulum calcium ATPase-2 (SERCA2) plays one of the central roles in myocardial contractility. Both, SERCA2 mRNA and protein are reduced in myocardial infarction (MI), but the correlation has not been always observed. MicroRNAs (miRNAs) act by targeting 3'-UTR mRNA, causing translational repression in physiological and pathological conditions, including cardiovascular diseases. One of the aims of our study was to identify miRNAs that could influence SERCA2 expression in human MI. The protein SERCA2 was decreased and 43 miRNAs were deregulated in infarcted myocardium compared to corresponding remote myocardium, analyzed by western blot and microRNA microarrays, respectively. All the samples were stored as FFPE tissue and in RNAlater. miRNAs binding prediction to SERCA2 including four prediction algorithms (TargetScan, PicTar, miRanda and mirTarget2) identified 213 putative miRNAs. TAM and miRNApath annotation of deregulated miRNAs identified 18 functional and 21 diseased states related to heart diseases, and association of the half of the deregulated miRNAs to SERCA2. Free-energy of binding and flanking regions (RNA22, RNAfold) was calculated for 10 up-regulated miRNAs from microarray analysis (miR-122, miR-320a/b/c/d, miR-574-3p/-5p, miR-199a, miR-140, and miR-483), and nine miRNAs deregulated from microarray analysis were used for validation with qPCR (miR-21, miR-122, miR-126, miR-1, miR-133, miR-125a/b, and miR-98). Based on qPCR results, the comparison between FFPE and RNAlater stored tissue samples, between Sybr Green and TaqMan approaches, as well as between different reference genes were also performed. Combing all the results, we identified certain miRNAs as potential regulators of SERCA2; however, further functional studies are needed for verification. Using qPCR, we confirmed deregulation of nine miRNAs in human MI, and show that qPCR normalization strategy is important for the outcome of miRNA expression analysis in human MI.

  1. Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-β assembly.

    Science.gov (United States)

    Ohnishi, Takayuki; Yanazawa, Masako; Sasahara, Tomoya; Kitamura, Yasuki; Hiroaki, Hidekazu; Fukazawa, Yugo; Kii, Isao; Nishiyama, Takashi; Kakita, Akiyoshi; Takeda, Hiroyuki; Takeuchi, Akihide; Arai, Yoshie; Ito, Akane; Komura, Hitomi; Hirao, Hajime; Satomura, Kaori; Inoue, Masafumi; Muramatsu, Shin-ichi; Matsui, Ko; Tada, Mari; Sato, Michio; Saijo, Eri; Shigemitsu, Yoshiki; Sakai, Satoko; Umetsu, Yoshitaka; Goda, Natsuko; Takino, Naomi; Takahashi, Hitoshi; Hagiwara, Masatoshi; Sawasaki, Tatsuya; Iwasaki, Genji; Nakamura, Yu; Nabeshima, Yo-ichi; Teplow, David B; Hoshi, Minako

    2015-08-11

    Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid β-protein (Aβ) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aβ oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na(+)/K(+)-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aβ-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.

  2. LOCALIZATION OF Na+, K+-ATPASE AND OTHER ENZYMES IN TELEOST PSEUDOBRANCH

    Science.gov (United States)

    Dendy, Leslie A.; Deter, Russell L.; Philpott, Charles W.

    1973-01-01

    In an effort to determine the subcellular localization of sodium- and potassium-activated adenosine triphosphatase (Na+, K+-ATPase) in the pseudobranch of the pinfish Lagodon rhomboides, this tissue was fractionated by differential centrifugation and the activities of several marker enzymes in the fractions were measured. Cytochrome c oxidase was found primarily in the mitochondrial-light mitochondrial (M+L) fraction. Phosphoglucomutase appeared almost exclusively in the soluble (S) fraction. Monoamine oxidase was concentrated in the nuclear (N) fraction, with a significant amount also in the microsomal (P) fraction but little in M+L or S. Na+, K+-ATPase and ouabain insensitive Mg2+-ATPase were distributed in N, M+L, and P, the former having its highest specific activity in P and the latter in M+L. Rate sedimentation analysis of the M+L fraction indicated that cytochrome c oxidase and Mg2+-ATPase were associated with a rapidly sedimenting particle population (presumably mitochondria), while Na+, K+-ATPase was found primarily in a slowly sedimenting component. At least 75% of the Na+, K+-ATPase in M+L appeared to be associated with structures containing no Mg2+-ATPase. Kinetic properties of the two ATPases were studied in the P fraction and were typical of these enzymes in other tissues. Na+, K+-ATPase activity was highly dependent on the ratio of Na+ and K+ concentrations but independent of absolute concentrations over at least a fourfold range. PMID:4349221

  3. Some assembly required: Contributions of Tom Stevens' lab to the V-ATPase field.

    Science.gov (United States)

    Graham, Laurie A; Finnigan, Gregory C; Kane, Patricia M

    2018-06-01

    Tom Stevens' lab has explored the subunit composition and assembly of the yeast V-ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V-ATPase proton pumps and led to the discovery of protein splicing of the V-ATPase catalytic subunit. The Vma - phenotype, characteristic of loss-of-V-ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics. V-ATPase subunit composition proved to be highly conserved among eukaryotes. Genetic screens for new vma mutants led to identification of a set of dedicated V-ATPase assembly factors and helped unravel the complex pathways for V-ATPase assembly. In later years, exploration of the evolutionary history of several V-ATPase subunits provided new information about the enzyme's structure and function. This review highlights V-ATPase work in the Stevens' lab between 1987 and 2017. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. 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)

    Marcelo da Cunha Amaral

    2001-03-01

    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.Este trabalho teve como objetivo conhecer os mecanismos de tolerância às variações de salinidade, pelas enzimas Mg++ - ATPase e Na+ K+ - ATPase, encontrada nas brânquias de Poecilia vivipara. No campo, foi observada a presença desta espécie em salinidades entre 0 e 28‰. No laboratório, os indivíduos foram mantidos em salinidade de 30‰ e responderam positivamente. Os indivíduos adultos, coletados em uma lagoa na praia dos Coqueiros, foram utilizados como matrizes. Nos experimentos foram usados alevinos que nasceram nos aquários testes. Para cada salinidade estudada foram feitas três réplicas com três espécimens em cada uma. Os alevinos foram mantidos em salinidades de 5, 10, 15, 20, 25, 30 e 35‰, durante um mês para total adaptação. As br

  5. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Directory of Open Access Journals (Sweden)

    Qingxi Yue

    Full Text Available Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the

  6. 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.

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

    Science.gov (United States)

    Qiu, Li Yan; Krieger, Elmar; Schaftenaar, Gijs; Swarts, Herman G P; Willems, Peter H G M; De Pont, Jan Joep H H M; Koenderink, Jan B

    2005-09-16

    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 started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.

  8. In and out of the cation pumps: P-type ATPase structure revisited

    DEFF Research Database (Denmark)

    Bublitz, Maike; Poulsen, Hanne; Morth, Jens Preben

    2010-01-01

    Active transport across membranes is a crucial requirement for life. P-type ATPases build up electrochemical gradients at the expense of ATP by forming and splitting a covalent phosphoenzyme intermediate, coupled to conformational changes in the transmembrane section where the ions are translocated....... The marked increment during the last three years in the number of crystal structures of P-type ATPases has greatly improved our understanding of the similarities and differences of pumps with different ion specificities, since the structures of the Ca2+-ATPase, the Na+,K+-ATPase and the H+-ATPase can now...

  9. Two types of essential carboxyl groups in Rhodospirillum rubrum proton ATPase

    International Nuclear Information System (INIS)

    Ceccarelli, E.; Vallejos, R.H.

    1983-01-01

    Two different types of essential carboxyl groups were detected in the extrinsic component of the proton ATPase of Rhodospirillum rubrum. Chemical modification of R. rubrum chromatophores or its solubilized ATPase by Woodward's reagent K resulted in inactivation of photophosphorylating and ATPase activities. The apparent order of reaction was nearly 1 with respect to reagent concentration and similar K1 were obtained for the soluble and membrane-bound ATPases suggesting that inactivation was associated with modification of one essential carboxyl group located in the soluble component of the proton ATPase. Inactivation was prevented by adenine nucleotides but not by divalent cations. Dicyclohexylcarbodiimide completely inhibited the solubilized ATPase with a K1 of 5.2 mM and a K2 of 0.81 min-1. Mg2+ afforded nearly complete protection with a Kd of 2.8 mM. Two moles of [14C]dicyclohexylcarbodiimide were incorporated per mole of enzyme for complete inactivation but in the presence of 30 mM MgCl2 only one mole was incorporated and there was no inhibition. The labeling was recovered mostly from the beta subunit. The incorporation of the labeled reagent into the ATPase was not prevented by previous modification with Woodward's reagent K. It is concluded that both reagents modified two different essential carboxyl groups in the soluble ATPase from R. rubrum

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

    African Journals Online (AJOL)

    A plasma membrane H + ATPase gene is germinationinduced in wheat embryos. ... African Journal of Biotechnology ... of a germination specific plasma membrane H+-ATPase was analyzed by RTPCR and in situ RNA hybridization methods.

  11. MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly.

    Science.gov (United States)

    Sharma, Stuti; Oot, Rebecca A; Wilkens, Stephan

    2018-05-12

    Vacuolar H+-ATPases (V-ATPases; V1Vo-ATPases) are rotary motor proton pumps that acidify intracellular compartments and in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo. H is required for MgATPase activity in holo V-ATPase, but also for stabilizing the MgADP inhibited state in membrane detached V1. However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunit H (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysis-driven conformational changes in V1 destabilized the interaction of H, or HNT, with V1ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V1 points to H's role in the mechanism of reversible disassembly. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  12. 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...

  13. Regulation of branchial V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 in response to acid and base infusions in the Pacific spiny dogfish (Squalus acanthias).

    Science.gov (United States)

    Tresguerres, Martin; Katoh, Fumi; Fenton, Heather; Jasinska, Edyta; Goss, Greg G

    2005-01-01

    To study the mechanisms of branchial acid-base regulation, Pacific spiny dogfish were infused intravenously for 24 h with either HCl (495+/- 79 micromol kg(-1) h(-1)) or NaHCO(3) (981+/-235 micromol kg(-1) h(-1)). Infusion of HCl produced a transient reduction in blood pH. Despite continued infusion of acid, pH returned to normal by 12 h. Infusion of NaHCO(3) resulted in a new steady-state acid-base status at approximately 0.3 pH units higher than the controls. Immunostained serial sections of gill revealed the presence of separate vacuolar proton ATPase (V-H(+)-ATPase)-rich or sodium-potassium ATPase (Na(+)/K(+)-ATPase)-rich cells in all fish examined. A minority of the cells also labeled positive for both transporters. Gill cell membranes prepared from NaHCO(3)-infused fish showed significant increases in both V-H(+)-ATPase abundance (300+/-81%) and activity. In addition, we found that V-H(+)-ATPase subcellular localization was mainly cytoplasmic in control and HCl-infused fish, while NaHCO(3)-infused fish demonstrated a distinctly basolateral staining pattern. Western analysis in gill membranes from HCl-infused fish also revealed increased abundance of Na(+)/H(+) exchanger 2 (213+/-5%) and Na(+)/K(+)-ATPase (315+/-88%) compared to the control.

  14. 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

    pumps are very homologous, and at least one of the phosphorylation sites is conserved, namely a cAMP activated protein kinase (PKA) site, which is important for regulating pumping activity, either by changing the cellular distribution of the ATPases or by directly altering the kinetic properties...

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

    Science.gov (United States)

    Huda, Kazi Md Kamrul; Banu, Mst Sufara Akhter; Pathi, Krishna Mohan; Tuteja, Narendra

    2013-01-01

    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 nature of this

  16. 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

  17. Thapsigargin defines the roles of cellular calcium in secretagogue-stimulated enzyme secretion from pancreatic acini.

    Science.gov (United States)

    Metz, D C; Patto, R J; Mrozinski, J E; Jensen, R T; Turner, R J; Gardner, J D

    1992-10-15

    In the present study we used thapsigargin (TG), an inhibitor of microsomal calcium ATPase, to evaluate the roles of free cytoplasmic calcium and intracellular stored calcium in secretagogue-stimulated enzyme secretion from rat pancreatic acini. Using microspectrofluorimetry of fura-2-loaded pancreatic acini, we found that TG caused a sustained increase in free cytoplasmic calcium by mobilizing calcium from inositol 1,4,5-trisphosphate-sensitive intracellular stores and by increasing influx of extracellular calcium. TG also caused a small increase in basal amylase secretion, inhibited the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate, and potentiated the stimulation of amylase secretion caused by 12-O-tetradecanoylphorbol-13-acetate or secretagogues that increase cyclic adenosine 3',5'-monophosphate. Bombesin, which like TG increased free cytoplasmic calcium, also potentiated the stimulation of amylase secretion caused by secretagogues that increase cyclic adenosine 3',5'-monophosphate, but did not inhibit the stimulation of amylase secretion caused by secretagogues that increase inositol 1,4,5-trisphosphate. Finally, TG inhibited the sustained phase of cholecystokinin-stimulated amylase secretion and potentiated the time course of vasoactive intestinal peptide-stimulated amylase secretion. The present findings indicate that stimulation of amylase secretion by secretagogues that increase inositol 1,4,5-trisphosphate does not depend on increased free cytoplasmic calcium per se. In contrast, TG-induced potentiation of the stimulation of secretagogues that increase cellular cyclic adenosine 3',5'-monophosphate appears to result from increased free cytoplasmic calcium per se.

  18. 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...

  19. FGF-23 dysregulates calcium homeostasis and electrophysiological properties in HL-1 atrial cells.

    Science.gov (United States)

    Kao, Yu-Hsun; Chen, Yao-Chang; Lin, Yung-Kuo; Shiu, Rong-Jie; Chao, Tze-Fan; Chen, Shih-Ann; Chen, Yi-Jen

    2014-08-01

    Fibroblast growth factor (FGF)-23 is a key regulator of phosphate homeostasis. Higher FGF-23 levels are correlated with poor outcomes in cardiovascular diseases. FGF-23 can produce cardiac hypertrophy and increase intracellular calcium, which can change cardiac electrical activity. However, it is not clear whether FGF-23 possesses arrhythmogenic potential through calcium dysregulation. Therefore, the purposes of this study were to evaluate the electrophysiological effects of FGF-23 and identify the underlying mechanisms. Patch clamp, confocal microscope with Fluo-4 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, calcium homeostasis and calcium regulatory proteins in HL-1 atrial myocytes with and without FGF-23 (10 and 25 ng/mL) incubation for 24 h. FGF-23 (25 ng/mL) increased L-type calcium currents, calcium transient and sarcoplasmic reticulum Ca(2+) contents in HL-1 cells. FGF-23 (25 ng/mL)-treated cells (n = 14) had greater incidences (57%, 17% and 15%, P calcium/calmodulin-dependent protein kinase IIδ and phospholamban (PLB) at threonine 17 but had similar phosphorylation extents of PLB at serine 16, total PLB and sarcoplasmic reticulum Ca(2+) -ATPase protein. Moreover, the FGF receptor inhibitor (PD173074, 10 nM), calmodulin inhibitor (W7, 5 μM) and phospholipase C inhibitor (U73122, 1 μM) attenuated the effects of FGF-23 on calcium/calmodulin-dependent protein kinase II phosphorylation. FGF-23 increases HL-1 cells arrhythmogenesis with calcium dysregulation through modulating calcium-handling proteins. © 2014 Stichting European Society for Clinical Investigation Journal Foundation.

  20. Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

    Science.gov (United States)

    Al-bataineh, Mohammad M.; Gong, Fan; Marciszyn, Allison L.; Myerburg, Michael M.

    2014-01-01

    The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress. PMID:24553431

  1. Regulation of glycolytic oscillations by mitochondrial and plasma membrane H+-ATPases

    DEFF Research Database (Denmark)

    Olsen, Lars Folke; Andersen, Ann Zahle; Lunding, Anita

    2009-01-01

    ,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol....../ATP antiporter and the mitochondrial F(0)F(1)-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F(0)F(1)-ATPase and plasma membrane H(+)-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations...

  2. Single-molecule analysis of inhibitory pausing states of V1-ATPase.

    Science.gov (United States)

    Uner, Naciye Esma; Nishikawa, Yoshihiro; Okuno, Daichi; Nakano, Masahiro; Yokoyama, Ken; Noji, Hiroyuki

    2012-08-17

    V(1)-ATPase, the hydrophilic V-ATPase domain, is a rotary motor fueled by ATP hydrolysis. Here, we found that Thermus thermophilus V(1)-ATPase shows two types of inhibitory pauses interrupting continuous rotation: a short pause (SP, 4.2 s) that occurred frequently during rotation, and a long inhibitory pause (LP, >30 min) that terminated all active rotations. Both pauses occurred at the same angle for ATP binding and hydrolysis. Kinetic analysis revealed that the time constants of inactivation into and activation from the SP were too short to represent biochemically predicted ADP inhibition, suggesting that SP is a newly identified inhibitory state of V(1)-ATPase. The time constant of inactivation into LP was 17 min, consistent with one of the two time constants governing the inactivation process observed in bulk ATPase assay. When forcibly rotated in the forward direction, V(1) in LP resumed active rotation. Solution ADP suppressed the probability of mechanical activation, suggesting that mechanical rotation enhanced inhibitory ADP release. These features were highly consistent with mechanical activation of ADP-inhibited F(1), suggesting that LP represents the ADP-inhibited state of V(1)-ATPase. Mechanical activation largely depended on the direction and angular displacement of forced rotation, implying that V(1)-ATPase rotation modulates the off rate of ADP.

  3. Long-term regulation of Na,K-ATPase pump during T-cell proliferation.

    Science.gov (United States)

    Karitskaya, Inna; Aksenov, Nikolay; Vassilieva, Irina; Zenin, Valerii; Marakhova, Irina

    2010-09-01

    The aim of the study was to elucidate the mechanism responsible for the proliferation-related regulation of Na,K-ATPase pump. Our data demonstrate that in mitogen-stimulated human blood lymphocytes, enhanced ouabain-sensitive Rb(K) fluxes in the middle/late stage of G(0)/G(1)/S transit are associated with the increased number of Na,K-ATPase pumps expressed at the cell surface (as determined by the [(3)H]ouabain binding). Analysis of total RNA (reverse transcription-polymerase chain reaction) and protein (Western blotting) showed a threefold increase in the level of Na,K-ATPase alpha1-subunit and beta1-subunit mRNAs and significant increase in the Na,K-ATPase alpha1-subunit protein during the first day of mitogen-induced proliferation. The elevated K transport as well as the increased expression of Na,K-ATPase is closely associated with the IL-2-dependent stage of T-cell response. The pharmacological inhibition of IL-2-induced MEK/ERK or JAK/STAT cascades suppressed the IL-2-induced proliferation and reduced the functional and protein expressions of Na,K-ATPase. It is concluded that during the lymphocyte transition from resting stage to proliferation, (1) long-term activation of Na,K-ATPase pump is due to the enhanced expression of Na,K-ATPase protein and mRNA, and (2) the cytokine signaling via the IL-2 receptor is necessary for the cell cycle-associated upregulation of Na,K-ATPase.

  4. Radioprotector modifying influence upon the ion transport ATPase activities

    International Nuclear Information System (INIS)

    Dvoretsky, A.I.; Egorova, E.G.; Ananieva, T.V.; Kulikova, I.A.

    1993-01-01

    The effects of aminothiol and biogenic amine radioprotectors (β-mercaptoethylamine, AET, serotonin, dopamine, histamine) on the basic ion transport enzymes, such as Na, K-ATP ase and Mg, Ca-ATPase activities were investigated in the tissues of numerous organs, with different radiosensitivity in the wistar rats. Experimental results showed that intraperitoneal injection of the used radioprotectors caused preliminary inhibition of the Na, K-ATPase activity in tissues from organs with different radioresistance, but had no influence on the Mg, Ca-ATPase activity in membranes of erythrocytes and rat brain cells. (2 tabs.)

  5. Porphyromonas gingivalis is highly sensitive to inhibitors of a proton-pumping ATPase.

    Science.gov (United States)

    Sekiya, Mizuki; Shimoyama, Yu; Ishikawa, Taichi; Sasaki, Minoru; Futai, Masamitsu; Nakanishi-Matsui, Mayumi

    2018-04-15

    Porphyromonas gingivalis is a well-known Gram-negative bacterium that causes periodontal disease. The bacterium metabolizes amino acids and peptides to obtain energy. An ion gradient across its plasma membrane is thought to be essential for nutrient import. However, it is unclear whether an ion-pumping ATPase responsible for the gradient is required for bacterial growth. Here, we report the inhibitory effect of protonophores and inhibitors of a proton-pumping ATPase on the growth of P. gingivalis. Among the compounds examined, curcumin and citreoviridin appreciably reduced the bacterial growth. Furthermore, these compounds inhibited the ATPase activity in the bacterial membrane, where the A-type proton-pumping ATPase (A-ATPase) is located. This study suggests that curcumin and citreoviridin inhibit the bacterial growth by inhibiting the A-ATPase in the P. gingivalis membrane. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Production and characterization of a monoclonal antibody to H+ATPase

    International Nuclear Information System (INIS)

    Yurko, M.; Fitch, F; Gluck, S.

    1986-01-01

    Acidification of endocytic vesicles is carried out by an ATP-dependent proton pump, H+ATPase, an FOF1 type enzyme comprised of at least 5 major subunits of 70, 56, 45, 35, and 17 kDa. A monoclonal antibody, H6.1, to H+ATPase from bovine kidney medulla, was raised to enable the structural characterization and localization of the pump. Several criteria were used to show that H6.1 recognized H+ATPase. 1.) H6.1 immunoprecipitated N-ethylmaleimide-sensitive and vanadate- and azide-insensitive solubilized ATPase activity (and GTPase activity) from both crude and purified enzyme preparations. 2.) H6.1 immunoprecipitated oligomycin-insensitive ATP-dependent proton transporting vesicles made from bovine kidney medulla, rat kidney, and CHO cells. 3.) H6.1 specifically immuno-precipitated the 5 subunits of H+ATPase from a partially purified preparation of the enzyme that had been labelled with I-125. H6.1 was then used as an immunocytochemical probe for the localization of H+ATPase. In bovine kidney medullary collecting duct, there was an intense apical staining of selected cells. In proximal tubule and in cultured CHO cells there was a granular pattern of staining characteristic of endocytic vesicles and lysosomes, suggesting that the kidney and CHO cell proton pumps are structurally related

  7. Cloning of a neonatal calcium atpase isoform (SERCA 1B) from extraocular muscle of adult blue marlin (Makaira nigricans).

    Science.gov (United States)

    Londraville, R L; Cramer, T D; Franck, J P; Tullis, A; Block, B A

    2000-10-01

    Complete cDNAs for the fast-twitch Ca2+ -ATPase isoform (SERCA 1) were cloned and sequenced from blue marlin (Makaira nigricans) extraocular muscle (EOM). Complete cDNAs for SERCA 1 were also cloned from fast-twitch skeletal muscle of the same species. The two sequences are identical over the coding region except for the last five codons on the carboxyl end; EOM SERCA 1 cDNA codes for 996 amino acids and the fast-twitch cDNAs code for 991 aa. Phylogenetic analysis revealed that EOM SERCA 1 clusters with an isoform of Ca2+ -ATPase normally expressed in early development of mammals (SERCA 1B). This is the first report of SERCA 1B in an adult vertebrate. RNA hybridization assays indicate that 1B expression is limited to extraocular muscles. Because EOM gives rise to the thermogenic heater organ in marlin, we investigated whether SERCA 1B may play a role in heat generation, or if 1B expression is common in EOM among vertebrates. Chicken also expresses SERCA 1B in EOM, but rat expresses SERCA 1A; because SERCA 1B is not specific to heater tissue we conclude it is unlikely that it plays a specific role in intracellular heat production. Comparative sequence analysis does reveal, however, several sites that may be the source of functional differences between fish and mammalian SERCAs.

  8. Different thermostabilities of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases from rabbit and trout muscles.

    Science.gov (United States)

    de Toledo, F G; Albuquerque, M C; Goulart, B H; Chini, E N

    1995-05-01

    Trout and rabbit (Ca2+ + Mg2+)-ATPases from sarcoplasmic reticulum were compared for differences in thermal inactivation and susceptibility to trypsin digestion. The trout ATPase is more heat-sensitive than the rabbit ATPase and is stabilized by Ca2+, Na+, K+ and nucleotides. Solubilization of both ATPases shows that the two ATPases have different protein-intrinsic inactivation kinetics. When digested by trypsin, the two ATPases display different cleavage patterns. The present results indicate that the trout and rabbit ATPases have dissimilarities in protein structure that may explain the differences in thermal inactivation kinetics.

  9. 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.

  10. Oxidative stress (glutathionylation and Na,K-ATPase activity in rat skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Carsten Juel

    Full Text Available Changes in ion distribution across skeletal muscle membranes during muscle activity affect excitability and may impair force development. These changes are counteracted by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for skeletal muscle function. The present study investigated the presence of oxidative stress (glutathionylation on the Na,K-ATPase in rat skeletal muscle membranes.Immunoprecipitation with an anti-glutathione antibody and subsequent immunodetection of Na,K-ATPase protein subunits demonstrated 9.0±1.3% and 4.1±1.0% glutathionylation of the α isoforms in oxidative and glycolytic skeletal muscle, respectively. In oxidative muscle, 20.0±6.1% of the β1 units were glutathionylated, whereas 14.8±2.8% of the β2-subunits appear to be glutathionylated in glycolytic muscle. Treatment with the reducing agent dithiothreitol (DTT, 1 mM increased the in vitro maximal Na,K-ATPase activity by 19% (P<0.05 in membranes from glycolytic muscle. Oxidized glutathione (GSSG, 0-10 mM increased the in vitro glutathionylation level detected with antibodies, and decreased the in vitro maximal Na,K-ATPase activity in a dose-dependent manner, and with a larger effect in oxidative compared to glycolytic skeletal muscle.This study demonstrates the existence of basal glutathionylation of both the α and the β units of rat skeletal muscle Na,K-ATPase. In addition, the study suggests a negative correlation between glutathionylation levels and maximal Na,K-ATPase activity.Glutathionylation likely contributes to the complex regulation of Na,K-ATPase function in skeletal muscle. Especially, glutathionylation induced by oxidative stress may have a role in Na,K-ATPase regulation during prolonged muscle activity.

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Characterization of the vacuolar H sup + -ATPase of higher plants

    Energy Technology Data Exchange (ETDEWEB)

    Manolson, M F

    1988-01-01

    The tonoplast H{sup +}-ATPase of Beta vulgaris L. was partially purified by Triton X-100 solubilization and Sepharose 4B chromatography resulting in the enrichment of two polypeptides. Kinetic analysis of ({alpha}-{sup 32}P) BzATP labeling identified the 57 kDa polypeptide as a nucleotide-binding subunit with a possible regulatory function. In addition, ({sup 14}C) DCCD-labeling identified a 16 kDa polypeptide as a putative transmembrane proton channel. It is concluded that the tonoplast H{sup +}-ATPase is a multimer composed of at least three polypeptides. Anti-57 and anti-67 kDa sera reacted with polypeptides of the corresponding size in bovine chromaffin granules, bovine clathrin-coated vesicles, and yeast vacuolar membranes, suggesting common structural features and common ancestry for endomembrane H{sup +}-ATPase of different organelles and different phyla. Anti-57 serum was used to isolate a cDNA encoding the corresponding subunit from Arabidopsis. Protein sequence analysis revealed homologies between endomembrane, F{sub 0}F{sub 1} and archaebacterial ATPases, suggesting that these different classes of ATPases have evolved from a common ancestor.

  14. Hormonal regulation of Na+/K+-dependent ATPase activity and pump function in corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin

    2011-10-01

    Na- and K-dependent ATPase (Na,K-ATPase) in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in these cells. Mouse corneal endothelial cells were exposed to dexamethasone or insulin. ATPase activity was evaluated by spectrophotometric measurement, and pump function was measured using an Ussing chamber. Western blotting and immunocytochemistry were performed to measure the expression of the Na,K-ATPase α1-subunit. Dexamethasone increased Na,K-ATPase activity and the pump function of endothelial cells. Western blot analysis indicated that dexamethasone increased the expression of the Na,K-ATPase α1-subunit but decreased the ratio of active to inactive Na,K-ATPase α1-subunit. Insulin increased Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were transient and blocked by protein kinase C inhibitors and inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A). Western blot analysis indicated that insulin decreased the amount of inactive Na,K-ATPase α1-subunit, but the expression of total Na,K-ATPase α1-subunit was unchanged. Immunocytochemistry showed that insulin increased cell surface expression of the Na,K-ATPase α1-subunit. Our results suggest that dexamethasone and insulin stimulate Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells was mediated by Na,K-ATPase synthesis and an increased enzymatic activity because of dephosphorylation of Na,K-ATPase α1-subunits. The effect of insulin is mediated by the protein kinase C, PP1, and/or PP2A pathways.

  15. Substrate Specificity of Na+,Cl-(HCO3-)-ATPase.

    Science.gov (United States)

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

    2016-09-01

    We studied substrate specificity of Na + ,Cl - (HCO 3 - )-ATPase. In most cases, replacement of ATP for other phosphate-containing substances resulted in not only pronounced suppression of phosphohydrolase reactions, but also dramatic changes of their responsiveness to the stimulating effect of monovalent ions. The data showed that Na + ,Cl - (HCO 3 - )-ATPase is a highly specific enzyme for ATP.

  16. Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosinetriphosphatase

    International Nuclear Information System (INIS)

    Teruel, J.A.; Inesi, G.

    1988-01-01

    The roles of the phosphorylation (phosphorylated enzyme intermediate) and nucleotide binding domains in calcium transport were studied by comparing acetyl phosphate and ATP as substrates for the Ca 2+ -ATPase of sarcoplasmic reticulum vesicles. The authors found that the maximal level of phosphoenzyme obtained with either substrate is approximately 4 nmol/mg of protein, corresponding to the stoichiometry of catalytic sites in their preparation. The initial burst of phosphoenzyme formation observed in the transient state, following addition of either substrate, is accompanied by internalization of 2 mol of calcium per mole of phosphoenzyme. The internalized calcium is then translocated with a sequential pattern, independent of the substrate used. Following a rate-limiting step, the phosphoenzyme undergoes hydrolytic cleavage and proceeds to the steady-state activity which is soon back inhibited by the rise of Ca 2+ concentration in the lumen of the vesicles. When the back inhibition is released by the addition of oxalate, substrate utilization and calcium transport occur with a ratio of 1:2, independent of the substrate and its concentration. When the nucleotide binding site is derivatized with FITP, the enzyme can still utilize acetyl phosphate (but not ATP) for calcium transport. These observations demonstrate that the basic coupling mechanism of catalysis and calcium transport involves the phosphorylation and calcium binding domains, and not the nucleotide binding domain. On the other hand, occupancy of the FITC-sensitive nucleotide site is involved in kinetic regulation not only with respect to utilization of substrate for the phosphoryl transfer reaction but also for subsequent steps related to calcium translocation and phosphoenzyme turnover

  17. Proton accumulation and ATPase activity in Golgi apparatus-enriched vesicles from rat liver

    International Nuclear Information System (INIS)

    Yeh, H.I.; van Rossum, G.D.

    1991-01-01

    We have studied the mechanism by which liver Golgi apparatus maintains the acidity of its contents, using a subcellular fraction from rat liver highly enriched in Golgi marker enzymes. Proton accumulation (measured by quenching of acridine-orange fluorescence) and anion-dependent ATPase were characterized and compared. Maximal ATPase and proton accumulation required ATP; GTP and other nucleotides gave 10% to 30% of maximal activity. Among anions, Cl- and Br- approximately doubled the activities; others were much less effective. Half-maximal increase of ATPase and H+ uptake required 55 mmol/L and 27 mmol/L Cl-, respectively. In predominantly chloride media, SCN- and NO3- markedly inhibited H+ uptake. Nitrate competitively inhibited both the chloride-dependent ATPase (apparent Ki 6 mmol/L) and proton uptake (apparent Ki 2 mmol/L). Nitrate and SCN- also inhibited uptake of 36Cl. Replacing K+ with Na+ had no effect on the initial rate of proton uptake but somewhat reduced the steady state attained. Replacement of K+ with NH4+ and choline reduced proton uptake without affecting ATPase. The ATPase and H+ uptake were supported equally well by Mg2+ or Mn2+. The ATPase was competitively inhibited by 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (apparent Ki 39 mumol/L). Other agents inhibiting both H+ uptake and ATPase were N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, chlorpromazine, diethylstilbestrol, Zn2+, Co2+ and Cu2+. In the Cl- medium, accumulated protons were released by ionophores at the relative rates, monensin = nigericin greater than valinomycin greater than carbonyl cyanide mchlorophenylhydrazone; the last of these also reduced ATPase activity. In the absence of Cl-, monensin and valinomycin both stimulated the ATPase. These results show a close association between ATPase activity and acidification of liver Golgi vesicles

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

    DEFF Research Database (Denmark)

    Juel, Carsten

    2016-01-01

    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...... 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...

  19. Regulation of α1 Na/K-ATPase Expression by Cholesterol*

    OpenAIRE

    Chen, Yiliang; Li, Xin; Ye, Qiqi; Tian, Jiang; Jing, Runming; Xie, Zijian

    2011-01-01

    We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol tr...

  20. Subcellular localization of H(+)-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation.

    Science.gov (United States)

    Scherer, G F

    1984-03-01

    A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H(+)-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H(+) pump was directly demonstrated. The H(+) pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a Δ pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K(+)-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because α-mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.

  1. A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations.

    Science.gov (United States)

    Calì, Tito; Lopreiato, Raffaele; Shimony, Joshua; Vineyard, Marisa; Frizzarin, Martina; Zanni, Ginevra; Zanotti, Giuseppe; Brini, Marisa; Shinawi, Marwan; Carafoli, Ernesto

    2015-06-26

    The particular importance of Ca(2+) signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca(2+) ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca(2+). A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca(2+) ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca(2+) transients generated by cell stimulation and impairs its Ca(2+) extrusion function under conditions of low resting cytosolic Ca(2+) as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca(2+)-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca(2+) homeostasis and the previous finding that PMCAs act as digenic modulators in Ca(2+)-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. A Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations*

    Science.gov (United States)

    Calì, Tito; Lopreiato, Raffaele; Shimony, Joshua; Vineyard, Marisa; Frizzarin, Martina; Zanni, Ginevra; Zanotti, Giuseppe; Brini, Marisa; Shinawi, Marwan; Carafoli, Ernesto

    2015-01-01

    The particular importance of Ca2+ signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca2+ ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca2+. A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca2+ ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca2+ transients generated by cell stimulation and impairs its Ca2+ extrusion function under conditions of low resting cytosolic Ca2+ as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca2+-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca2+ homeostasis and the previous finding that PMCAs act as digenic modulators in Ca2+-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype. PMID:25953895

  3. Epididymosomes: transfer of fertility-modulating proteins to the sperm surface.

    Science.gov (United States)

    Martin-DeLeon, Patricia A

    2015-01-01

    A variety of glycosylphosphatidylinositol (GPI)-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca 2 + -ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca 2 + efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4's interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca 2 + -dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.

  4. Epididymosomes: transfer of fertility-modulating proteins to the sperm surface

    Directory of Open Access Journals (Sweden)

    Patricia A Martin-DeLeon

    2015-01-01

    Full Text Available A variety of glycosylphosphatidylinositol (GPI-linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane (TM proteins, including plasma membrane Ca 2 + -ATPase 4 (PMCA4. Synthesized in the testis, PMCA4 is an essential protein and the major Ca 2 + efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4′s interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca 2 + -dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.

  5. [Properties and localization of Mg- and Ca-ATpase activities in wheat embryo cell nuclei].

    Science.gov (United States)

    Vasil'eva, N A; Belkina, G G; Stepanenko, S Y; Atalykova, F I; Oparin, A I

    1978-05-01

    The isolated nuclei of wheat embryo possess the ATPase activity. The addition of Mg2+ and Ca2+ significantly increases the activities of nuclear ATPases, whereas Hg2+, Cu2+ and Mn2+ inhibit the activity. The activating effect of Mg2+ is enhanced by an addition of Na and K ions. The activity of wheat embryo nuclear Mg-ATPase is higher than its Ca-ATPase activity; both ATPases also differ in their pH optima. Separation of total nuclear protein according to the solubility of its individual protein components in wheat and strong salt solutions, using the detergents, as well as ammonium sulfate precipitation and dialysis do not result in separation of Mg-activated and Ca-activated ATPases, although their levels of activities and ratios change in the course of fractionation. The Mg- and Ca-ATPase activities of the wheat embryo nuclei were found in the nuclear fraction of albumin, in nonhistone proteins and nuclear membranes. In the albumin nuclear fraction and subfractions of non-histone proteins the higher level of activity is observed in Ca-ATPase, whereas in the nuclei and soluble fractions of residual proteins in Mg-ATPase.

  6. Comparative properties of caveolar and noncaveolar preparations of kidney Na+/K+-ATPase.

    Science.gov (United States)

    Liu, Lijun; Ivanov, Alexander V; Gable, Marjorie E; Jolivel, Florent; Morrill, Gene A; Askari, Amir

    2011-10-11

    To evaluate previously proposed functions of renal caveolar Na(+)/K(+)-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na(+)/K(+)-ATPase (α,β,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na(+)/K(+)-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme. As minor constituents, both caveolar and noncaveolar pools also contained Src, EGFR, PI3K, and several other proteins known to be involved in stimulous-induced signaling by Na(+)/K(+)-ATPase, indicating that signaling function is not limited to the caveolar pool. Endogenous Src was active in both pools but was not further activated by ouabain, calling into question direct interaction of Src with native Na(+)/K(+)-ATPase. Chemical cross-linking, co-immunoprecipitation, and immunodetection studies showed that in the caveolar pool, caveolin-1 oligomers, annexin-2 tetramers, and oligomers of the α,β,γ-protomers of Na(+)/K(+)-ATPase form a large multiprotein complex. In conjunction with known roles of E-cadherin and the β-subunit of Na(+)/K(+)-ATPase in cell adhesion and noted intercellular β,β-contacts within the structure of Na(+)/K(+)-ATPase, our findings suggest that interacting caveolar Na(+)/K(+)-ATPases located at renal adherens junctions maintain contact of two adjacent cells, conduct essential ion pumping, and are capable of locus-specific signaling in junctional cells.

  7. Improved biolistic transfection of hair cells.

    Directory of Open Access Journals (Sweden)

    Hongyu Zhao

    Full Text Available Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C and PMCA2 (ATP2B2; plasma-membrane Ca(2+-ATPase isoform 2 to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells.

  8. 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

    the curcuminoids, demethoxycurcumin was the most potent inhibitor of all tested P-type ATPases from fungal (Pma1p; H+-ATPase), plant (AHA2; H+-ATPase) and animal (SERCA; Ca2+-ATPase) cells. All three curcuminoids acted as non-competitive antagonist to ATP and hence may bind to a highly conserved allosteric site...

  9. Characterization of Na+K+-ATPase in bovine sperm.

    Science.gov (United States)

    Hickey, Katie D; Buhr, Mary M

    2012-04-15

    Existing as a ubiquitous transmembrane protein, Na(+)K(+)-ATPase affects sperm fertility and capacitation through ion transport and a recently identified signaling function. Functional Na(+)K(+)-ATPase is a dimer of α and β subunits, each with isoforms (four and three, respectively). Since specific isoform pairings and locations may influence or indicate function, the objective of this study was to identify and localize subunits of Na(+)K(+)-ATPase in fresh bull sperm by immunoblotting and immunocytochemistry using antibodies against α1 and 3, and all β isoforms. Relative quantity of Na(+)K(+)-ATPase in head plasma membranes (HPM's) from sperm of different bulls was determined by densitometry of immunoblot bands, and compared to bovine kidney. Sperm and kidney specifically bound all antibodies at kDa equivalent to commercial controls, and to additional lower kDa bands in HPM. Immunofluorescence of intact sperm confirmed that all isoforms were present in the head region of sperm and that α3 was also uniformly distributed post-equatorially. Permeabilization exposing internal membranes typically resulted in an increase in fluorescence, indicating that some antibody binding sites were present on the inner surface of the HPM or the acrosomal membrane. Deglycosylation of β1 reduced the kDa of bands in sperm, rat brain and kidney, with the kDa of the deglycosylated bands differing among tissues. Two-dimensional blots of β1 revealed three distinct spots. Based on the unique quantity, location and structure Na(+)K(+)-ATPase subunits in sperm, we inferred that this protein has unique functions in sperm. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Characterisation of the sarcolemmal calcium pump in guinea pig hearts and its influence through g-strophanthin. Charakterisierung der sarkolemmalen Calciumpumpe des Meerschweinchenherzens und ihre Beeinflussung durch g-Strophanthin

    Energy Technology Data Exchange (ETDEWEB)

    Lell, R.

    1982-11-25

    In this work the sarcolemmal calcium transport of the guinea pig heart is studied with the help of the micropore filtration method on the sarcolemmal vesicles. The calcium ATPase could be better characterised by its ability to accumulate calcium in the sarcolemmal vesicles. The calcium pump is magnesium-dependent and positively activated by sodium and potassium. The pH-optimum of the activity lies around 6.8-7.0 depending on intracellular conditions. Up until a concentration of 30 micrograms protein per ml of reaction mixture the calcium accumulation increased proportionally. With higher concentrations it came to a relative decrease. The addition of digitoxin and g-strophanthin to the reaction mixture did not change the activity of the calcium pump. With a perfusion of the heart with a toxic concentration of 10/sup 06/ M g-strophanthin a glycoside intoxication on the beating heart was caused and a time-dependent inhibition of the calcium pump of sarcolemmal vesicles was detectable.

  11. 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.

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

    DEFF Research Database (Denmark)

    Lyons, Joseph; Laban, Milena; Mikkelsen, Stine

    2017-01-01

    P4-ATPases are members of the P-type ATPase superfamily that drive the inward translocation (flipping) of lipids within the membrane. These lipid flippase largely function as binary complexes with an auxiliary protein from the CDC50 family. The bulk of our knowledge has derived genetic and bioche......P4-ATPases are members of the P-type ATPase superfamily that drive the inward translocation (flipping) of lipids within the membrane. These lipid flippase largely function as binary complexes with an auxiliary protein from the CDC50 family. The bulk of our knowledge has derived genetic...... 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...... and bATP8A2/CDC50A, has yielded comparable low-resolution envelopes of these two transporters, highlighting the bulk architecture of the complex. Current efforts and progress on the functional characterization and cryo-EM analysis of both lipid transporters reconstituted in Salipro are described...

  13. Targeting vacuolar H+-ATPases as a new strategy against cancer.

    Science.gov (United States)

    Fais, Stefano; De Milito, Angelo; You, Haiyan; Qin, Wenxin

    2007-11-15

    Growing evidence suggests a key role of tumor acidic microenvironment in cancer development, progression, and metastasis. As a consequence, the need for compounds that specifically target the mechanism(s) responsible for the low pH of tumors is increasing. Among the key regulators of the tumor acidic microenvironment, vacuolar H(+)-ATPases (V-ATPases) play an important role. These proteins cover a number of functions in a variety of normal as well as tumor cells, in which they pump ions across the membranes. We discuss here some recent results showing that a molecular inhibition of V-ATPases by small interfering RNA in vivo as well as a pharmacologic inhibition through proton pump inhibitors led to tumor cytotoxicity and marked inhibition of human tumor growth in xenograft models. These results propose V-ATPases as a key target for new strategies in cancer treatment.

  14. Purification, characterization and crystallization of the F-ATPase from Paracoccus denitrificans.

    Science.gov (United States)

    Morales-Rios, Edgar; Watt, Ian N; Zhang, Qifeng; Ding, Shujing; Fearnley, Ian M; Montgomery, Martin G; Wakelam, Michael J O; Walker, John E

    2015-09-01

    The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex. © 2015 The Authors.

  15. Association with β-COP Regulates the Trafficking of the Newly Synthesized Na,K-ATPase*

    Science.gov (United States)

    Morton, Michael J.; Farr, Glen A.; Hull, Michael; Capendeguy, Oihana; Horisberger, Jean-Daniel; Caplan, Michael J.

    2010-01-01

    Plasma membrane expression of the Na,K-ATPase requires assembly of its α- and β-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase α-subunit and the coat protein, β-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase β-subunit, the Na,K-ATPase α-subunit interacts with β-COP, is retained in the endoplasmic reticulum, and is targeted for degradation. In the presence of the Na,K-ATPase β-subunit, the α-subunit does not interact with β-COP and traffics to the plasma membrane. Pulse-chase experiments demonstrate that in cells expressing both the Na,K-ATPase α- and β-subunits, newly synthesized α-subunit associates with β-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the α- and β-subunits to form the pump holoenzyme. The interaction with β-COP was reduced by mutating a dibasic motif at Lys54 in the Na,K-ATPase α-subunit. This mutant α-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase β-subunit expression. Although the Lys54 α-subunit reaches the cell surface without need for β-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the β-subunit. PMID:20801885

  16. Na+/K+-ATPase: Activity and inhibition

    Science.gov (United States)

    Čolović, M.; Krstić, D.; Krinulović, K.; Momić, T.; Savić, J.; Vujačić, A.; Vasić, V.

    2009-09-01

    The aim of the study was to give an overview of the mechanism of inhibition of Na+/K+-ATPase activity induced by some specific and non specific inhibitors. For this purpose, the effects of some ouabain like compounds (digoxin, gitoxin), noble metals complexes ([PtCl2DMSO2], [AuCl4]-, [PdCl4]2-, [PdCl(dien)]+, [PdCl(Me4dien)]+), transition metal ions (Cu2+, Zn2+, Fe2+, Co2+), and heavy metal ions (Hg2+, Pb2+, Cd2+) on the activity of Na+/K+-ATPase from rat synaptic plasma membranes (SPM), porcine cerebral cortex and human erythrocytes were discussed.

  17. In and out of the cation pumps: P-type ATPase structure revisited

    DEFF Research Database (Denmark)

    Bublitz, Maike; Poulsen, Hanne; Morth, Jens Preben

    2010-01-01

    . The marked increment during the last three years in the number of crystal structures of P-type ATPases has greatly improved our understanding of the similarities and differences of pumps with different ion specificities, since the structures of the Ca2+-ATPase, the Na+,K+-ATPase and the H+-ATPase can now......Active transport across membranes is a crucial requirement for life. P-type ATPases build up electrochemical gradients at the expense of ATP by forming and splitting a covalent phosphoenzyme intermediate, coupled to conformational changes in the transmembrane section where the ions are translocated...... be compared directly. Mechanisms for ion gating, charge neutralization and backflow prevention are starting to emerge from comparative structural analysis; and in combination with functional studies of mutated pumps this provides a framework for speculating on how the ions are bound and released as well...

  18. Probing the functional subunits of the tonoplast H+-ATPase

    International Nuclear Information System (INIS)

    Randall, S.K.; Lai, S.; Sze, H.

    1986-01-01

    The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kDa. The 16 kDA polypeptide covalently binds N,N'-dicyclohexylcarbodiimide and is postulated to be a component of the proton channel. Initial studies to identify other subunits indicate that both the 72 and 60 kDa subunits covalently bind 14 C]-7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and [ 14 C]N-ethylamleimide, inhibitors of the tonoplast ATPase. ATP prevents binding of these inhibitors suggesting that both the 72 and 60 kDa subunits are involved in substrate binding. Polyclonal antibody has been made to the 72 kDa subunit. Western blot analysis of tonoplast vesicles reveals single reactive polypeptide (72 kDa). The antibody shows no cross-reactivity towards either the mitochondrial F 1 -ATPase or the plasma membrane ATPase. This antibody specifically inhibits ATP hydrolysis and ATP-dependent H + pumping in native tonoplast vesicles. The authors conclude that the 72 kDa subunit is intimately associated with the catalytic (or ATP-binding) site

  19. Effect of TGFβ on Na+/K+ ATPase activity in megakaryocytes

    International Nuclear Information System (INIS)

    Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina; Laufer, Stefan; Borst, Oliver; Gawaz, Meinrad; Lang, Florian

    2014-01-01

    Highlights: • TGFß1 markedly up-regulates Na + /K + 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 + /K + ATPase generates the Na + and K + 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 + /K + 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 + /K + 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 + /K + ATPase activity determined from K + induced current utilizing whole cell patch clamp. The pump current (I pump ) was determined in the absence and presence of Na + /K + 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 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, TGFß1 is a powerful regulator of megakaryocytic Na + /K + ATPase activity

  20. The AAA protein spastin possesses two levels of basal ATPase activity.

    Science.gov (United States)

    Fan, Xiangyu; Lin, Zhijie; Fan, Guanghui; Lu, Jing; Hou, Yongfei; Habai, Gulijiazi; Sun, Linyue; Yu, Pengpeng; Shen, Yuequan; Wen, Maorong; Wang, Chunguang

    2018-04-30

    The AAA ATPase spastin is a microtubule-severing enzyme that plays important roles in various cellular events including axon regeneration. Herein, we found that the basal ATPase activity of spastin is negatively regulated by spastin concentration. By determining a spastin crystal structure, we demonstrate the necessity of intersubunit interactions between spastin AAA domains. Neutralization of the positive charges in the microtubule-binding domain (MTBD) of spastin dramatically decreases the ATPase activity at low concentration, although the ATP-hydrolyzing potential is not affected. These results demonstrate that, in addition to the AAA domain, the MTBD region of spastin is also involved in regulating ATPase activity, making interactions between spastin protomers more complicated than expected. © 2018 Federation of European Biochemical Societies.

  1. [Changes induced by hypertonic solutions in the transportation of calcium by the cardiac reticular sarcoplasma].

    Science.gov (United States)

    Sierra, M; Holguín, J A

    1979-01-01

    In the sarcoplasmic reticulum of the myocardium, celular organell which function is to regulate the cytoplasmic concentration of calcium in contraction and relaxation, we have studied the effect of hypertonic solutions of sucrose between 1 and 6.96 times the normal tonicity in order to observe the behavior of the internal linked or free calcium of this structure, as well as to prove the hypothesis that hypertonic solutions encourage the calcium exit of the sarcoplasmatic reticulum with the resulting signs of contractures. The following results were obtained: 1. The ATP hydrolisis and calcium transport rate are 14% and 90% respectively of the maximum speeds of 10(-5) M in calcium, while for concentrations of 10(-7) M or ess of the said cation, the transport rates and the ATPase do not reach 5% of the maximum values. 2. Between 1 and 2.54 times of the normal tonicity the calcium uptake remains between 400 and 500 nmoles of calcium/mg protein/min, the transported amount of calcium varies between 14 and 16 nmoles/mg protein and the rate of the ATP hydrolysis increases a 37% to 0.4 M in sucrose. 3. Between 0.4 and 1.2 M in sucrose of 2.54 to 6.96 times the isotonicity, the calcium transport rate velocity as well as the ATP hydrolisis are strongly inhibited. The vesicles volume minimizes and the amount of linked calcium remains within the control values, proving that the capacity of linking this cathion is independent from sarcoplasmic reticulum volume. These results show that the sarcoplasmic reticulum is involved in the contractures induced by hypertonic solutions in intact cells, since the osmolarity increase produces changes of volume which results in a decrease of the calcium transportation velocity or in an increase of the exit of said cathion.

  2. One Dimensional Finite Element Method Approach to Study Effect of Ryanodine Receptor and Serca Pump on Calcium Distribution in Oocytes

    Science.gov (United States)

    Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

    2013-11-01

    Oocyte is a female gametocyte or germ cell involved in reproduction. Calcium ions (Ca2+) impact nearly all aspects of cellular life as they play an important role in a variety of cellular functions. Calcium ions contributes to egg activation upon fertilization. Since it is the internal stores which provide most of the calcium signal, much attention has been focused on the intracellular channels. There are mainly two types of calcium channels which release calcium from the internal stores to the cytoplasm in many cell types. These channels are IP3-Receptor and Ryanodine Receptor (RyR). Further it is essential to maintain low cytosolic calcium concentration, the cell engages the Serco/Endoplasmic reticulum Ca2+ ATPases (SERCA) present on the ER or SR membrane for the re-uptake of cytosolic calcium at the expense of ATP hydrolysis. In view of above an attempt has been made to study the effect of the Ryanodine receptor (RyR) and the SERCA pump on the calcium distribution in oocytes. The main aim of this paper is to study the calcium concentration in absence and presence of these parameters. The FEM is used to solve the proposed Mathematical model under appreciate initial and boundary conditions. The program has been developed in MATLAB 7.10 for the entire problem to get numerical results.

  3. 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.

  4. Effect of therapeutic concentration of lithium on live HEK293 cells; increase of Na+/K+-ATPase, change of overall protein composition and alteration of surface layer of plasma membrane.

    Science.gov (United States)

    Vosahlikova, Miroslava; Ujcikova, Hana; Chernyavskiy, Oleksandr; Brejchova, Jana; Roubalova, Lenka; Alda, Martin; Svoboda, Petr

    2017-05-01

    The effect of long-term exposure of live cells to lithium cations (Li) was studied in HEK293 cells cultivated in the presence of 1mM LiCl for 7 or 21days. The alteration of Na + /K + -ATPase level, protein composition and biophysical state of plasma membrane was determined with the aim to characterize the physiological state of Li-treated cells. Na + /K + -ATPase level was determined by [ 3 H]ouabain binding and immunoblot assays. Overall protein composition was determined by 2D electrophoresis followed by proteomic analysis by MALDI-TOF MS/MS and LFQ. Li interaction with plasma membrane was characterized by fluorescent probes DPH, TMA-DPH and Laurdan. Na + /K + -ATPase was increased in plasma membranes isolated from cells exposed to Li. Identification of Li-altered proteins by 2D electrophoresis, MALDI-TOF MS/MS and LFQ suggests a change of energy metabolism in mitochondria and cytosol and alteration of cell homeostasis of calcium. Measurement of Laurdan generalized polarization indicated a significant alteration of surface layer of isolated plasma membranes prepared from both types of Li-treated cells. Prolonged exposure of HEK293 cells to 1mM LiCl results in up-regulation of Na + /K + -ATPase expression, reorganization of overall cellular metabolism and alteration of the surface layer/polar head-group region of isolated plasma membranes. Our findings demonstrate adaptation of live HEK293 cell metabolism to prolonged exposure to therapeutic concentration of Li manifested as up-regulation of Na + /K + -ATPase expression, alteration of protein composition and change of the surface layer of plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Inhibition of ecto-ATPase activities impairs HIV-1 infection of macrophages.

    Science.gov (United States)

    Schachter, Julieta; Delgado, Kelly Valcárcel; Barreto-de-Souza, Victor; Bou-Habib, Dumith Chequer; Persechini, Pedro Muanis; Meyer-Fernandes, José Roberto

    2015-05-01

    Nucleotides and nucleosides are secreted into extracellular media at different concentrations as a consequence of different physiologic and pathological conditions. Ecto-nucleotidases, enzymes present on the surface of most cells, hydrolyze these extracellular nucleotides and reduce the concentration of them, thus affecting the activation of different nucleotide and nucleoside receptors. Also, ecto-nucleotidases are present in a number of microorganisms and play important roles in host-pathogen interactions. Here, we characterized the ecto-ATPase activities present on the surface of HIV-1 particle and human macrophages as well. We found that the kinetic properties of HIV-1 and macrophage ecto-ATPases are similar, suggesting that the enzyme is the same. This ecto-ATPase activity was increased in macrophages infected in vitro with HIV-1. Using three different non-related ecto-ATPase inhibitors-POM-1, ARL67156 and BG0-we showed that the inhibition of these macrophage and viral ecto-ATPase activities impairs HIV-1 infection. In addition, we also found that elevated extracellular concentrations of ATP inhibit HIV-1 production by infected macrophages. Copyright © 2014 Elsevier GmbH. All rights reserved.

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

    International Nuclear Information System (INIS)

    Miles, Andrew J.; Fedosova, Natalya U.; Hoffmann, Søren V.; Wallace, B.A.; Esmann, Mikael

    2013-01-01

    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

  7. 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.

  8. Biochar and lignite affect H+-ATPase and H+-PPase activities in root tonoplast and nutrient contents of mung bean under salt stress.

    Science.gov (United States)

    Torabian, Shahram; Farhangi-Abriz, Salar; Rathjen, Judith

    2018-05-31

    This research was conducted to evaluate effects of biochar (50 and 100 g kg -1 soil) and lignite (50 and 100 g kg -1 soil) treatments on H + -ATPase and H + -PPase activity of root tonoplast, nutrient content, and performance of mung bean under salt stress. High saline conditions increased H + -ATPase and H + -PPase activities in root tonoplast, sodium (Na) content, reactive oxygen species (H 2 O 2 and O 2 - ) generation, relative electrolyte leakage (REL) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) activity in root and leaf, but decreased relative water content (RWC), chlorophyll content index, leaf area, potassium (K), calcium (Ca), magnesium (Mg), zinc (Zn) and iron (Fe) content of plant tissues, root and shoot dry weight of mung bean. Lignite and biochar treatments decreased the H + -ATPase and H + -PPase activities of root tonoplast under salt stress. Moreover, these treatments increased the cation exchange capacity of soil and nutrient values in plant tissues. Biochar and lignite diminished the generation of reactive oxygen species and DPPH activity in root and leaf cells, and these superior effects improved chlorophyll content index, leaf area and growth of mung bean under both conditions. In general, the results of this study demonstrated that biochar and lignite decreased the entry of Na ion into the cells, enriched plant cells with nutrients, and consequently improved mung bean performance under salt toxicity. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  9. Stabilization of the H,K-ATPase M5M6 membrane hairpin by K+ ions. Mechanistic significance for p2-type atpases.

    Science.gov (United States)

    Gatto, C; Lutsenko, S; Shin, J M; Sachs, G; Kaplan, J H

    1999-05-14

    The integral membrane protein, the gastric H,K-ATPase, is an alpha-beta heterodimer, with 10 putative transmembrane segments in the alpha-subunit and one such segment in the beta-subunit. All transmembrane segments remain within the membrane domain following trypsinization of the intact gastric H,K-ATPase in the presence of K+ ions, identified as M1M2, M3M4, M5M6, and M7, M8, M9, and M10. Removal of K+ ions from this digested preparation results in the selective loss of the M5M6 hairpin from the membrane. The release of the M5M6 fragment is directed to the extracellular phase as evidenced by the accumulation of the released M5M6 hairpin inside the sealed inside out vesicles. The stabilization of the M5M6 hairpin in the membrane phase by the transported cation as well as loss to the aqueous phase in the absence of the transported cation has been previously observed for another P2-type ATPase, the Na, K-ATPase (Lutsenko, S., Anderko, R., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940). Thus, the effects of the counter-transported cation on retention of the M5M6 segment in the membrane as compared with the other membrane pairs may be a general feature of P2-ATPase ion pumps, reflecting a flexibility of this region that relates to the mechanism of transport.

  10. Effect of triorganotin compounds on calcium transport mechanisms in rat cardiac sarcoplasmic reticulum

    International Nuclear Information System (INIS)

    Cameron, J.A.; Kodavanti, P.R.S.; Yallapragada, P.R.; Desaiah, D.

    1990-01-01

    Although organotin compounds, in general, are neurotoxicants, recent studies indicate that these tin compounds affect heme metabolism as well as cardiovascular system. Sarcoplasmic reticulum (SR) calcium pump together with phosphorylation of phospholamban has an important role in myocardial contraction and relaxation. Since organotin compounds interfere with cardiovascular system, we have studied the in vitro as well as in vivo effects of tributyltin bromide (TBT), triethyltin bromide (TET) and trimethyltin chloride (TMT) on cardiac SR Ca 2+ -pump activity, in order to know the relative potency of these tin compounds. SR was isolated from heart ventricles of male Sprague-Dawley rats and used for in vitro studies. For in vivo studies, rats were treated orally in corn oil for 6 days with different doses of TET (0.5, 1.0 and 1.5 mg/kg/d), TMT (0.75, 1.5 and 2.5 mg/kg/d) and TBT (0.75, 1.5 and 2.5 mg/kg/d). Rats were sacrificed 24 hr after the last dosage and cardiac SR was prepared. Cardiac SR Ca 2+ -ATPase and 45 Ca-uptake were measured. All the three tin compounds inhibited Ca 2+ -ATPase and 45 Ca-uptake in vitro in a concentration dependent manner. The order of potency for Ca 2+ -ATPase as determined IC 50 , is TBT (2 uM) > TET (63 uM) > TMT (280 uM). For 45 Ca-uptake, if followed the same order i.e., TBT (0.35 uM) > TET (10 uM) > TMT (440 uM). In agreement with in vitro results, both SR Ca 2+ -ATPase and 45 Ca-uptake were significantly inhibited in rats treated with these tin compounds. These studies indicate that triorganotin compounds affect Ca 2+ -pumping mechanisms and thereby alter cardiac contraction-relaxation process

  11. Plasma Membrane H(+)-ATPase Regulation in the Center of Plant Physiology.

    Science.gov (United States)

    Falhof, Janus; Pedersen, Jesper Torbøl; Fuglsang, Anja Thoe; Palmgren, Michael

    2016-03-07

    The plasma membrane (PM) H(+)-ATPase is an important ion pump in the plant cell membrane. By extruding protons from the cell and generating a membrane potential, this pump energizes the PM, which is a prerequisite for growth. Modification of the autoinhibitory terminal domains activates PM H(+)-ATPase activity, and on this basis it has been hypothesized that these regulatory termini are targets for physiological factors that activate or inhibit proton pumping. In this review, we focus on the posttranslational regulation of the PM H(+)-ATPase and place regulation of the pump in an evolutionary and physiological context. The emerging picture is that multiple signals regulating plant growth interfere with the posttranslational regulation of the PM H(+)-ATPase. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  12. A novel interaction between calcium-modulating cyclophilin ligand and Basigin regulates calcium signaling and matrix metalloproteinase activities in human melanoma cells.

    Science.gov (United States)

    Long, Tingting; Su, Juan; Tang, Wen; Luo, Zhongling; Liu, Shuang; Liu, Zhaoqian; Zhou, Honghao; Qi, Min; Zeng, Weiqi; Zhang, Jianglin; Chen, Xiang

    2013-10-01

    Intracellular free calcium is a ubiquitous second messenger regulating a multitude of normal and pathogenic cellular responses, including the development of melanoma. Upstream signaling pathways regulating the intracellular free calcium concentration ([Ca2+]i) may therefore have a significant impact on melanoma growth and metastasis. In this study, we demonstrate that the endoplasmic reticulum (ER)-associated protein calcium-modulating cyclophilin ligand (CAML) is bound to Basigin, a widely expressed integral plasma membrane glycoprotein and extracellular matrix metalloproteinase inducer (EMMPRIN, or CD147) implicated in melanoma proliferation, invasiveness, and metastasis. This interaction between CAML and Basigin was first identified using yeast two-hybrid screening and further confirmed by co-immunoprecipitation. In human A375 melanoma cells, CAML and Basigin were co-localized to the ER. Knockdown of Basigin in melanoma cells by siRNA significantly decreased resting [Ca2+]i and the [Ca2+]i increase induced by the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG), indicating that the interaction between CAML and Basigin regulates ER-dependent [Ca2+]i signaling. Meanwhile upregulating the [Ca2+]i either by TG or phorbol myristate acetate (PMA) could stimulate the production of MMP-9 in A375 cells with the expression of Basigin. Our study has revealed a previously uncharacterized [Ca2+]i signaling pathway that may control melanoma invasion, and metastasis. Disruption of this pathway may be a novel therapeutic strategy for melanoma treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  13. Carbonylation Modification Regulates Na/K-ATPase Signaling and Salt Sensitivity: A Review and a Hypothesis.

    Science.gov (United States)

    Shah, Preeya T; Martin, Rebecca; Yan, Yanling; Shapiro, Joseph I; Liu, Jiang

    2016-01-01

    Na/K-ATPase signaling has been implicated in different physiological and pathophysiological conditions. Accumulating evidence indicates that oxidative stress not only regulates the Na/K-ATPase enzymatic activity, but also regulates its signaling and other functions. While cardiotonic steroids (CTS)-induced increase in reactive oxygen species (ROS) generation is an intermediate step in CTS-mediated Na/K-ATPase signaling, increase in ROS alone also stimulates Na/K-ATPase signaling. Based on literature and our observations, we hypothesize that ROS have biphasic effects on Na/K-ATPase signaling, transcellular sodium transport, and urinary sodium excretion. Oxidative modulation, in particular site specific carbonylation of the Na/K-ATPase α1 subunit, is a critical step in proximal tubular Na/K-ATPase signaling and decreased transcellular sodium transport leading to increases in urinary sodium excretion. However, once this system is overstimulated, the signaling, and associated changes in sodium excretion are blunted. This review aims to evaluate ROS-mediated carbonylation of the Na/K-ATPase, and its potential role in the regulation of pump signaling and sodium reabsorption in the renal proximal tubule (RPT).

  14. Further investigations on the inorganic phosphate binding site of beef heart mitochondrial F1-ATPase

    International Nuclear Information System (INIS)

    Pougeois, R.; Lauquin, G.J.

    1985-01-01

    The possibility that 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive derivative of inorganic phosphate (P /sub i/ ), could mimic ATP was investigated. ANPP was hydrolyzed in the dark by sarcoplasmic reticulum Ca 2+ -ATPase in the presence of Ca 2+ but not in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANPP was not hydrolyzed by purified mitochondrial F1-ATPase; however, ADP and ATP protected F1-ATPase against ANPP photoinactivation. On the other hand, the trinitrophenyl nucleotide analogues (TNP-ADP, TNP-ATP, and TNP-AMP-PNP), which bind specifically at the two catalytic sites of F1-ATPase, abolished P /sub i/ binding on F1-ATPase; they do not protect F1-ATPase against ANPP photoinactivation. Furthermore, ANPP-photoinactivated F1-ATPase binds the TNP analogues in the same way as the native enzyme. The Pi binding site of F1-ATPase, which is shown to be photolabeled by ANPP, does not appear to be at the gamma-phosphate position of the catalytic sites

  15. The modulation of erythrocyte Na+/K+-ATPase activity by curcumin

    Directory of Open Access Journals (Sweden)

    Prabhakar Singh

    2015-11-01

    Full Text Available Curcumin, an active biphenolic molecule present in turmeric (Curcuma longa, has been reported to elicit plethora of health protective effects. The present study was carried out in vitro, in vivo and in silico to investigate the modulatory effects of curcumin on erythrocyte membrane Na+/K+-ATPase activity. In vitro curcumin (10−5 M to 10−8 M was incubated with human erythrocytes membrane. In vivo curcumin (340 mg/kg b.w. and 170 mg/kg b.w. was supplemented to wistar rats for 21 days. In silico, catalytic unit α of Na+/K+-ATPase (3b8e.pdb protein was used as a receptor for the natural ligand ATP to study curcumin-mediated docking simulation using AutoDock4. The in vitro effect of curcumin on the Na+/K+-ATPase activity in human erythrocytes was biphasic. An inhibitory response was observed at 10−5 M (p < 0.001. An activation of the Na+/K+-ATPase activity was observed at 10−7 and 10−8 M (p < 0.001 and p < 0.01. In vivo, curcumin supplementation to rats increased the Na+/K+-ATPase activity at doses 340 mg/kg b.w. (p < 0.001 as well as at 170 mg/kg b.w., (p < 0.01. AutoDock4 docking simulation study showed that both ligands curcumin and ATP actively interacted with amino acids Glu214, Ser215, Glu216, Thr371, Asn377, Arg378, Met379, Arg438, Val440, Ala444, Lys451 and Asp586 at the catalytic cavity of Na+/K+-ATPase. ATP had more H bonding and hydrophobic interaction with active site amino acid residues compared to curcumin. These finding may explain some of the health beneficial properties of curcumin associated with deregulated Na+/K+-ATPase activity or ions homeostasis.

  16. 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.

  17. 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.

  18. Human and rodent muscle Na(+)-K(+)-ATPase in diabetes related to insulin, starvation, and training

    DEFF Research Database (Denmark)

    Schmidt, T A; Hasselbalch, S; Farrell, P A

    1994-01-01

    cerebral cortex Na(+)-K(+)-ATPase concentration as a result of diabetes, semistarvation, or insulin treatment. In human subjects, Na(+)-K(+)-ATPase concentration in vastus lateralis muscle biopsies was 17 and 22% greater (P dependent diabetes...... mellitus (n = 24) and insulin-dependent diabetes mellitus (n = 7) than in control subjects (n = 8). A positive linear correlation between muscle Na(+)-K(+)-ATPase and plasma insulin concentrations was observed (r = 0.50, P = 0.006; n = 29). Thus, insulin seems a regulator of muscle Na......(+)-K(+)-ATPase concentration, reduction of muscle Na(+)-K(+)-ATPase concentration with untreated diabetes bears similarities with undernourishment, and physical conditioning may ameliorate the muscle Na(+)-K(+)-ATPase concentration decrease induced by diabetes....

  19. General and specific lipid-protein interactions in Na,K-ATPase.

    Science.gov (United States)

    Cornelius, F; Habeck, M; Kanai, R; Toyoshima, C; Karlish, S J D

    2015-09-01

    The molecular activity of Na,K-ATPase and other P2 ATPases like Ca(2+)-ATPase is influenced by the lipid environment via both general (physical) and specific (chemical) interactions. Whereas the general effects of bilayer structure on membrane protein function are fairly well described and understood, the importance of the specific interactions has only been realized within the last decade due particularly to the growing field of membrane protein crystallization, which has shed new light on the molecular details of specific lipid-protein interactions. It is a remarkable observation that specific lipid-protein interactions seem to be evolutionarily conserved, and conformations of specifically bound lipids at the lipid-protein surface within the membrane are similar in crystal structures determined with different techniques and sources of the protein, despite the rather weak lipid-protein interaction energy. Studies of purified detergent-soluble recombinant αβ or αβFXYD Na,K-ATPase complexes reveal three separate functional effects of phospholipids and cholesterol with characteristic structural selectivity. The observations suggest that these three effects are exerted at separate binding sites for phophatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and saturated PC or sphingomyelin/cholesterol (inhibitory), which may be located within three lipid-binding pockets identified in recent crystal structures of Na,K-ATPase. The findings point to a central role of direct and specific interactions of different phospholipids and cholesterol in determining both stability and molecular activity of Na,K-ATPase and possible implications for physiological regulation by membrane lipid composition. This article is part of a special issue titled "Lipid-Protein Interactions." Copyright © 2015 Elsevier B.V. All rights reserved.

  20. V-ATPase is a candidate therapeutic target for Ewing sarcoma.

    Science.gov (United States)

    Avnet, Sofia; Di Pompo, Gemma; Lemma, Silvia; Salerno, Manuela; Perut, Francesca; Bonuccelli, Gloria; Granchi, Donatella; Zini, Nicoletta; Baldini, Nicola

    2013-08-01

    Suppression of oxidative phosphorylation combined with enhanced aerobic glycolysis and the resulting increased generation of protons are common features of several types of cancer. An efficient mechanism to escape cell death resulting from intracellular acidification is proton pump activation. In Ewing sarcoma (ES), although the tumor-associated chimeric gene EWS-FLI1 is known to induce the accumulation of hypoxia-induced transcription factor HIF-1α, derangements in metabolic pathways have been neglected so far as candidate pathogenetic mechanisms. In this paper, we observed that ES cells simultaneously activate mitochondrial respiration and high levels of glycolysis. Moreover, although the most effective detoxification mechanism of proton intracellular storage is lysosomal compartmentalization, ES cells show a poorly represented lysosomal compartment, but a high sensitivity to the anti-lysosomal agent bafilomycin A1, targeting the V-ATPase proton pump. We therefore investigated the role of V-ATPase in the acidification activity of ES cells. ES cells with the highest GAPDH and V-ATPase expression also showed the highest acidification rate. Moreover, the localization of V-ATPase was both on the vacuolar and the plasma membrane of all ES cell lines. The acidic extracellular pH that we reproduced in vitro promoted high invasion ability and clonogenic efficiency. Finally, targeting V-ATPase with siRNA and omeprazole treatments, we obtained a significant selective reduction of tumor cell number. In summary, glycolytic activity and activation of V-ATPase are crucial mechanisms of survival of ES cells and can be considered as promising selective targets for the treatment of this tumor. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Na,K-ATPase structure/function relationships probed by the denaturant urea.

    Science.gov (United States)

    Esmann, Mikael; Fedosova, Natalya U; Olesen, Claus

    2015-05-01

    Urea interacts with the Na,K-ATPase, leading to reversible as well as irreversible inhibition of the hydrolytic activity. The enzyme purified from shark rectal glands is more sensitive to urea than Na,K-ATPase purified from pig kidney. An immediate and reversible inhibition under steady-state conditions of hydrolytic activity at 37°C is demonstrated for the three reactions studied: the overall Na,K-ATPase activity, the Na-ATPase activity observed in the absence of K+ as well as the K+-dependent phosphatase reaction (K-pNPPase) seen in the absence of Na+. Half-maximal inhibition is seen with about 1M urea for shark enzyme and about 2M urea for pig enzyme. In the presence of substrates there is also an irreversible inhibition in addition to the reversible process, and we show that ATP protects against the irreversible inhibition for both the Na,K-ATPase and Na-ATPase reaction, whereas the substrate paranitrophenylphosphate leads to a slight increase in the rate of irreversible inhibition of the K-pNPPase. The rate of the irreversible inactivation in the absence of substrates is much more rapid for shark enzyme than for pig enzyme. The larger number of potentially urea-sensitive hydrogen bonds in shark enzyme compared to pig enzyme suggests that interference with the extensive hydrogen bonding network might account for the higher urea sensitivity of shark enzyme. The reversible inactivation is interpreted in terms of domain interactions and domain accessibilities using as templates the available crystal structures of Na,K-ATPase. It is suggested that a few interdomain hydrogen bonds are those mainly affected by urea during reversible inactivation. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis

    NARCIS (Netherlands)

    Sakamoto, K; van Veen, HW; Saito, H; Kobayashi, H; Konings, WN

    2002-01-01

    The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 muM hop compounds. The

  3. ROS and calcium signaling mediated pathways involved in stress responses of the marine microalgae Dunaliella salina to enhanced UV-B radiation.

    Science.gov (United States)

    Zhang, Xinxin; Tang, Xuexi; Wang, Ming; Zhang, Wei; Zhou, Bin; Wang, You

    2017-08-01

    UV-B ray has been addressed to trigger common metabolic responses on marine microalgae, however, the upstream events responsible for these changes in marine microalgae are poorly understood. In the present study, a species of marine green microalgae Dunaliella salina was exposed to a series of enhanced UV-B radiation ranging from 0.25 to 1.00 KJ·m -2 per day. The role of ROS and calcium signaling in the D. salina responses to UV-B was discussed. Results showed that enhanced UV-B radiation markedly decreased the cell density in a dose-dependent manner, but the contents of protein and glycerol that were essential for cell growth increased. It suggested that it was cell division instead of cell growth that UV-B exerted negative effects on. The subcellular damages on nuclei and plasmalemma further evidenced the hypothesis. The nutrient absorption was affected with UV-B exposure, and the inhibition on PO 4 3- uptake was more serious compared to NO 3 - uptake. UV-B radiation promoted reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) contents, decreased the redox status and altered the antioxidant enzyme activities. The addition of the ROS scavenger and the glutathione biosynthesis precursor N-acetyl-l-cysteine (NAC) alleviated the stress degree, implying ROS-mediated pathway was involved in the stress response to UV-B radiation. Transient increase in Ca 2+ -ATPase was triggered simultaneously with UV-B exposure. Meanwhile, the addition of an intracellular free calcium chelator aggravated the damage of cell division, but exogenous calcium and ion channel blocker applications did not, inferring that endogenously initiated calcium signaling played roles in response to UV-B. Cross-talk analysis showed a relatively clear relationship between ROS inhibition and Ca 2+ -ATPase suppression, and a relation between Ca 2+ inhibition and GPx activity change was also observed. It was thus presumed that ROS-coupled calcium signaling via the

  4. Effect of endurance swimming on rat cardiac myofibrillar ATPase with experimental diabetes.

    Science.gov (United States)

    Belcastro, A N; Maybank, P; Rossiter, M; Secord, D

    1985-09-01

    Diabetes is characterized by depressed cardiac functional properties attributed to Ca2+-activated ATPase activity. In contrast, endurance swimming enhances the cardiac functional properties and Ca2+-activated myofibril ATPase. Thus, the purpose of this study was to observe if the changes associated with experimental diabetes can be ameliorated with training. Diabetes was induced with a single i.v. injection of streptozotocin (60 mg/kg). Blood and urine glucose concentrations were 802 +/- 44 and 6965 +/- 617 mg/dL, respectively. The training control and training diabetic animals were made to swim (+/- 2% body weight) 4 days/week for 8 weeks. Cardiac myofibril, at 10 microM free Ca2+ concentration was reduced by 54% in the sedentary diabetics compared with sedentary control animals (p less than 0.05). Swim training enhanced the Ca2+-activated myofibril ATPase activities for the normal animals. The diabetic animals, which swam for 8 weeks, had further reduced their Ca2+-activated myofibril ATPase activity when compared with sedentary diabetics (p less than 0.05). Similarly, the Mg2+-stimulated myofibril ATPase activity was depressed by 31% in diabetics following endurance swimming. It is concluded that the depressed Ca2+-activated myofibril ATPase activity of diabetic hearts is not reversible with endurance swimming.

  5. Molecular architecture of the N-type ATPase rotor ring from Burkholderia pseudomallei.

    Science.gov (United States)

    Schulz, Sarah; Wilkes, Martin; Mills, Deryck J; Kühlbrandt, Werner; Meier, Thomas

    2017-04-01

    The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N-type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N 1 N o -type ATPase and investigated the structure and ion specificity of its membrane-embedded c-ring rotor by single-particle electron cryo-microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low-density, low-CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c-ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c 17 ring is H + specific, demonstrating that the ATPase is proton-coupled. The c 17 ring stoichiometry results in a very high ion-to-ATP ratio of 5.7. We propose that this N-ATPase is a highly efficient proton pump that helps these melioidosis-causing bacteria to survive in the hostile, acidic environment of phagosomes. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  6. 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.

  7. Copper-transporting P-type ATPases use a unique ion-release pathway

    DEFF Research Database (Denmark)

    Andersson, Magnus; Mattle, Daniel; Sitsel, Oleg

    2014-01-01

    Heavy metals in cells are typically regulated by PIB-type ATPases. The first structure of the class, a Cu(+)-ATPase from Legionella pneumophila (LpCopA), outlined a copper transport pathway across the membrane, which was inferred to be occluded. Here we show by molecular dynamics simulations...... that extracellular water solvated the transmembrane (TM) domain, results indicative of a Cu(+)-release pathway. Furthermore, a new LpCopA crystal structure determined at 2.8-Å resolution, trapped in the preceding E2P state, delineated the same passage, and site-directed-mutagenesis activity assays support...... a functional role for the conduit. The structural similarities between the TM domains of the two conformations suggest that Cu(+)-ATPases couple dephosphorylation and ion extrusion differently than do the well-characterized PII-type ATPases. The ion pathway explains why certain Menkes' and Wilson's disease...

  8. Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells

    International Nuclear Information System (INIS)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-01-01

    Aldosterone and insulin stimulate Na + transport through mechanisms involving protein synthesis. Na + -K + -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na + -K + -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na + -K + -[ 32 P]ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na + -K + -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na + entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na + -K + -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/

  9. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-08-01

    Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

  10. The effects of dexamethasone on the Na,K-ATPase activity and pump function of corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin; Yamada, Masakazu; Mochizuki, Hiroshi; Shiraishi, Atsushi; Joko, Takeshi; Nishida, Teruo

    2009-05-01

    The Na(+)- and K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. We investigated the possible role of dexamethasone in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Confluent monolayers of mouse corneal endothelial cells were exposed to dexamethasone. ATPase activity of the cells was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with the use of an Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis was examined to measure the expression of the Na,K-ATPase alpha(1)-subunit. Dexamethasone (1 or 10 microM) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of dexamethasone were blocked by cycloheximide, a protein synthesis inhibitor. Western blot analysis also indicated that dexamethasone increased the expression of the Na,K-ATPase alpha(1)-subunit, whereas it decreased the expression of the phospho-Na,K-ATPase alpha(1)-subunit. Our results suggest that dexamethasone stimulates Na,K-ATPase activity in mouse corneal endothelial cells. The effect of dexamethasone activation in these cells is mediated by Na,K-ATPase synthesis and increase in an enzymatic activity by dephosphorylation of Na,K-ATPase alpha(1)-subunits.

  11. The influence of blood plasma of irradiated animals on activity of Ca2+ - ATPase and Mg2+ - ATPase in plasma membrane of thymocytes

    International Nuclear Information System (INIS)

    Dreval', V.I.

    1994-01-01

    Rats were irradiated at doses 1.5, 4.0, 7.0 and 10 Gy. After 1, 8, 15, 22 and 30 days the effect of blood plasma on activity of Ca 2+ -ATPase and Mg 2+ -ATPase in plasma membrane of thymocytes was investigated. It was found that the raise of irradiation dose leads to increasing of blood plasma effect on membrane-bound enzymes

  12. Protein import into chloroplasts requires a chloroplast ATPase

    International Nuclear Information System (INIS)

    Pain, D.; Blobel, G.

    1987-01-01

    The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the [ 35 S]methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H + , K + , Na + , or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors

  13. Protein import into chloroplasts requires a chloroplast ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Pain, D.; Blobel, G.

    1987-05-01

    The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the (/sup 35/S)methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H/sup +/, K/sup +/, Na/sup +/, or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors.

  14. Structural and functional studies of heavy metal ATPases

    DEFF Research Database (Denmark)

    Sitsel, Oleg

    2015-01-01

    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......Copper and zinc are trace elements that are crucial for the well-being of all cells and are an indispensable part of many proteins. At the same time, the intracellular levels of these metals require careful regulation, as an excess or deficiency may be lethal. P1B-ATPases are key players in Cu......+ and Zn2+ homeostasis that belong to the superfamily of P-type ATPases, transmembrane proteins which are present in virtually all lifeforms, with functions ranging from membrane potential generation to muscle relaxation. The goal of this thesis is to improve our understanding of P1B-ATPases by focusing...

  15. High fat diet disrupts endoplasmic reticulum calcium homeostasis in the rat liver.

    Science.gov (United States)

    Wires, Emily S; Trychta, Kathleen A; Bäck, Susanne; Sulima, Agnieszka; Rice, Kenner C; Harvey, Brandon K

    2017-11-01

    Disruption to endoplasmic reticulum (ER) calcium homeostasis has been implicated in obesity, however, the ability to longitudinally monitor ER calcium fluctuations has been challenging with prior methodologies. We recently described the development of a Gaussia luciferase (GLuc)-based reporter protein responsive to ER calcium depletion (GLuc-SERCaMP) and investigated the effect of a high fat diet on ER calcium homeostasis. A GLuc-based reporter cell line was treated with palmitate, a free fatty acid. Rats intrahepatically injected with GLuc-SERCaMP reporter were fed a cafeteria diet or high fat diet. The liver and plasma were examined for established markers of steatosis and compared to plasma levels of SERCaMP activity. Palmitate induced GLuc-SERCaMP release in vitro, indicating ER calcium depletion. Consumption of a cafeteria diet or high fat pellets correlated with alterations to hepatic ER calcium homeostasis in rats, shown by increased GLuc-SERCaMP release. Access to ad lib high fat pellets also led to a corresponding decrease in microsomal calcium ATPase activity and an increase in markers of hepatic steatosis. In addition to GLuc-SERCaMP, we have also identified endogenous proteins (endogenous SERCaMPs) with a similar response to ER calcium depletion. We demonstrated the release of an endogenous SERCaMP, thought to be a liver esterase, during access to a high fat diet. Attenuation of both GLuc-SERCaMP and endogenous SERCaMP was observed during dantrolene administration. Here we describe the use of a reporter for in vitro and in vivo models of high fat diet. Our results support the theory that dietary fat intake correlates with a decrease in ER calcium levels in the liver and suggest a high fat diet alters the ER proteome. Lay summary: ER calcium dysregulation was observed in rats fed a cafeteria diet or high fat pellets, with fluctuations in sensor release correlating with fat intake. Attenuation of sensor release, as well as food intake was observed during

  16. Regulatory Mechanisms in the P4-ATPase Complex

    DEFF Research Database (Denmark)

    Costa, Sara

    . The functionality on the P4-ATPase complex is essential for several cellular processes, such as vesicle-mediated transport. However, the specific role of flippase activity in vesicle biogenesis and the regulatory mechanism behind this process is still poorly understood. In these studies, we identified...... affordable alternative using a microscope-based cytometer. This system can simultaneously provide information on flippase activity and expression levels. Taken together, the findings described in this thesis provide new tools for P4-ATPase characterization and valuable insights into the regulation...

  17. Study on the changes in the levels of membrane-bound ATPases ...

    African Journals Online (AJOL)

    An attempt has been made to determine the deleterious effects of λ cyhalothrin- induced in fresh water tilapia (Oreochromis mossambicus) with respect to changes in the activities of membrane-bound ATPases (Na+/K+, Mg+ and Ca2+ ATPase) and mineral status ...

  18. Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3'-to-5' DNA translocase and helicase that prefers to unwind 3'-tailed RNA:DNA hybrids.

    Science.gov (United States)

    Ordonez, Heather; Shuman, Stewart

    2013-05-17

    We are interested in the distinctive roster of helicases of Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis Lhr as the exemplar of a novel clade of superfamily II helicases, by virtue of its biochemical specificities and signature domain organization. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3'-to-5' translocation along single strand DNA and to unwind duplexes en route. The ATPase is more active in the presence of calcium than magnesium. ATP hydrolysis is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for a DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence. Lhr is 8-fold better at unwinding an RNA:DNA hybrid than it is at displacing a DNA:DNA duplex of identical nucleobase sequence. The truncated derivative Lhr-(1-856) is an autonomous ATPase, 3'-to-5' translocase, and RNA:DNA helicase. Lhr-(1-856) is 100-fold better RNA:DNA helicase than DNA:DNA helicase. Lhr homologs are found in bacteria representing eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria (including Escherichia coli).

  19. Relationship between serum adiponectin level and ATPase activity of erythrocyte membrance in patients with 2-type diabetes

    International Nuclear Information System (INIS)

    Song Jiejin

    2008-01-01

    Objective: To explore the possible mechanism of development nephrosis as related to changes of serum adiponectin levels and alteration of activities of Na + ·K + -ATPase and Ca +2 ·Mg +2 -ATPase of erythrocyte membrance in patients with 2-type diabetes. Methods: Serum adiponectin levels (with RIA) and erythrocyte membrance (prepared with Reilnila method) Na + ·K + - ATPase and Ca +2 ·Mg +2 -ATPase activity were determined in 45 DM2 patients without nephropathy, 31 DM2 patients with nephropathy and 30 controls. Results: Serum adiponectin levels in the diabetic patients were significantly lower than those in controls (P + ·K + -ATPase and Ca +2 ·Mg +2 -ATPase activities were also significantly lower than those in controls (P + ·K + -ATPase and Ca +2 ·Mg +2 -ATPase activities of erythrocyte membrance. (authors)

  20. Increasing acidification of nonreplicating Lactococcus lactis Delta thyA mutants by incorporating ATPase activity

    DEFF Research Database (Denmark)

    Pedersen, Martin Bastian; Købmann, Brian Jensen; Jensen, Peter Ruhdal

    2002-01-01

    % of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing...... nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by noureplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We...... concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis....

  1. Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S. cerevisiae

    DEFF Research Database (Denmark)

    Rudashevskaya, Elena; Ye, Juanying; Young, Clifford

     It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H...... of heterologous system of yeast cells, expressing plant proton pump. Therefore identification of possible regulatory effects by phosphorylation events in plant H+-ATPase in the system is significant. A number of putative phosphorylation sites at regulatory C-domain of H+-ATPase (AHA2) have been point...... functioning of the residues and suggests, that plant H+-ATPase could be regulated by phosphorylation at several sites being in yeast cells. Plant H+-ATPase purified from yeast cells by his-tag affinity chromatography was subjected to IMAC and TiO2 for enrichment of phosphopeptides. The phosphopeptides were...

  2. Bcl-2 overexpression: effects on transmembrane calcium movement

    International Nuclear Information System (INIS)

    Rangaswami, Arun A.; Premack, Brett; Walleczek, Jan; Killoran, Pamela; Gardner, Phyllis; Knox, Susan J.

    1996-01-01

    Purpose/Objective: High levels of expression of the proto-oncogene bcl-2 and its 26 kD protein product Bcl-2 have been correlated with the inhibition of apoptosis and the increased resistance of tumor cells to cytotoxic drugs and ionizing radiation. Unfortunately, the specific mechanism of action of Bcl-2 remains poorly understood. In the studies described here, the role of intracellular calcium fluxes and plasma membrane calcium cycling in the induction of apoptosis, and the effect of Bcl-2 expression on the modulation of transmembrane calcium fluxes following treatment of cells with cytotoxic agents were studied. The relationship between intracellular calcium release, capacitive calcium entry, and the plasma membrane potential were also investigated. Materials and Methods: Human B-cell lymphoma (PW) and human promyelocytic leukemia (HL60) cell lines were transfected with Bcl-2 and a control vector. The Bcl-2 transfectants over expressed the Bcl-2 onco-protein and were more resistant to irradiation than the control cells. Cells were loaded with fluorescent indicators indo-1 and fura-2 AM to quantify the cytosolic calcium concentration and subsequent calcium responses to a variety of cytotoxic stimuli, including the microsomal ATPase inhibitor, thapsigargin, using fluorometric measurements. Comparisons of resting and stimulated cytosolic calcium concentrations were made between the parental, neomycin control, and bcl-2 transfected cells. In order to determine the actual calcium influx rate, cells were loaded with either indo-1 or fura-2 and then exposed to 0.1 mM extracellular manganese, which enters the cells through calcium influx channels and quenches the fluorescent signal in proportion to the calcium influx rate. In order to determine the role of the membrane potential in driving calcium influx, cells were treated with either 0.1 μM Valinomycin or isotonic potassium chloride to either hyper polarize or depolarize the resting membrane potential, and the

  3. 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.

  4. 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

  5. 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...... resolution 3D structure the mechanism behind is only poorly understood. This thesis aimed at illuminating the autoinhibitory mechanism in plant and yeast PM H+-ATPases and below some of our main findings will be highlighted. The two terminal domains of the PM H+-ATPases have several amino acid residues...... that can be phosphorylated, and it has been demonstrated that these phosphorylation sites in both plant and yeast are highly involved in the regulation of terminal autoinhibition. In this study we used a phylogenetic analysis to investigate the evolutionary development of these phosphorylation sites...

  6. Congruence between PM H+-ATPase and NADPH oxidase during root growth: a necessary probability.

    Science.gov (United States)

    Majumdar, Arkajo; Kar, Rup Kumar

    2018-07-01

    Plasma membrane (PM) H + -ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O 2 ˙ - , respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H + -ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H + -ATPase inhibitor). Conversely, H + -ATPase activity retarded in response to different ROS scavengers [CuCl 2 , N, N' -dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl 2 and diphenyleneiodonium (DPI)], while H 2 O 2 promoted PM H + -ATPase activity at lower concentrations. Repressing effects of Ca +2 antagonists (La +3 and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H + -ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H + -ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.

  7. Characteristics of a (Na+K+)-ATPase inhibitor in extracts of tea

    International Nuclear Information System (INIS)

    Sagnella, G.A.; MacGregor, G.A.

    1984-01-01

    Extracts of tea were examined for inhibitors of the sodium-potassium pump by investigating the effect of the extracts on 1) isolated preparations of (Na + -K + )-ATPase from hog brain and human blood cells; 2) the displacement of radioactive ouabain from its specific receptor on red blood cells, and 3) the uptake of radioactive rubidium in intact red blood cells. It has been found that extracts of tea were potent inhibitors of the purified hog brain (Na + -K + )-ATPase. However, the inhibition was not specific for the (Na + -K + )-ATPase and the extract of tea did not displace 3 H-ouabain in a specific ouabain-receptor assay. Additionally, the tea extracts displayed only a small inhibitory effect on the uptake of 86 Rb in intact red blood cells. These observations suggest that the material is not like digitalis and that, unlike cardiac glycosides, it may inhibit the activity of the (Na + -K + )-ATPase by interacting with the enzyme at intracellular sites

  8. The ATPases of cohesin interface with regulators to modulate cohesin-mediated DNA tethering

    Science.gov (United States)

    Çamdere, Gamze; Guacci, Vincent; Stricklin, Jeremiah; Koshland, Douglas

    2015-01-01

    Cohesin tethers together regions of DNA, thereby mediating higher order chromatin organization that is critical for sister chromatid cohesion, DNA repair and transcriptional regulation. Cohesin contains a heterodimeric ATP-binding Cassette (ABC) ATPase comprised of Smc1 and Smc3 ATPase active sites. These ATPases are required for cohesin to bind DNA. Cohesin’s DNA binding activity is also promoted by the Eco1 acetyltransferase and inhibited by Wpl1. Recently we showed that after cohesin stably binds DNA, a second step is required for DNA tethering. This second step is also controlled by Eco1 acetylation. Here, we use genetic and biochemical analyses to show that this second DNA tethering step is regulated by cohesin ATPase. Furthermore, our results also suggest that Eco1 promotes cohesion by modulating the ATPase cycle of DNA-bound cohesin in a state that is permissive for DNA tethering and refractory to Wpl1 inhibition. DOI: http://dx.doi.org/10.7554/eLife.11315.001 PMID:26583750

  9. Vacuolar-type H+-ATPase and Na+, K+-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water

    DEFF Research Database (Denmark)

    Seidelin, Michel; Brauner, Colin J; Jensen, Frank Bo

    2001-01-01

    Changes in branchial vacuolar-type H+-ATPase B-subunit mRNA and Na+, K+-ATPase alpha- and beta-subunit mRNA and ATP hydrolytic activity were examined in smolting Atlantic salmon exposed to hyperoxic and/or hypercapnic fresh water. Pre-smolts, smolts, and post-smolts were exposed for 1 to 4 days...... Na+, K+-ATPase activity was generally unaffected by the experimental treatments. We suggest that the reduced expression of branchial vacuolar-type H(+)-ATPase B-subunit mRNA observed during internal hypercapnic acidosis may lead to reduction of functional V-type H+-ATPase abundance as a compensatory...

  10. 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.

  11. The V-ATPase a2-subunit as a putative endosomal pH-sensor.

    Science.gov (United States)

    Marshansky, V

    2007-11-01

    V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

  12. Myofibril ATPase activity of cardiac and skeletal muscle of exhaustively exercised rats.

    Science.gov (United States)

    Belcastro, A N; Turcotte, R; Rossiter, M; Secord, D; Maybank, P E

    1984-01-01

    The activation characteristics of Mg-ATP and Ca2+ on cardiac and skeletal muscle myofibril ATPase activity were studied in rats following a run to exhaustion. In addition, the effect of varying ionic strength was determined on skeletal muscle from exhausted animals. The exhausted group (E) ran at a speed of 25 m min-1 with an 8% incline. Myofibril ATPase activities for control (C) and E were determined with 1, 3 and 5 mM Mg-ATP and 1 and 10 microM Ca2+ at pH 7.0 and 30 degrees C. For control skeletal muscle, at 1 and 10 microM Ca2+, there was an increase in ATPase activity from 1 to 5 mM Mg-ATP (P less than 0.05). For E animals the myofibril ATPase activities at 10 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ the activities at 3 and 5 mM Mg-ATP were greater for the E animals (P less than 0.05). Increasing KCl concentrations resulted in greater inhibition for E animals. With cardiac muscle, the myofibril ATPase activities at 1.0 microM free Ca2+ were lower for E at all Mg-ATP levels (P less than 0.05). In contrast, at 10 microM Ca2+, the E group exhibited an elevated myofibril ATPase activity. The results indicate that Mg-ATP and Ca2+ activation of cardiac and skeletal muscle myofibril ATPase is altered with exhaustive exercise.

  13. Review: The HSP90 molecular chaperone-an enigmatic ATPase.

    Science.gov (United States)

    Pearl, Laurence H

    2016-08-01

    The HSP90 molecular chaperone is involved in the activation and cellular stabilization of a range of 'client' proteins, of which oncogenic protein kinases and nuclear steroid hormone receptors are of particular biomedical significance. Work over the last two decades has revealed a conformational cycle critical to the biological function of HSP90, coupled to an inherent ATPase activity that is regulated and manipulated by many of the co-chaperones proteins with which it collaborates. Pharmacological inhibition of HSP90 ATPase activity results in degradation of client proteins in vivo, and is a promising target for development of new cancer therapeutics. Despite this, the actual function that HSP90s conformationally-coupled ATPase activity provides in its biological role as a molecular chaperone remains obscure. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 594-607, 2016. © 2016 The Authors. Biopolymers Published by Wiley Periodicals, Inc.

  14. Calcium uptake by sarcoplasmic reticulum isolated from hearts of septic rats

    International Nuclear Information System (INIS)

    McDonough, K.H.

    1988-01-01

    Myocardial sarcoplasmic reticulum (SR) plays a critical role in the regulation of the cytosolic calcium fluctuations that occur during the cardiac cycle. One function of the SR is to lower the calcium concentration so that myocardial relaxation and thus ventricular filling can occur. The aim of the present study was to determine if hyperdynamic sepsis induced a decrease in the capacity of SR to take up calcium. This defect would result in decreased ventricular filling and thus decreased cardiac output, as has previously been shown in isolated perfused working hearts removed from septic rats. Therefore, rats were anesthetized with ether, and sepsis was induced by the injection of an aliquot of a fecal homogenate into the peritoneal cavity. Control animals either underwent surgery and received an aliquot of sterilized fecal inoculum (sham) or were untreated (no surgery). On day 2 after surgery, animals were anesthetized with pentobarbital, and hearts were removed, weighted, and SR isolated. The rate of uptake of 45 Ca 2+ by SR from septic rats was not depressed compared to controls but in fact was elevated. Maximum 45 Ca 2+ accumulated by the SR and Ca 2+ -stimulated ATPase activity were similar in SR from control and septic hearts. These results suggest that the contractile dysfunction noted in the myocardium in early sepsis is probably not due to inadequate SR removal of Ca 2+ during diastole

  15. 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

  16. 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.

  17. The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease.

    Science.gov (United States)

    Clausen, Michael V; Hilbers, Florian; Poulsen, Hanne

    2017-01-01

    The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

  18. Calcium signaling in closely related protozoan groups (Alveolata): non-parasitic ciliates (Paramecium, Tetrahymena) vs. parasitic Apicomplexa (Plasmodium, Toxoplasma).

    Science.gov (United States)

    Plattner, H; Sehring, I M; Mohamed, I K; Miranda, K; De Souza, W; Billington, R; Genazzani, A; Ladenburger, E-M

    2012-05-01

    The importance of Ca2+-signaling for many subcellular processes is well established in higher eukaryotes, whereas information about protozoa is restricted. Recent genome analyses have stimulated such work also with Alveolates, such as ciliates (Paramecium, Tetrahymena) and their pathogenic close relatives, the Apicomplexa (Plasmodium, Toxoplasma). Here we compare Ca2+ signaling in the two closely related groups. Acidic Ca2+ stores have been characterized in detail in Apicomplexa, but hardly in ciliates. Two-pore channels engaged in Ca2+-release from acidic stores in higher eukaryotes have not been stingently characterized in either group. Both groups are endowed with plasma membrane- and endoplasmic reticulum-type Ca2+-ATPases (PMCA, SERCA), respectively. Only recently was it possible to identify in Paramecium a number of homologs of ryanodine and inositol 1,3,4-trisphosphate receptors (RyR, IP3R) and to localize them to widely different organelles participating in vesicle trafficking. For Apicomplexa, physiological experiments suggest the presence of related channels although their identity remains elusive. In Paramecium, IP3Rs are constitutively active in the contractile vacuole complex; RyR-related channels in alveolar sacs are activated during exocytosis stimulation, whereas in the parasites the homologous structure (inner membrane complex) may no longer function as a Ca2+ store. Scrutinized comparison of the two closely related protozoan phyla may stimulate further work and elucidate adaptation to parasitic life. See also "Conclusions" section. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. 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......, these data establish the relative orientations of ATPases with respect to the 20S proteasome....

  20. Electrostatic control by lipids upon the membrane-bound (Na+ + K+)-ATPase.

    Science.gov (United States)

    Ahrens, M L

    1981-04-06

    In this paper, the membrane-bound (Na+ + K+)-ATPase from bovine brain is shown to be controlled by electrostatic alterations of the charged lipids surrounding the enzyme. The properties under investigation are the enzymatic activity, activation energy and the response of the enzymatic system to temperature. Arrhenius plots of the ATPase activity are biphasic with a break at temperature Ti. The temperature Ti, the activation energies at temperatures above and below Ti, and the enzymatic activity at any constant temperature have been shown to depend upon the concentrations of alkali and alkaline-earth metal ions in the solution. These electrolyte dependencies are ascribed to changes of electrostatic conditions at the lipids surrounding the ATPase. If the higher electrostatic screening ability of divalent ions is taken into account, the results in the presence of mono- and divalent ions become virtually the same. As a result of this work, it is concluded that electrostatic alterations are transmitted to the ATPase from the lipids of the membrane in which the enzyme is embedded. Inhibition and activation of the enzyme by mono-and divalent metal ions may thus be explained without any auxiliary hypothesis, particularly without postulating specific binding sites for the different ionic species at the protein. In addition, the specific lipid requirement of the ATPase may be understood better in the light of this interpretation.

  1. Role of insulin in regulation of Na+-/K+-dependent ATPase activity and pump function in corneal endothelial cells.

    Science.gov (United States)

    Hatou, Shin; Yamada, Masakazu; Akune, Yoko; Mochizuki, Hiroshi; Shiraishi, Atsushi; Joko, Takeshi; Nishida, Teruo; Tsubota, Kazuo

    2010-08-01

    The Na(+)-/K(+)-dependent ATPase (Na,K-ATPase) expressed in the basolateral membrane of corneal endothelial cells plays an important role in the pump function of the corneal endothelium. The role of insulin in the regulation of Na,K-ATPase activity and pump function in corneal endothelial cells was investigated. Confluent monolayers of mouse corneal endothelial cells were exposed to insulin. ATPase activity was evaluated by spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate; Na,K-ATPase activity was defined as the portion of total ATPase activity sensitive to ouabain. Pump function was measured with the use of a Ussing chamber; pump function attributable to Na,K-ATPase activity was defined as the portion of the total short-circuit current sensitive to ouabain. Western blot analysis and immunocytochemistry were performed to measure the expression of the Na,K-ATPase alpha(1)-subunit. Insulin increased the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. These effects were blocked by protein kinase C (PKC) inhibitors and protein phosphatases 1 and 2A inhibitor. Western blot analysis indicated that insulin decreased the ratio of the inactive Na,K-ATPase alpha(1)-subunit. Immunocytochemistry indicated that insulin increased the cell surface expression of the Na,K-ATPase alpha(1)-subunit. These results suggest that insulin increases the Na,K-ATPase activity and pump function of cultured corneal endothelial cells. The effect of insulin is mediated by PKC and presumably results in the activation of PP1, 2A, or both, which are essential for activating Na,K-ATPase by alpha(1)-subunit dephosphorylation.

  2. Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket.

    NARCIS (Netherlands)

    Koenderink, J.B.; Geibel, S.; Grabsch, E.; Pont, J.J.H.H.M. de; Bamberg, E.; Friedrich, T.

    2003-01-01

    Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp,

  3. Changes in acetylcholinesterase, Na+,K+-ATPase, and Mg2+-ATPase activities in the frontal cortex and the hippocampus of hyper- and hypothyroid adult rats.

    Science.gov (United States)

    Carageorgiou, Haris; Pantos, Constantinos; Zarros, Apostolos; Stolakis, Vasileios; Mourouzis, Iordanis; Cokkinos, Dennis; Tsakiris, Stylianos

    2007-08-01

    The thyroid hormones (THs) are crucial determinants of normal development and metabolism, especially in the central nervous system. The metabolic rate is known to increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na+,K+)- and Mg2+-adenosinetriphosphatase (ATPase) in the frontal cortex and the hippocampus of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25 microg/100 g body weight) once daily for 14 days, and hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. A region-specific behavior was observed concerning the examined enzyme activities in hyper- and hypothyroidism. In hyperthyroidism, AChE activity was significantly increased only in the hippocampus (+22%), whereas Na+,K+-ATPase activity was significantly decreased in the hyperthyroid rat hippocampus (-47%) and remained unchanged in the frontal cortex. In hypothyroidism, AChE activity was significantly decreased in the frontal cortex (-23%) and increased in the hippocampus (+21%). Na+,K+-ATPase activity was significantly decreased in both the frontal cortex (-35%) and the hippocampus (-43%) of hypothyroid rats. Mg2+-ATPase remained unchanged in the regions of both hyper- and hypothyroid rat brains. Our data revealed that THs affect the examined adult rat brain parameters in a region- and state-specific way. The TH-reduced Na+,K+-ATPase activity may increase the synaptic acetylcholine release and, thus, modulate AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems in the examined brain regions.

  4. Radionuclide assay of membrane Na+, K+-ATPase activity of peserved red blood cells

    International Nuclear Information System (INIS)

    Trusov, V.V.; Zelenin, A.A.; Marizin, S.A.

    1986-01-01

    The radionuclide tests were used to investigate the influence of varying blood preservatives on erythrocylic membrane Na + , K + -ATPase activity in samples of whole blood and packed red blood cells from normal donors prepared by standard methods. The tests were performed before and after seven days of preservation under standard conditions. It was found that blood preservations lowered membrane Na + , K + -ATPase activity: its minimum reduction was recorded with citroglucopnosphate, while glugicir induced a significant drop in Na + , K + -ATPase activity of preserved red blood cells regardless of the type of the blood transfusion solution. The assay of membrane Na + , K + -ATPase activity of preserved red blood cells with the use of 86 Rb could be recommended as an evaluation test for preserved blood and its components

  5. Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System

    NARCIS (Netherlands)

    Koenderink, J.B.; Swarts, H.G.

    2016-01-01

    P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed

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

    Directory of Open Access Journals (Sweden)

    Jiarong Zhang

    2017-10-01

    Full Text Available 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.

  7. Effect of hindlimb unweighting on single soleus fiber maximal shortening velocity and ATPase activity

    Science.gov (United States)

    Mcdonald, K. S.; Fitts, R. H.

    1993-01-01

    The effect of hindlimb unweighting (HU) for 1 to 3 wks on the shortening velocity of a soleus fiber, its ATPase content, and the relative contents of the slow and fast myosin was investigated by measuring fiber force, V(0), ATPase activity, and myosin content in SDS protein profiles of a single rat soleus fiber suspended between a motor arm and a transducer. It was found that HU induces a progressive increase in fiber V(0) that is likely caused, at least in part, by an increase in the fiber's myofibrillar ATPase activity. The HU-induced increases in V(0) and ATPase were associated with the presence of a greater percentage of fast type IIa fibers. However, a large population of fibers after 1, 2, and 3 wks of HU showed increases in V(0) and ATPase but displayed the same myosin protein profile on SDS gels as control fibers.

  8. The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease

    Directory of Open Access Journals (Sweden)

    Michael V. Clausen

    2017-06-01

    Full Text Available The sodium and potassium gradients across the plasma membrane are used by animal cells for numerous processes, and the range of demands requires that the responsible ion pump, the Na,K-ATPase, can be fine-tuned to the different cellular needs. Therefore, several isoforms are expressed of each of the three subunits that make a Na,K-ATPase, the alpha, beta and FXYD subunits. This review summarizes the various roles and expression patterns of the Na,K-ATPase subunit isoforms and maps the sequence variations to compare the differences structurally. Mutations in the Na,K-ATPase genes encoding alpha subunit isoforms have severe physiological consequences, causing very distinct, often neurological diseases. The differences in the pathophysiological effects of mutations further underline how the kinetic parameters, regulation and proteomic interactions of the Na,K-ATPase isoforms are optimized for the individual cellular needs.

  9. Data on Na,K-ATPase in primary cultures of renal proximal tubule cells treated with catecholamines

    Directory of Open Access Journals (Sweden)

    Mary Taub

    2016-03-01

    Full Text Available This data article is concerned with chronic regulation of Na,K-ATPase by catecholamines. After a chronic treatment, inhibition of Na,K-ATPase activity was observed in cultures with dopamine, while a stimulation was observed in cultures treated with norepinephrine. Following a chronic incubation with guanabenz, an α adrenergic agonist, an increase in Na,K-ATPase α and β subunit mRNAs was observed. This data supports the research article entitled, “Renal proximal tubule Na, K-ATPase is controlled by CREB regulated transcriptional coactivators as well as salt inducible kinase 1” (Taub et al. 2015 [1]. Keywords: Catecholamines, Kidney, Proximal tubule, Na,K-ATPase, Chronic

  10. Plant P4-ATPases: lipid translocators with a role in membrane traficking

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura

    a large family of membrane proteins involved in pumping different physiologically-relevant substrates across biological membranes [4]. The members of the P4 subfamily (also known as flippases) catalyze the energy-driven translocation of lipids necessary for establishing transbilayer lipid asymmetry [5......], a feature necessary for correct functioning of the cells [6,7]. Deletion of one or more P4-ATPase genes causes defects in vesicle budding in various organisms [8-10] and some members of the yeast family have been shown to interact with the vesiculation machinery [11,12]. Thus, unraveling the key features...... of P4-ATPase functioning is crucial to understand the mechanisms underlying the whole secretory and endocytic pathways. In the model plant Arabidopsis, 12 members of the P4-ATPase family have been described (ALA1-ALA12, for Aminophospholipid ATPase) [4]. In the past years, we have characterized several...

  11. Nucleotide binding to Na+/K+-ATPase

    Czech Academy of Sciences Publication Activity Database

    Kubala, Martin; Lánský, Zdeněk; Ettrich, R.; Plášek, J.; Teisinger, Jan; Amler, Evžen

    2005-01-01

    Roč. 272, č. S1 (2005), s. 191-191 E-ISSN 1742-4658. [FEBS Congress /30./ and IUBMB Conference /9./. 02.07.2005-07.07.2005, Budapest] Keywords : Na+/K+- ATPase * ATP binding * TNP-ATP Subject RIV: BO - Biophysics

  12. Increased leucocyte Na-K ATPase in obesity: reversal following weight loss

    International Nuclear Information System (INIS)

    Turaihi, K.; Baron, D.N.; Dandona, P.

    1987-01-01

    Ouabain-sensitive 86 Rb influx and [ 3 H] ouabain binding capacity were investigated in the leucocytes of 17 obese patients and 15 control subjects. Both were significantly increased in the obese when compared with controls. Following dietary restriction and a 4% to 5% weight reduction in the obese over 2 weeks, [ 3 H] ouabain binding and ouabain-sensitive 86 Rb influx (a model for K+ influx) decreased to levels similar to those in controls. This shows that the number of Na-K ATPase sites on leucocyte membranes of the obese are significantly increased and that this is associated with accelerated 86 Rb transport. Since both of these indices decreased following 4% to 5% reduction in body weight while the patients were still obese, increased Na-K ATPase is neither a marker of nor cardinal to the pathogenesis of obesity. We conclude that (1) increase in Na-K ATPase units and 86 Rb influx are not characteristic of obesity itself and (2) dietary restriction over the short-term with limited weight reduction restores Na-K ATPase units and 86 Rb influx to normal

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

    Science.gov (United States)

    Miles, Andrew J; Fedosova, Natalya U; Hoffmann, Søren V; Wallace, B A; Esmann, Mikael

    2013-05-31

    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. Copyright © 2013 The Author. Published by Elsevier Inc. All rights reserved.

  14. Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities.

    Science.gov (United States)

    Hsieh, Ying-Hsin; Huang, Ying-Ju; Jin, Jin-Shan; Yu, Liyan; Yang, Hsiuchin; Jiang, Chun; Wang, Binghe; Tai, Phang C

    2014-11-14

    SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg(2+), mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Structure and function of the latent F0-F1-ATPase complex of Micrococcus lysodeikticus

    International Nuclear Information System (INIS)

    Chung, Y.S.

    1988-01-01

    The latent F 0 F 1 -ATPase from Micrococcus luteus (lysodeikticus) has been purified to homogeneity, and nine distinct subunit bands were observed on SDS-PAGE. Five of nine bands corresponded to the F 1 subunits and the other four bands are likely to be subunits a, a', b, and c of the F 0 segment of the complex. The subunit designated as a' probably arises from proteolytic cleavage of the 25,5000 Mr subunit a. The F 0 F 1 -ATPase complex has a molecular weight of approximately 1,060,000, as determined by Fast Protein Liquid Chromatography (FPLC). It is assumed that the F 0 F 1 -ATPase peak obtained by FPLC was a dimer and that molecular weight of the F 0 F 1 -ATPase monomer was accordingly 530,000. The stoichiometry of the subunits was determined with 14 C-labeled F 0 F 1 -ATPase prepared from cells grown on medium containing 14 C-amino acids. Antibodies to the native and SDS-denatured F 1 and F 0 F 1 -ATPase as well as to individual SDS-dissociated subunits have been generated for immunochemical analysis. The arrangement of the subunits in F 1 and F 0 F 1 -ATPase have been investigated using bifunctional chemical cross-linking agents

  16. 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.

  17. 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.

  18. 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

    rats, cardiomyopathic hamsters, and human subjects. These methods have earlier been shown to quantify the Na+,K+-ATPase concentration in muscle tissue with high accuracy. When rats were swim trained for six weeks the heart ventricular muscle Na+,K+-ATPase concentration was increased by 20% (p less than...... was increased by up to 46% (p less than 0.001) and decreased by up to 30% (p less than 0.005) after training and immobilisation respectively. Cardiomyopathic hamsters showed a reduction of 33% (p less than 0.005) in the heart ventricular Na+,K+-ATPase concentration compared with normal hamsters. This decrease...

  19. Tributyltin (TBT) inhibition of oligomycin-sensitive Mg-ATPase activity in mussel mitochondria.

    Science.gov (United States)

    Pagliarani, Alessandra; Bandiera, Patrizia; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Nesci, Salvatore; Borgatti, Anna Rosa

    2008-06-01

    Tributyltin (TBT), one of the most toxic lipophilic aquatic pollutants, can be efficiently incorporated from sea water and sediments by filter-feeding molluscs. As far as we are aware TBT effects on the mitochondrial oligomycin-sensitive Mg-ATPase, the enzymatic core of energy production and a known target of TBT toxicity in mammals, have not been yet investigated in molluscs; thus the hydrolytic capability of the mitochondrial complex in the presence of micromolar concentrations of TBT was assayed in the mussel Mytilus galloprovincialis. Gill and mantle ATPase activities were progressively depressed by increasing TBT doses up to a maximal inhibition (82% in the gills and 74% in the mantle) at 0.62 microM TBT. Non-cooperative inhibition kinetics (n(H) approximately -1) and a non-competitive mechanism with respect to ATP substrate were pointed out. The mitochondrial Mg-ATPase susceptivity to TBT in the marine mussel was consistent with the formation of a TBT-Mg-ATPase complex, apparently more stable in the gills than in the mantle. The complex shape of the dose-response curve and the partial release of Mg-ATPase inhibition within the 0.6-34.4 microM TBT range suggest multiple interactions of TBT with the enzyme complex putatively related to its molecular mechanism of toxicity.

  20. Cryo-EM studies of the structure and dynamics of vacuolar-type ATPases

    Science.gov (United States)

    Mazhab-Jafari, Mohammad T.; Rubinstein, John L.

    2016-01-01

    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. PMID:27532044

  1. Novel Role for Na,K-ATPase in Phosphatidylinositol 3-Kinase Signaling and Suppression of Cell Motility

    OpenAIRE

    Barwe, Sonali P.; Anilkumar, Gopalakrishnapillai; Moon, Sun Y.; Zheng, Yi; Whitelegge, Julian P.; Rajasekaran, Sigrid A.; Rajasekaran, Ayyappan K.

    2005-01-01

    The Na,K-ATPase, consisting of α- and β-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase β1-subunit (Na,K-β) in highly motile Moloney sarcoma virus-transforme...

  2. Functional analysis of a potential regulatory K+-binding site in the Na+, K+-ATPase

    DEFF Research Database (Denmark)

    Schack, Vivien Rodacker; Vilsen, Bente

    The Na+, K+-ATPase functions by actively transporting 3 Na+ ions out of and 2 K+ ions into the cell, thereby creating ion gradients crucial for many physiological processes. Recently, a combined structural and functional study of the closely related Ca2+-ATPase indicated the presence...... of a regulatory K+-binding site in the P-domain of the enzyme, identifying E732 as being of particular importance (Sorensen, Clausen et al. 2004). In addition, P709 is thought to play a significant role in the structural organization of this site. Both E732 and P709 are highly conserved among P-type ATPases (E732...... is present as either glutamic acid or aspartic acid), which supports their importance and additionally raises the question whether this site may play a general role among P-type ATPases. In Na+, K+-ATPase, K+ functions directly as a substrate for membrane binding sites, however, an additional regulatory...

  3. Plasmalemmal V-H+-ATPases regulate intracellular pH in human lung microvascular endothelial cells

    International Nuclear Information System (INIS)

    Rojas, Jose D.; Sennoune, Souad R.; Maiti, Debasish; Martinez, Gloria M.; Bakunts, Karina; Wesson, Donald E.; Martinez-Zaguilan, Raul

    2004-01-01

    The lung endothelium layer is exposed to continuous CO 2 transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na + /H + exchanger and HCO 3 - -dependent H + -transporting mechanisms regulate intracellular pH (pH cyt ) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H + -ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na + /H + exchanger and HCO 3 - -based H + -transporting mechanisms, to maintain pH cyt homeostasis. Immunocytochemical studies revealed V-H + -ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na + and HCO 3 - that were similar to those observed in the presence of either Na + , or Na + and HCO 3 - . The Na + - and HCO 3 - -independent pH cyt recovery was inhibited by bafilomycin A 1 , a V-H + -ATPase inhibitor. These studies show a Na + - and HCO 3 - -independent pH cyt regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases

  4. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    Energy Technology Data Exchange (ETDEWEB)

    Kohio, Hinissan P.; Adamson, Amy L., E-mail: aladamso@uncg.edu

    2013-09-15

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.

  5. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    International Nuclear Information System (INIS)

    Kohio, Hinissan P.; Adamson, Amy L.

    2013-01-01

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells

  6. The role of Na,K-ATPase/Src-kinase signaling pathway in the vascular wall contaction

    DEFF Research Database (Denmark)

    Bouzinova, Elena

    Aim: Na,K-ATPase is essential for maintaining the transmembrane ion gradient and might initiate various intracellular signaling. These signals possibly act through a modification of the local ion concentrations or via Src-kinase activation. It is known that inhibition of the α-2 isoform of Na......,K-ATPase by ouabain elevates blood pressure. Consequently, ouabain was shown to potentiate arterial contraction in vitro. In contrast, we have demonstrated that siRNA-induced down-regulation of the α-2 isoform Na,K-ATPase expression reduced arterial sensitivity to agonist stimulation and prevented the effect......) phosphorylation assay. Down-regulation of the α-2 isoform Na,K-ATPase prevented the inhibitory effect of Src inhibitors on arterial contraction. Conclusions: The pro-contractile action of ouabain-sensitive Na,K-ATPase inhibition is associated with Src-kinase inhibition suggesting the role of this signaling...

  7. Glutamate transporter activity promotes enhanced Na+/K+-ATPase -mediated extracellular K+ management during neuronal activity

    DEFF Research Database (Denmark)

    Larsen, Brian R; Holm, Rikke; Vilsen, Bente

    2016-01-01

    , in addition, Na+ /K+ -ATPase-mediated K+ clearance could be governed by astrocytic [Na+ ]i . During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+ -coupled glutamate transporters, thereby elevating [Na+ ]i . It thus remains unresolved whether...... the different Na+ /K+ -ATPase isoforms are controlled by [K+ ]o or [Na+ ]i during neuronal activity. Hippocampal slice recordings of stimulus-induced [K+ ]o transients with ion-sensitive microelectrodes revealed reduced Na+ /K+ -ATPase-mediated K+ management upon parallel inhibition of the glutamate transporter......+ affinity to the α1 and α2 isoforms than the β2 isoform. In summary, enhanced astrocytic Na+ /K+ -ATPase-dependent K+ clearance was obtained with parallel glutamate transport activity. The astrocytic Na+ /K+ -ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na+ ]i...

  8. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

    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...

  9. 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...

  10. Influence of kaempferol, a flavonoid compound, on membrane-bound ATPases in streptozotocin-induced diabetic rats.

    Science.gov (United States)

    Al-Numair, Khalid S; Veeramani, Chinnadurai; Alsaif, Mohammed A; Chandramohan, Govindasamy

    2015-01-01

    Kaempferol is a flavonoid found in many edible plants (e.g. tea, cabbage, beans, tomato, strawberries, and grapes) and in plants or botanical products commonly used in traditional medicine. Numerous preclinical studies have shown that kaempferol have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and antidiabetic activities. The present study investigates the effect of kaempferol on membrane-bound ATPases in erythrocytes and in liver, kidney, and heart of streptozotocin (STZ)-induced diabetic rats. Diabetes was induced into adult male albino rats of the Wistar strain, by intraperitoneal administration of STZ (40 mg/kg body weight (BW)). Kaempferol (100 mg/kg BW) or glibenclamide (600 µg/kg BW) was administered orally once daily for 45 d to normal and STZ-induced diabetic rats. The effects of kaempferol on membrane-bound ATPases (total ATPase, Na(+)/K(+)-ATPase, Ca(2+)-ATPase, and Mg(2+)-ATPase) activity in erythrocytes and in liver, kidney, and heart were determined. In our study, diabetic rats had significantly (p kaempferol (100 mg/kg BW) or glibenclamide (600 µg/kg BW) for a period of 45 d resulted in significant (p kaempferol has the potential to restore deranged activity of membrane-bound ATPases in STZ-induced diabetic rats. Further detailed investigation is necessary to discover kaempferol's action mechanism.

  11. Quantitative measurement of membrane Na+-K+ ATPase activity using thallium-201: comparison with rubidium-86

    International Nuclear Information System (INIS)

    Lee, Jae Tae; Shon, Sang Kyun; Lee, Kyu Bo; Lee, In Kyu

    1998-01-01

    Na + -K + ATPase activity has been estimated by the degree of inhibition of cation transport by cardiac glycosides (ouabain) using Rb-86 as a substrate. The biological characteristics of Tl-201 is known to be similar to those of potassium as a transport substrate in the presence of glucose, insulin or phobol myristate acetate (PMA). The purpose of this study was to measure ouabain sensitive Na + -K + ATPase activity using Tl-201 and compare with that using Rb-86. Smooth muscle cells isolated from rat aorta or human placental umbilical artery were cultured, and used to measure cellular Na + -K + ATPase activity. Na + -K + ATPase activity was measured as a percentage decrease in cellular uptake of Tl-201 or Rb-86 by ouabain under the presence of glucose, insulin or PMA in media. Na + -K + ATPase activity measured with Tl-201, as a transport substrate, was not different from those measured with Rb-86 in rat or human smooth muscle cell preparation. Incubation with high concentration glucose resulted in about 30% decrease in enzyme activity. In contrast, insulin or PMA resulted in 50-70% or 28% increase from baseline activity, respectively. These results suggests that Tl-201 could replace Rb-86 in measurement of ouabain sensitive Na + -K + ATPase activity in vitro. High level of glucose concentration decreased cellular Na + -K + ATPase activity, but insulin or PMA increased it

  12. Na+,K+-ATPase as the Target Enzyme for Organic and Inorganic Compounds

    Directory of Open Access Journals (Sweden)

    Tatjana Momić

    2008-12-01

    Full Text Available This paper gives an overview of the literature data concerning specific and non specific inhibitors of Na+,K+-ATPase receptor. The immobilization approaches developed to improve the rather low time and temperature stability of Na+,K+-ATPase, as well to preserve the enzyme properties were overviewed. The functional immobilization of Na+,K+-ATPase receptor as the target, with preservation of the full functional protein activity and access of various substances to an optimum number of binding sites under controlled conditions in the combination with high sensitive technology for the detection of enzyme activity is the basis for application of this enzyme in medical, pharmaceutical and environmental research.

  13. 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

  14. “Oxygen sensing” by Na,K-ATPase: these miraculous thiols

    Directory of Open Access Journals (Sweden)

    Anna Bogdanova

    2016-08-01

    Full Text Available Control over the Na,K-ATPase function plays a central role in adaptation of the organisms to hypoxic and anoxic conditions. As the enzyme itself does not possess O2 binding sites its oxygen-sensitivity is mediated by a variety of redox-sensitive modifications including S-glutathionylation, S-nitrosylation and redox-sensitive phosphorylation. This is an overview of the current knowledge on the plethora of molecular mechanisms tuning the activity of the ATP-consuming Na,K-ATPase to the cellular metabolic activity. Recent findings suggest that oxygen-derived free radicals and H2O2, NO, and oxidised glutathione are the signalling messengers that make the Na,K-ATPase oxygen-sensitive. This very ancient signalling pathway targeting thiols of all three subunits of the Na,K-ATPase as well as redox-sensitive kinases sustains the enzyme activity at the optimal level avoiding terminal ATP depletion and maintaining the transmembrane ion gradients in cells of anoxia-tolerant species. We acknowledge the complexity of the underlying processes as we characterise the sources of reactive oxygen and nitrogen species production in hypoxic cells, and identify their targets, the reactive thiol groups which, upon modification, impact the enzyme activity. Structured accordingly, this review presents a summery on (i the sources of free radical production in hypoxic cells, (ii localisation of regulatory thiols within the Na,K-ATPase and the role reversible thiol modifications play in responses of the enzymes to a variety of stimuli (hypoxia, receptors’ activation control of the enzyme activity (iii redox-sensitive regulatory phosphorylation, and (iv the role of fine modulation of the Na,K-ATPase function in survival success under hypoxic conditions. The co-authors attempted to cover all the contradictions and standing hypotheses in the field and propose the possible future developments in this dynamic area of research, the importance of which is hard to overestimate

  15. Direct interaction of beta-amyloid with Na,K-ATPase as a putative regulator of the enzyme function

    Science.gov (United States)

    Petrushanko, Irina Yu.; Mitkevich, Vladimir A.; Anashkina, Anastasia A.; Adzhubei, Alexei A.; Burnysheva, Ksenia M.; Lakunina, Valentina A.; Kamanina, Yulia V.; Dergousova, Elena A.; Lopina, Olga D.; Ogunshola, Omolara O.; Bogdanova, Anna Yu.; Makarov, Alexander A.

    2016-06-01

    By maintaining the Na+ and K+ transmembrane gradient mammalian Na,K-ATPase acts as a key regulator of neuronal electrotonic properties. Na,K-ATPase has an important role in synaptic transmission and memory formation. Accumulation of beta-amyloid (Aβ) at the early stages of Alzheimer’s disease is accompanied by reduction of Na,K-ATPase functional activity. The molecular mechanism behind this phenomenon is not known. Here we show that the monomeric Aβ(1-42) forms a tight (Kd of 3 μM), enthalpy-driven equimolar complex with α1β1 Na,K-ATPase. The complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Aβ(1-42) is localized in the “gap” between the alpha- and beta-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Aβ(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activity and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increase of the Aβ(1-42) level. However prolonged increase of Aβ(1-42) level under pathological conditions could lead to chronical inhibition of Na,K-ATPase and disruption of neuronal function. Taken together, our data suggest the role of beta-amyloid as a novel physiological regulator of Na,K-ATPase.

  16. Increased leucocyte Na-K ATPase in obesity: reversal following weight loss

    Energy Technology Data Exchange (ETDEWEB)

    Turaihi, K.; Baron, D.N.; Dandona, P.

    1987-09-01

    Ouabain-sensitive /sup 86/Rb influx and (/sup 3/H) ouabain binding capacity were investigated in the leucocytes of 17 obese patients and 15 control subjects. Both were significantly increased in the obese when compared with controls. Following dietary restriction and a 4% to 5% weight reduction in the obese over 2 weeks, (/sup 3/H) ouabain binding and ouabain-sensitive /sup 86/Rb influx (a model for K+ influx) decreased to levels similar to those in controls. This shows that the number of Na-K ATPase sites on leucocyte membranes of the obese are significantly increased and that this is associated with accelerated /sup 86/Rb transport. Since both of these indices decreased following 4% to 5% reduction in body weight while the patients were still obese, increased Na-K ATPase is neither a marker of nor cardinal to the pathogenesis of obesity. We conclude that (1) increase in Na-K ATPase units and /sup 86/Rb influx are not characteristic of obesity itself and (2) dietary restriction over the short-term with limited weight reduction restores Na-K ATPase units and /sup 86/Rb influx to normal.

  17. Evaluation of Na+, K+-ATPase activity in the brain of young rats after acute administration of fenproporex.

    Science.gov (United States)

    Rezin, Gislaine T; Scaini, Giselli; Gonçalves, Cinara L; Ferreira, Gabriela K; Cardoso, Mariane R; Ferreira, Andréa G K; Cunha, Maira J; Schmitz, Felipe; Varela, Roger B; Quevedo, João; Wyse, Angela T S; Streck, Emilio L

    2014-01-01

    Fenproporex is an amphetamine-based anorectic which is rapidly converted into amphetamine in vivo. Na+, K+-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that the effects of fenproporex on brain metabolism are poorly known and that Na+, K+-ATPase is essential for normal brain function, this study sought to evaluate the effect of this drug on Na+, K+-ATPase activity in the hippocampus, hypothalamus, prefrontal cortex, and striatum of young rats. Young male Wistar rats received a single injection of fenproporex (6.25, 12.5, or 25 mg/kg intraperitoneally) or polysorbate 80 (control group). Two hours after the last injection, the rats were killed by decapitation and the brain was removed for evaluation of Na+, K+-ATPase activity. Fenproporex decreased Na+, K+-ATPase activity in the striatum of young rats at doses of 6.25, 12.5, and 25 mg/kg and increased enzyme activity in the hypothalamus at the same doses. Na+, K+-ATPase activity was not affected in the hippocampus or prefrontal cortex. Fenproporex administration decreased Na+, K+-ATPase activity in the striatum even in low doses. However, in the hypothalamus, Na+, K+-ATPase activity was increased. Changes in this enzyme might be the result of the effects of fenproporex on neuronal excitability.

  18. Role of Na+/K+-ATPase in Natriuretic Effect of Prolactin in a Model of Cholestasis of Pregnancy.

    Science.gov (United States)

    Abramicheva, P A; Balakina, T A; Bulaeva, O A; Guseva, A A; Lopina, O D; Smirnova, O V

    2017-05-01

    Participation of Na+/K+-ATPase in the natriuretic effect of prolactin in a cholestasis of pregnancy model was investigated. The Na+/K+-ATPase activity in rat kidney medulla, where active sodium reabsorption occurs, decreased in the model of cholestasis of pregnancy and other hyperprolactinemia types compared with intact animals. This effect was not connected with the protein level of α1- and β-subunits of Na+/K+-ATPase measured by Western blotting in the kidney medulla. Decrease in Na+/K+-ATPase activity in the kidney cortex was not significant, as well as decrease in the quantity of mRNA and proteins of the α1- and β-subunits of Na+/K+-ATPase. There were no correlations between the Na+/K+-ATPase activity and sodium clearance, although sodium clearance increased significantly in the model of cholestasis of pregnancy and other hyperprolactinemia groups under conditions of stable glomerular filtration rate measured by creatinine clearance. We conclude that the Na+/K+-ATPase is not the only mediator of the natriuretic effect of prolactin in the model of cholestasis of pregnancy.

  19. 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 Na(+)-K(+)-ATPase plays a central role in the clearance of K(+) from the extracellular fluid, therefore maintaining blood [K(+)]. Na(+)-K(+)-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high-fat diet (HFD) and exercise......(+)-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 ... phospholemman. Phospholemman mRNA and protein expression were increased after HFD and restored to control levels after ET. Insulin-stimulated translocation of the alpha(2)-subunit to plasma membrane was impaired by HFD, whereas alpha(1)-subunit translocation remained unchanged. Alterations in sodium pump...

  20. Functional interaction of nicotinic acetylcholine receptors and Na+/K+ ATPase from Locusta migratoria manilensis (Meyen).

    Science.gov (United States)

    Bao, Haibo; Sun, Huahua; Xiao, Youxin; Zhang, Yixi; Wang, Xin; Xu, Xiaoyong; Liu, Zewen; Fang, Jichao; Li, Zhong

    2015-03-06

    Associated proteins are important for the correct functioning of nicotinic acetylcholine receptors (nAChRs). In the present study, a neonicotinoid-agarose affinity column was used to isolate related proteins from a solubilized membrane preparation from the nervous system of Locusta migratoria manilensis (Meyen). 1530 peptides were identified and most of them were involved in the membranous structure, molecular interaction and cellular communication. Among these peptides, Na(+)/K(+) ATPase had the highest MASCOT score and were involved in the molecular interaction, which suggested that Na(+)/K(+) ATPase and nAChRs might have strong and stable interactions in insect central nervous system. In the present study, functional interactions between nAChRs and Na(+)/K(+) ATPase were examined by heterologous expression in Xenopus oocytes. The results showed that the activated nAChRs increased pump currents of Na(+)/K(+) ATPase, which did not require current flow through open nAChRs. In turn, Na(+)/K(+) ATPase significantly increased agonist sensitivities of nAChRs in a pump activity-independent manner and reduced the maximum current (Imax) of nAChRs. These findings provide novel insights concerning the functional interactions between insect nAChRs and Na(+)/K(+) ATPase.

  1. Temperature-specific inhibition of human red cell Na+/K+ ATPase by 2450-MHz microwave radiation

    Energy Technology Data Exchange (ETDEWEB)

    Allis, J.W.; Sinha-Robinson, B.L.

    1987-01-01

    The ATPase activity in human red blood cell membranes was investigated in vitro as a function of temperature and exposure to 2450-MHz continuous wave microwave radiation to confirm and extend a report of Na+ transport inhibition under certain conditions of temperature and exposure. Assays were conducted spectrophotometrically during microwave exposure with a custom-made spectrophotometer-waveguide apparatus. Temperature profiles of total ATPase and Ca+2 ATPase (ouabain-inhibited) activity between 17 and 31 degrees C were graphed as an Arrhenius plot. Each data set was fitted to two straight lines which intersect between 23 and 24 degrees C. The difference between the total and Ca+2 ATPase activities, which represented the Na+/K+ ATPase activity, was also plotted and treated similarly to yield an intersection near 25 degrees C. Exposure of membrane suspensions to electromagnetic radiation, at a dose rate of 6 W/kg and at five temperatures between 23 and 27 degrees C, resulted in an activity change only for the Na+/K+ ATPase at 25 degrees C. The activity decreased by approximately 35% compared to sham-irradiated samples. A possible explanation for the unusual temperature/microwave interaction is proposed.

  2. 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.

  3. Formation of oriented membrane multilayers of Na/K-ATPase

    International Nuclear Information System (INIS)

    Pachence, J.M.; Knott, R.; Edelman, I.S.; Schoenborn, B.P.; Wallace, B.A.

    1982-01-01

    The isolated membrane-bound enzyme retains its ouabain-sensitive ATP hydrolysis activity, and produces ATP-dependent Na + and K + fluxes when incorporated into phospholipid vesicles. The ultimate goal of this work is to determine its low resolution structure using both X-ray and neutron diffraction. A number of methods were used to impart lamellar stacking order to highly purified pig Na/K-ATPase membranes. Upon partial dehydration, x-ray diffraction from Na/K-ATPase membrane multilayers at 98% relative humidity yielded discrete reflections of 118 A periodicity, diffracting to 1/14.8 A -1 , additionally, continuous diffraction to 1/10 A -1 was obtained. Subjecting the membrane multilayers to high magnetic fields improved the quality of the lamellar diffraction dramatically. Neutron diffraction studies of the partially dehydrated Na/K-ATPase membrane multilayers detected a mosaic spread of 2 0 when the samples were subjected to a magnetic field of 5 Tesla perpendicular to the membrane surface; the reflections were narrower than the camera line width; hence, the lattice disorder has also decreased significantly, although only four orders were measured

  4. Enhanced Store-Operated Calcium Entry in Platelets is Associated with Peripheral Artery Disease in Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Weijie Xia

    2015-11-01

    Full Text Available Background/Aims: Platelet dysfunction plays an important role in thrombosis in diabetes with peripheral artery disease (PAD. Store-operated calcium entry (SOCE and stromal interaction molecule 1 (STIM1 regulate platelet activity by modulating calcium influx. We hypothesized that enhanced SOCE in platelets is associated with diabetes with PAD. Methods: We studied the activity of platelets from healthy participants and from type 2 diabetic patients. Platelet calcium influx and protein expression of STIM1 and sarcoendoplasmic reticulum Ca2+-ATPase 3 (SERCA3 were investigated. Results: Compared with platelets from diabetic patients without PAD, platelets from diabetic patients with PAD exhibited significantly increased SOCE . Menthol administration completely inhibited calcium influx in platelets from diabetic patients without PAD, but this effect was blunted in those from diabetic patients with PAD. Furthermore, the increase in SOCE was correlated with the ankle brachial index (ABI in diabetic patients. High glucose significantly up-regulated STIM1 and SERCA3 protein expression and induced the phosphorylation of phospholipase C (PLC in platelets from healthy participants. This effect was attenuated in the presence of menthol or U73122, an inhibitor of PLC. Similarly, significant increases in STIM1 and SERCA3 protein expression were found in platelets from diabetic patients compared to those from healthy participants. Conclusion: Platelets from diabetic patients with PAD exhibited enhanced Store-operated calcium influx, which was associated with elevated STIM1/SERCA3 expression via a PLC-dependent pathway and was inhibited by menthol.

  5. [Physiological prion and activity of plasma membrane Na+,K(+)- and Ca(2+)-ATPase in the medulla oblongata cells of rats of different ages].

    Science.gov (United States)

    Kushkevych, M V; Vlizlo, V V; Martyn, Iu V

    2013-01-01

    Based on the results of immunohistochemical analysis of the rat medulla tissue the localization of physiological prion has been established. Specifically, in rats aged one month they are placed in the gray matter near the bodies of neurons and mikrohliocytes and in animals of six and thirty months--in olive kernel core and upward path bodies. Physiological prion is localized along the nerve processes and is absent in the neuron bodies. In the medulla oblongata of animals aged six months its amount is the highest compared to animals of other age. The activity of plasma membrane ATPases in this tissue decreases with age, the content of sodium and calcium ions increases, while that of potassium is almost unchanged.

  6. Isolation and characterization of a specific endogenous Na+, K+-ATPase inhibitor from bovine adrenal

    International Nuclear Information System (INIS)

    Tamura, M.; Lam, T.T.; Inagami, T.

    1988-01-01

    In order to identify a specific endogenous Na + ,K + -ATPase inhibitor which could possibly be related to salt-dependent hypertension, the authors looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na + ,K + -ATPase; (ii) competitive displacing activity against [ 3 H]ouabain binding to the enzyme; (iii) inhibitory activity for 86 Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C 18 open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na + ,K + -ATPase. These results strongly suggest that this water-soluble nonpeptidic Na + ,K + -ATPase inhibitor may be a specific endogenous regulator for the ATPase

  7. Asn792 participates in the hydrogen bond network around the K+-binding pocket of gastric H,K-ATPase.

    NARCIS (Netherlands)

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

    2005-01-01

    Asn792 present in M5 of gastric H,K-ATPase is highly conserved within the P-type ATPase family. A direct role in K+ binding was postulated for Na,K-ATPase but was not found in a recent model for gastric H,K-ATPase (Koenderink, J. B., Swarts, H. G. P., Willems, P. H. G. M., Krieger, E., and De Pont,

  8. Visualizing the mapped ion pathway through the Na,K-ATPase pump.

    Science.gov (United States)

    Takeuchi, Ayako; Reyes, Nicolás; Artigas, Pablo; Gadsby, David C

    2009-11-01

    The Na(+),K(+)-ATPase pump achieves thermodynamically uphill exchange of cytoplasmic Na(+) ions for extracellular K(+) ions by using ATP-mediated phosphorylation, followed by autodephosphorylation, to power conformational changes that allow ion access to the pump's binding sites from only one side of the membrane at a time. Formally, the pump behaves like an ion channel with two tightly coupled gates that are constrained to open and close alternately. The marine agent palytoxin disrupts this coupling, allowing both gates to sometimes be open, so temporarily transforming a pump into an ion channel. We made a cysteine scan of Na(+),K(+)-ATPase transmembrane (TM) segments TM1 to TM6, and used recordings of Na(+) current flow through palytoxin-bound pump-channels to monitor accessibility of introduced cysteine residues via their reaction with hydrophilic methanethiosulfonate (MTS) reagents. To visualize the open-channel pathway, the reactive positions were mapped onto a homology model of Na(+),K(+)-ATPase based on the structure of the related sarcoplasmicand endoplasmic-reticulum (SERCA) Ca(2+)-ATPase in a BeF(3)(-)-trapped state,(1,2) in which the extra-cytoplasmic gate is wide open (although the cytoplasmic access pathway is firmly shut). The results revealed a single unbroken chain of reactive positions that traverses the pump from the extracellular surface to the cytoplasm, comprises residues from TM1, TM2, TM4 and TM6, and passes through the equivalent of cation binding site II in SERCA, but not through site I. Cavity search analysis of the homology model validated its use for mapping the data by yielding a calculated extra-cytoplasmic pathway surrounded by MTS-reactive residues. As predicted by previous experimental results, that calculated extra-cytoplasmic pathway abruptly broadens above residue T806, at the outermost end of TM6 that forms the floor of the extracellular-facing vestibule. These findings provide a structural basis for further understanding cation

  9. Visualizing the mapped ion pathway through the Na,K-ATPase pump

    Science.gov (United States)

    Takeuchi, Ayako; Reyes, Nicolás; Artigas, Pablo; Gadsby, David C.

    2009-01-01

    The Na+,K+-ATPase pump achieves thermodynamically uphill exchange of cytoplasmic Na+ ions for extracellular K+ ions by using ATP-mediated phosphorylation, followed by autodephosphorylation, to power conformational changes that allow ion access to the pump's binding sites from only one side of the membrane at a time. Formally, the pump behaves like an ion channel with two tightly coupled gates that are constrained to open and close alternately. The marine agent palytoxin disrupts this coupling, allowing both gates to sometimes be open, so temporarily transforming a pump into an ion channel. We made a cysteine scan of Na+,K+-ATPase transmembrane (TM) segments TM1 to TM6, and used recordings of Na+ current flow through palytoxin-bound pump-channels to monitor accessibility of introduced cysteine residues via their reaction with hydrophilic methanethiosulfonate (MTS) reagents. To visualize the open-channel pathway, the reactive positions were mapped onto a homology model of Na+,K+-ATPase based on the structure of the related sarcoplasmic- and endoplasmic-reticulum (SERCA) Ca2+-ATPase in a BeF3−-trapped state1,2, in which the extra-cytoplasmic gate is wide open (although the cytoplasmic access pathway is firmly shut). The results revealed a single unbroken chain of reactive positions that traverses the pump from the extracellular surface to the cytoplasm, comprises residues from TM1, TM2, TM4, and TM6, and passes through the equivalent of cation binding site II in SERCA, but not through site I. Cavity search analysis of the homology model validated its use for mapping the data by yielding a calculated extra-cytoplasmic pathway surrounded by MTS-reactive residues. As predicted by previous experimental results, that calculated extra-cytoplasmic pathway abruptly broadens above residue T806, at the outermost end of TM6 which forms the floor of the extracellular-facing vestibule. These findings provide a structural basis for further understanding cation translocation by

  10. 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...

  11. Preparation of a highly concentrated, completely monomeric, active sarcoplasmic reticulum Ca2+-ATPase.

    Science.gov (United States)

    Lüdi, H; Hasselbach, W

    1985-11-21

    Sarcoplasmic reticulum vesicles from fast skeletal muscle were partially delipidated with sodium cholate at high ionic strength and sedimented in a discontinuous sucrose gradient. Phospholipid content was reduced from 0.777 mumol/mg protein to 0.242 mumol/mg protein. As judged from gel electrophoresis and high pressure liquid gel chromatography, accessory proteins were removed during centrifugation and the Ca2+-ATPase was obtained in an almost pure form. Addition of myristoylglycerophosphocholine (1 mg/mg protein) reactivates ATPase and dinitrophenylphosphatase activity to the same degree obtained with native vesicles. Using the analytical ultracentrifuge it could be demonstrated that the reactivated Ca2+-ATPase was present exclusively in a monomeric state. These results were obtained at high and low ionic strength and up to a protein concentration of 10 mg/ml. Therefore this preparation should be very useful to investigate differences between oligomeric and monomeric Ca2+-ATPase.

  12. Inhibition of partially purified K+/H+-ATPase from guinea-pig isolated and enriched parietal cells by substituted benzimidazoles.

    Science.gov (United States)

    Beil, W.; Sewing, K. F.

    1984-01-01

    The cellular and subcellular distributions of adenosinetriphosphatases (ATPases) were examined in guinea-pig gastric mucosal cells. All cell types displayed Mg2+-ATPase and bicarbonate (HCO3-)-stimulated ATPase activity. K+-ATPase was located only in fractions derived from parietal cells. Differential and density-gradient centrifugation of material prepared from parietal cells revealed that K+-ATPase activity was located in a tubulo-vesicular membrane fraction. Enzyme activity was ten fold greater in this fraction than in a crude parietal cell homogenate. The substituted benzimidazoles, omeprazole and picoprazole, inhibited K+-ATPase (IC50 1.8 +/- 0.5 mumol l-1 and 3.1 +/- 0.4 mumol l-1, respectively). Detailed kinetic analysis indicated that these compounds were non-competitive and reversible inhibitors of the enzyme. In contrast cimetidine and verapamil were without effect on the enzyme. The relevance of the inhibition of K+-ATPase to the antisecretory activity of the benzimidazoles, in experimental animals and man, is discussed. PMID:6146367

  13. Cellular localization of Na(+), K(+)-ATPase in the mammalian vestibular system

    Science.gov (United States)

    Kerr, T. P.

    1984-01-01

    Two different, but complementary, procedures for cellular localization of Na+, K+-ATPase in the guinea pig vestibular system were employed. One of these techniques, devised by Stirling, depends upon the well documented ability of the specific inhibitor ouabain to bind selectively to Na+,K+-ATPase, blocking catalytic activity. Microdisected vestibular tissues are incubated with tritium-labelled (3H-) ouabain, and regions with a high concentration of Na+,K+-ATPase are subsequently identified by light microscope autoradiography. A second method, originated by Ernst, detects inorganic phosphate released from an artificial substrate (nitrophenyl phosphate) by catalytic activity of the enzyme. In the presence of strontium ion, phosphate is precipitated near regions of high activity, then converted to a product which may finally be visualized in the electron microscope. This cytochemical enzymatic reaction is inhibited by ouabain.

  14. Mechanism of photoinactivation of plant plasma membrane ATPases

    International Nuclear Information System (INIS)

    Imbrie, C.W.; Murphy, T.M.

    1984-01-01

    UV radiation at 290 and 365 nm inactivates two forms of the K + -stimulated ATPase associated with the plasma membrane of suspension-cultured cells of Rosa damascena. One form is 15 and 36 times more sensitive than the other to 290 and 365 nm, respectively. For both forms, the inactivation requires oxygen, is inhibited by azide and diazobicyclo(2.2.2.2)octane, but not glycerol, and is enhanced up to 7.5 times in deuterium oxide solvent. Inactivation occurs concomitantly with loss of absorbance at 290 nm. Cs + and NO 3 - , quenchers of tryptophan fluorescence, inhibit inactivation. The results suggest that inactivation involves singlet-oxygen mediated destruction of tryptophans in the ATPases. (author)

  15. 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.

  16. Binding of the Inhibitor Protein IF1 to Bovine F1-ATPase

    Science.gov (United States)

    Bason, John V.; Runswick, Michael J.; Fearnley, Ian M.; Walker, John E.

    2011-01-01

    In the structure of bovine F1-ATPase inhibited with residues 1–60 of the bovine inhibitor protein IF1, the α-helical inhibitor interacts with five of the nine subunits of F1-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF1 destabilizes the interaction of the inhibitor with F1-ATPase and may assist in removing the inhibitor from its binding site when F1Fo-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF1 and the C-terminal domains of the βDP-subunit and βTP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the βDP-subunit. Several conserved charged amino acids in the long α-helix of IF1 are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F1-ATPase and occupy aqueous cavities in F1-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme. PMID:21192948

  17. Renal aging in WKY rats: changes in Na+,K+ -ATPase function and oxidative stress.

    Science.gov (United States)

    Silva, E; Pinto, V; Simão, S; Serrão, M P; Afonso, J; Amaral, J; Pinho, M J; Gomes, P; Soares-da-Silva, P

    2010-12-01

    It has been suggested that alterations in Na(+),K(+)-ATPase mediate the development of several aging-related pathologies, such as hypertension and diabetes. Thus, we evaluated Na(+),K(+)-ATPase function and H(2)O(2) production in the renal cortex and medulla of Wistar Kyoto (WKY) rats at 13, 52 and 91 weeks of age. Creatinine clearance, proteinuria, urinary excretion of Na(+) and K(+) and fractional excretion of Na(+) were also determined. The results show that at 91 weeks old WKY rats had increased creatinine clearance and did not have proteinuria. Despite aging having had no effect on urinary Na(+) excretion, urinary K(+) excretion was increased and fractional Na(+) excretion was decreased with age. In renal proximal tubules and isolated renal cortical cells, 91 week old rats had decreased Na(+),K(+)-ATPase activity when compared to 13 and 52 week old rats. In renal medulla, 91 week old rats had increased Na(+),K(+)-ATPase activity, paralleled by an increase in protein expression of α(1)-subunit of Na(+),K(+)-ATPase. In addition, renal H(2)O(2) production increased with age and at 91 weeks of age renal medulla H(2)O(2) production was significantly higher than renal cortex production. The present work demonstrates that although at 91 weeks of age WKY rats were able to maintain Na(+) homeostasis, aging was accompanied by alterations in renal Na(+),K(+)-ATPase function. The observed increase in oxidative stress may account, in part, for the observed changes. Possibly, altered Na(+),K(+)-ATPase renal function may precede the development of age-related pathologies and loss of renal function. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. The promiscuous phosphomonoestearase activity of Archaeoglobus fulgidus CopA, a thermophilic Cu+ transport ATPase.

    Science.gov (United States)

    Bredeston, Luis M; González Flecha, F Luis

    2016-07-01

    Membrane transport P-type ATPases display two characteristic enzymatic activities: a principal ATPase activity provides the driving force for ion transport across biological membranes, whereas a promiscuous secondary activity catalyzes the hydrolysis of phosphate monoesters. This last activity is usually denoted as the phosphatase activity of P-ATPases. In the present study, we characterize the phosphatase activity of the Cu(+)-transport ATPase from Archaeglobus fulgidus (Af-CopA) and compare it with the principal ATPase activity. Our results show that the phosphatase turnover number was 20 times higher than that corresponding to the ATPase activity, but it is compensated by a high value of Km, producing a less efficient catalysis for pNPP. This secondary activity is enhanced by Mg(2+) (essential activator) and phospholipids (non-essential activator), and inhibited by salts and Cu(+). Transition state analysis of the catalyzed and noncatalyzed hydrolysis of pNPP indicates that Af-CopA enhances the reaction rates by a factor of 10(5) (ΔΔG(‡)=38 kJ/mol) mainly by reducing the enthalpy of activation (ΔΔH(‡)=30 kJ/mol), whereas the entropy of activation is less negative on the enzyme than in solution. For the ATPase activity, the decrease in the enthalpic component of the barrier is higher (ΔΔH(‡)=39 kJ/mol) and the entropic component is small on both the enzyme and in solution. These results suggest that different mechanisms are involved in the transference of the phosphoryl group of p-nitrophenyl phosphate and ATP. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Na,K-ATPase biostimulation by low-energy laser irradiation: comparative effects in membrane, solubilized and proteoliposomes enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Rigos, C.F.; Tedesco, A.C.; Ciancaglini, P. [Universidade de Sao Paulo (USP), Ribeirao Preto, SP (Brazil). Faculdade de Filosofia, Ciencias e Letras. Dept. de Quimica; Santos, H.L. [Universidade Federal de Sao Joao Del Rei (UFSJ), MG (Brazil)

    2008-07-01

    Full text: The mechanism of laser irradiation action on living cells is not yet understood. The role of membrane ATPases as possible targets has been analyzed. In our group we have been working with Na,K-ATPase. This enzyme is a member of the P-type family of active cation transport proteins. Thus, the aim of the present work was to investigate the effect of low-energy laser irradiation (685 nm, 35 mW) on the ATPase activity of different forms of the Na,K-ATPase. Membrane-bound and solubilized (ab)2 form of Na,K-ATPase was obtained from the rabbit kidney and DPPC:DPPE-proteoliposomes were prepared by the co-solubilization method. Irradiations were carried out at 685 nm. The ATPase activity of the membrane fraction was not altered with exposition to irradiation doses between 4 and 24 J/c m2. With irradiation doses ranging from 32 to 40 J/c m2, a 28% increase on the ATPase activity was observed while when using up to 50 J/c m2 no additional enhancement was observed. When bio stimulation was done using the purified or the reconstituted enzyme, an increase of about 36-40% on the ATPase activity was observed using only 4-8 J/c m2. With irradiation above these values (24 J/c m2) no additional increase in the activity appeared. These studies revealed that the bio stimulation of ATPase activity from different forms of the Na,K -ATPase is dose dependent in different ranges of irradiation exposure. The stimulation promoted by visible laser doses was modulated and the process was reverted after 2 h for the enzyme present in the membrane and after about 5 h for the solubilized or the reconstituted in DPPC:DPPE-liposomes.

  20. Na,K-ATPase biostimulation by low-energy laser irradiation: comparative effects in membrane, solubilized and proteoliposomes enzyme

    International Nuclear Information System (INIS)

    Rigos, C.F.; Tedesco, A.C.; Ciancaglini, P.

    2008-01-01

    Full text: The mechanism of laser irradiation action on living cells is not yet understood. The role of membrane ATPases as possible targets has been analyzed. In our group we have been working with Na,K-ATPase. This enzyme is a member of the P-type family of active cation transport proteins. Thus, the aim of the present work was to investigate the effect of low-energy laser irradiation (685 nm, 35 mW) on the ATPase activity of different forms of the Na,K-ATPase. Membrane-bound and solubilized (ab)2 form of Na,K-ATPase was obtained from the rabbit kidney and DPPC:DPPE-proteoliposomes were prepared by the co-solubilization method. Irradiations were carried out at 685 nm. The ATPase activity of the membrane fraction was not altered with exposition to irradiation doses between 4 and 24 J/c m2. With irradiation doses ranging from 32 to 40 J/c m2, a 28% increase on the ATPase activity was observed while when using up to 50 J/c m2 no additional enhancement was observed. When bio stimulation was done using the purified or the reconstituted enzyme, an increase of about 36-40% on the ATPase activity was observed using only 4-8 J/c m2. With irradiation above these values (24 J/c m2) no additional increase in the activity appeared. These studies revealed that the bio stimulation of ATPase activity from different forms of the Na,K -ATPase is dose dependent in different ranges of irradiation exposure. The stimulation promoted by visible laser doses was modulated and the process was reverted after 2 h for the enzyme present in the membrane and after about 5 h for the solubilized or the reconstituted in DPPC:DPPE-liposomes

  1. Transient expression of P-type ATPases in tobacco epidermal cells

    DEFF Research Database (Denmark)

    Pedas, Lisbeth Rosager; Palmgren, Michael Broberg; Lopez Marques, Rosa Laura

    2016-01-01

    Transient expression in tobacco cells is a convenient method for several purposes such as analysis of protein-protein interactions and the subcellular localization of plant proteins. A suspension of Agrobacterium tumefaciens cells carrying the plasmid of interest is injected into the intracellula...... for example protein-protein interaction studies. In this chapter, we describe the procedure to transiently express P-type ATPases in tobacco epidermal cells, with focus on subcellular localization of the protein complexes formed by P4-ATPases and their β-subunits....

  2. Modelled microgravity alters the Na+, K+-ATPase activity in rat heart homogenates

    Science.gov (United States)

    Peana, Alessandra T.; Pippia, Proto; Paci, Silvia; Tognacini, Christina; Assaretti, Anna Rita; Meloni, Antonietta M.; Galleri, Grazia; Bernardini, Federico

    2005-08-01

    This study was aimed at establishing whether modeled microgravity conditions, created in a three-dimensional clinostat (Random Positioning Machine, RPM), influence the membrane-associated Na+, K+- and Mg2+- ATPase activities in heart homogenates from rats (ex- posed to RPM for 48 hours). The experimental data indicate that modeled low g significantly decreased the total ATPase (p<0.01) and Na+, K+ -ATPase activities (p<0.05) with no change of the Mg2+-ATPase activity, compared to the respective rat control groups (ground). This Na+, K+- pump inhibition could cause a digital- like effect in response to several modifications of many physiological processes even if this inhibition might also be causally related to the physiological environment induced by RPM. The exact mechanism by which total A TPase and Na+, K+ -A TPase activities decrease in response to RPM conditions remains to be established. We cannot rule out that a reduced intracellular ATP production, previously demonstrated in other cellular systems submitted to modeled microgravity conditions, could be responsible for the effects reported here.

  3. An Arginine Finger Regulates the Sequential Action of Asymmetrical Hexameric ATPase in the Double-Stranded DNA Translocation Motor.

    Science.gov (United States)

    Zhao, Zhengyi; De-Donatis, Gian Marco; Schwartz, Chad; Fang, Huaming; Li, Jingyuan; Guo, Peixuan

    2016-10-01

    Biological motors are ubiquitous in living systems. Currently, how the motor components coordinate the unidirectional motion is elusive in most cases. Here, we report that the sequential action of the ATPase ring in the DNA packaging motor of bacteriophage ϕ29 is regulated by an arginine finger that extends from one ATPase subunit to the adjacent unit to promote noncovalent dimer formation. Mutation of the arginine finger resulted in the interruption of ATPase oligomerization, ATP binding/hydrolysis, and DNA translocation. Dimer formation reappeared when arginine mutants were mixed with other ATPase subunits that can offer the arginine to promote their interaction. Ultracentrifugation and virion assembly assays indicated that the ATPase was presenting as monomers and dimer mixtures. The isolated dimer alone was inactive in DNA translocation, but the addition of monomer could restore the activity, suggesting that the hexameric ATPase ring contained both dimer and monomers. Moreover, ATP binding or hydrolysis resulted in conformation and entropy changes of the ATPase with high or low DNA affinity. Taking these observations together, we concluded that the arginine finger regulates sequential action of the motor ATPase subunit by promoting the formation of the dimer inside the hexamer. The finding of asymmetrical hexameric organization is supported by structural evidence of many other ATPase systems showing the presence of one noncovalent dimer and four monomer subunits. All of these provide clues for why the asymmetrical hexameric ATPase gp16 of ϕ29 was previously reported as a pentameric configuration by cryo-electron microscopy (cryo-EM) since the contact by the arginine finger renders two adjacent ATPase subunits closer than other subunits. Thus, the asymmetrical hexamer would appear as a pentamer by cryo-EM, a technology that acquires the average of many images. Copyright © 2016 Zhao et al.

  4. Effect of near-UV light on Na-K-ATPase of the rat lens

    Energy Technology Data Exchange (ETDEWEB)

    Torriglia, A.; Zigman, S.

    1988-06-01

    The influence of in vitro near-UV radiation exposure on the physical state of the rat lens and on its membrane-bound Na-K-ATPase activity was investigated. Lens swelling was correlated to the appearance of opacities and the inactivation of the enzyme. The results show a significant decrease in the Na-K-ATPase activity which may be an early change leading to osmotic type cataracts. The dose-effect curves obtained for cortical and epithelial enzymes were different. Since the data do not follow a mono-exponential function, the existence of two forms of Na-K-ATPase in the lens is discussed.

  5. Evaluation of Na+, K+-ATPase activity in the brain of young rats after acute administration of fenproporex

    Directory of Open Access Journals (Sweden)

    Gislaine T. Rezin

    2014-05-01

    Full Text Available Objectives: Fenproporex is an amphetamine-based anorectic which is rapidly converted into amphetamine in vivo. Na+, K+-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that the effects of fenproporex on brain metabolism are poorly known and that Na+, K+-ATPase is essential for normal brain function, this study sought to evaluate the effect of this drug on Na+, K+-ATPase activity in the hippocampus, hypothalamus, prefrontal cortex, and striatum of young rats. Methods: Young male Wistar rats received a single injection of fenproporex (6.25, 12.5, or 25 mg/kg intraperitoneally or polysorbate 80 (control group. Two hours after the last injection, the rats were killed by decapitation and the brain was removed for evaluation of Na+, K+-ATPase activity. Results: Fenproporex decreased Na+, K+-ATPase activity in the striatum of young rats at doses of 6.25, 12.5, and 25 mg/kg and increased enzyme activity in the hypothalamus at the same doses. Na+, K+-ATPase activity was not affected in the hippocampus or prefrontal cortex. Conclusion: Fenproporex administration decreased Na+, K+-ATPase activity in the striatum even in low doses. However, in the hypothalamus, Na+, K+-ATPase activity was increased. Changes in this enzyme might be the result of the effects of fenproporex on neuronal excitability.

  6. 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...... 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....

  7. ATPase and morphologic changes induced by UVB on Langerhans cells in guinea pigs

    International Nuclear Information System (INIS)

    Hanau, D.; Fabre, M.; Lepoittevin, J.P.; Stampf, J.L.; Grosshans, E.; Benezra, C.

    1985-01-01

    The authors have devised, in guinea pigs, an improved ATPase technique which enables one to proceed from light to electron microscope study while preserving, on the ultrastructural level, the various membranous structures, in particular the Langerhans cell (LC) granules. Using this method, they have been able to confirm the action of acute, low-dose UVB on the surface enzymatic marker, ATPase. Moreover, this study has shown that the ATPase-negative LC contain abnormal LC granules or, more often, are deficient in LC granules. In a previous work, the authors have shown that, after epicutaneous application of a hapten, one successively observes an extensive adsorptive pinocytosis process, the disappearance of the membranous ATPase system, and the appearance of LC granules in the cytoplasm. Therefore, the authors may suppose that, after UVB irradiation, the disappearance of the ATPase system and/or the possible alteration of the adsorptive pinocytosis process interrupts or alters the formation of LC granules. These successive events might play a vital role in the formation of the hapten--carrier protein-Ia antigen complex. In their absence in a large number of LC, following UV irradiation, epicutaneous application of a hapten would lead to the development of a state of immune tolerance

  8. 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

  9. Salt-induced Na+/K+-ATPase-α/β expression involves soluble adenylyl cyclase in endothelial cells.

    Science.gov (United States)

    Mewes, Mirja; Nedele, Johanna; Schelleckes, Katrin; Bondareva, Olga; Lenders, Malte; Kusche-Vihrog, Kristina; Schnittler, Hans-Joachim; Brand, Stefan-Martin; Schmitz, Boris; Brand, Eva

    2017-10-01

    High dietary salt intake may lead to vascular stiffness, which predicts cardiovascular diseases such as heart failure, and myocardial and cerebral infarctions as well as renal impairment. The vascular endothelium is a primary target for deleterious salt effects leading to dysfunction and endothelial stiffness. We hypothesize that the Ca 2+ - and bicarbonate-activated soluble adenylyl cyclase (sAC) contributes to Na + /K + -ATPase expression regulation in vascular endothelial cells and is an important regulator of endothelial stiffness. In vitro stimulation of vascular endothelial cells with high sodium (150 mM Na + )-induced Na + /K + -ATPase-α and Na + /K + -ATPase-β protein expression determined by western blot. Promoter analyses revealed increased cAMP response element (CRE)-mediated Na + /K + -ATPase-α transcriptional activity under high sodium concentrations. Inhibition of sAC by the specific inhibitor KH7 or siRNA reduced the sodium effects. Flame photometry revealed increased intracellular sodium concentrations in response to high sodium stimulations, which were paralleled by elevated ATP levels. Using atomic force microscopy, a nano-technique that measures cellular stiffness and deformability, we detected significant endothelial stiffening under increased sodium concentrations, which was prevented by inhibition of sAC using KH7 and Na + /K + -ATPase using ouabain. Furthermore, analysis of primary aortic endothelial cells in an in vitro aging model revealed an impaired Na + /K + -ATPase-α sodium response and elevated intracellular sodium levels with cellular aging. We conclude that sAC mediates sodium-induced Na + /K + -ATPase expression in vascular endothelium and is an important regulator of endothelial stiffness. The reactivity of Na + /K + -ATPase-α expression regulation in response to high sodium seems to be impaired in aging endothelial cells and might be a component of endothelial dysfunction.

  10. Radiation inactivation analysis of chloroplast CF0-CF1 ATPase

    International Nuclear Information System (INIS)

    Wang, M.Y.; Chien, L.F.; Pan, R.L.

    1988-01-01

    Radiation inactivation technique was employed to measure the functional size of adenosine triphosphatase of spinach chloroplasts. The functional size for acid-base-induced ATP synthesis was 450 +/- 24 kilodaltons; for phenazine methosulfate-mediated ATP synthesis, 613 +/- 33 kilodaltons; and for methanol-activated ATP hydrolysis, 280 +/- 14 kilodaltons. The difference (170 +/- 57 kilodaltons) between 450 +/- 24 and 280 +/- 14 kilodaltons is explained to be the molecular mass of proton channel (coupling factor 0) across the thylakoid membrane. Our data suggest that the stoichiometry of subunits I, II, and III of coupling factor 0 is 1:2:15. Ca2+- and Mg2+-ATPase activated by methanol, heat, and trypsin digestion have a similar functional size. However, anions such as SO 3 (2-) and CO 3 (2-) increased the molecular mass for both ATPase's (except trypsin-activated Mg2+-ATPase) by 12-30%. Soluble coupling factor 1 has a larger target size than that of membrane-bound. This is interpreted as the cold effect during irradiation

  11. Single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Ulaszewski, S.; Van Herck, J.C.; Dufour, J.P.; Kulpa, J.; Nieuwenhuis, B.; Goffeau, A.

    1987-01-01

    A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma- 32 P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors

  12. The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily.

    Science.gov (United States)

    Schwartz, Chad; De Donatis, Gian Marco; Fang, Huaming; Guo, Peixuan

    2013-08-15

    The AAA+ superfamily of proteins is a class of motor ATPases performing a wide range of functions that typically exist as hexamers. The ATPase of phi29 DNA packaging motor has long been a subject of debate in terms of stoichiometry and mechanism of action. Here, we confirmed the stoichiometry of phi29 motor ATPase to be a hexamer and provide data suggesting that the phi29 motor ATPase is a member of the classical hexameric AAA+ superfamily. Native PAGE, EMSA, capillary electrophoresis, ATP titration, and binomial distribution assay show that the ATPase is a hexamer. Mutations in the known Walker motifs of the ATPase validated our previous assumptions that the protein exists as another member of this AAA+ superfamily. Our data also supports the finding that the phi29 DNA packaging motor uses a revolution mechanism without rotation or coiling (Schwartz et al., this issue). Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  13. 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.

  14. Pro-contractile action of the Na,K-ATPase/Src-kinase signaling pathway in the vascular wall

    DEFF Research Database (Denmark)

    Bouzinova, Elena; Aalkjær, Christian; Matchkov, Vladimir

    Aim: Na,K-ATPase is essential for maintaining the transmembrane ion gradient and might initiate various intracellular signaling. These signals possibly act through a modification of the local ion concentrations or via Src-kinase activation. It is known that inhibition of the α-2 isoform of Na......,K-ATPase by ouabain elevates blood pressure. Consequently, ouabain was shown to potentiate arterial contraction in vitro. In contrast, we have demonstrated that siRNA-induced down-regulation of the α-2 isoform Na,K-ATPase expression reduced arterial sensitivity to agonist stimulation and prevented the effect......) phosphorylation assay. Down-regulation of the α-2 isoform Na,K-ATPase prevented the inhibitory effect of Src inhibitors on arterial contraction. Conclusions: The pro-contractile action of ouabain-sensitive Na,K-ATPase inhibition is associated with Src-kinase inhibition suggesting the role of this signaling...

  15. Biochemical characterization of the plasma membrane H+ - ATPase from red beet (Beta vulgaris) hypocotyl tissue

    International Nuclear Information System (INIS)

    Oleski, N.A.

    1986-01-01

    Several biochemical techniques including selective solubilization followed by gel filtration or various types of affinity chromatography, and antibody production were employed in an attempt to purify the plasma membrane H + - ATPase from red beet hypocotyl tissue. While the enzyme could not be purified using any of these methods, it was possible to successfully conduct a more detailed biochemical analysis of the H + - ATPase. The molecular weight and isoelectric point of the enzyme were determined using N,N'dicyclohexylcarbodiimide (DCCD) and a H + - ATPase antibody. When plasma membrane vesicles were incubated with 20 μM [ 14 C]-DCCD at 0 C, a single 97,000 dalton protein was apparent on a fluorograph. A close correlation between [ 14 C]-DCCD labelling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentrations suggests that this 97,000 dalton protein is a component of the plasma membrane H + - ATPase. An antibody raised against the plasma membrane H + - ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the enzyme to be pH 6.5

  16. Spontaneous shaker rat mutant - a new model for X-linked tremor/ataxia.

    Science.gov (United States)

    Figueroa, Karla P; Paul, Sharan; Calì, Tito; Lopreiato, Raffaele; Karan, Sukanya; Frizzarin, Martina; Ames, Darren; Zanni, Ginevra; Brini, Marisa; Dansithong, Warunee; Milash, Brett; Scoles, Daniel R; Carafoli, Ernesto; Pulst, Stefan M

    2016-05-01

    The shaker rat is an X-linked recessive spontaneous model of progressive Purkinje cell (PC) degeneration exhibiting a shaking ataxia and wide stance. Generation of Wistar Furth (WF)/Brown Norwegian (BN) F1 hybrids and genetic mapping of F2 sib-sib offspring using polymorphic markers narrowed the candidate gene region to 26 Mbp denoted by the last recombinant genetic marker DXRat21 at 133 Mbp to qter (the end of the long arm). In the WF background, the shaker mutation has complete penetrance, results in a stereotypic phenotype and there is a narrow window for age of disease onset; by contrast, the F2 hybrid phenotype was more varied, with a later age of onset and likely non-penetrance of the mutation. By deep RNA-sequencing, five variants were found in the candidate region; four were novel without known annotation. One of the variants caused an arginine (R) to cysteine (C) change at codon 35 of the ATPase, Ca(2+) transporting, plasma membrane 3 (Atp2b3) gene encoding PMCA3 that has high expression in the cerebellum. The variant was well supported by hundreds of overlapping reads, and was found in 100% of all affected replicas and 0% of the wild-type (WT) replicas. The mutation segregated with disease in all affected animals and the amino acid change was found in an evolutionarily conserved region of PMCA3. Despite strong genetic evidence for pathogenicity, in vitro analyses of PMCA3(R35C) function did not show any differences to WT PMCA3. Because Atp2b3 mutation leads to congenital ataxia in humans, the identified Atp2b3 missense change in the shaker rat presents a good candidate for the shaker rat phenotype based on genetic criteria, but cannot yet be considered a definite pathogenic variant owing to lack of functional changes. © 2016. Published by The Company of Biologists Ltd.

  17. Spontaneous shaker rat mutant – a new model for X-linked tremor/ataxia

    Directory of Open Access Journals (Sweden)

    Karla P. Figueroa

    2016-05-01

    Full Text Available The shaker rat is an X-linked recessive spontaneous model of progressive Purkinje cell (PC degeneration exhibiting a shaking ataxia and wide stance. Generation of Wistar Furth (WF/Brown Norwegian (BN F1 hybrids and genetic mapping of F2 sib-sib offspring using polymorphic markers narrowed the candidate gene region to 26 Mbp denoted by the last recombinant genetic marker DXRat21 at 133 Mbp to qter (the end of the long arm. In the WF background, the shaker mutation has complete penetrance, results in a stereotypic phenotype and there is a narrow window for age of disease onset; by contrast, the F2 hybrid phenotype was more varied, with a later age of onset and likely non-penetrance of the mutation. By deep RNA-sequencing, five variants were found in the candidate region; four were novel without known annotation. One of the variants caused an arginine (R to cysteine (C change at codon 35 of the ATPase, Ca2+ transporting, plasma membrane 3 (Atp2b3 gene encoding PMCA3 that has high expression in the cerebellum. The variant was well supported by hundreds of overlapping reads, and was found in 100% of all affected replicas and 0% of the wild-type (WT replicas. The mutation segregated with disease in all affected animals and the amino acid change was found in an evolutionarily conserved region of PMCA3. Despite strong genetic evidence for pathogenicity, in vitro analyses of PMCA3R35C function did not show any differences to WT PMCA3. Because Atp2b3 mutation leads to congenital ataxia in humans, the identified Atp2b3 missense change in the shaker rat presents a good candidate for the shaker rat phenotype based on genetic criteria, but cannot yet be considered a definite pathogenic variant owing to lack of functional changes.

  18. Spontaneous shaker rat mutant – a new model for X-linked tremor/ataxia

    Science.gov (United States)

    Figueroa, Karla P.; Paul, Sharan; Calì, Tito; Lopreiato, Raffaele; Karan, Sukanya; Frizzarin, Martina; Ames, Darren; Zanni, Ginevra; Brini, Marisa; Dansithong, Warunee; Milash, Brett; Scoles, Daniel R.; Carafoli, Ernesto; Pulst, Stefan M.

    2016-01-01

    ABSTRACT The shaker rat is an X-linked recessive spontaneous model of progressive Purkinje cell (PC) degeneration exhibiting a shaking ataxia and wide stance. Generation of Wistar Furth (WF)/Brown Norwegian (BN) F1 hybrids and genetic mapping of F2 sib-sib offspring using polymorphic markers narrowed the candidate gene region to 26 Mbp denoted by the last recombinant genetic marker DXRat21 at 133 Mbp to qter (the end of the long arm). In the WF background, the shaker mutation has complete penetrance, results in a stereotypic phenotype and there is a narrow window for age of disease onset; by contrast, the F2 hybrid phenotype was more varied, with a later age of onset and likely non-penetrance of the mutation. By deep RNA-sequencing, five variants were found in the candidate region; four were novel without known annotation. One of the variants caused an arginine (R) to cysteine (C) change at codon 35 of the ATPase, Ca2+ transporting, plasma membrane 3 (Atp2b3) gene encoding PMCA3 that has high expression in the cerebellum. The variant was well supported by hundreds of overlapping reads, and was found in 100% of all affected replicas and 0% of the wild-type (WT) replicas. The mutation segregated with disease in all affected animals and the amino acid change was found in an evolutionarily conserved region of PMCA3. Despite strong genetic evidence for pathogenicity, in vitro analyses of PMCA3R35C function did not show any differences to WT PMCA3. Because Atp2b3 mutation leads to congenital ataxia in humans, the identified Atp2b3 missense change in the shaker rat presents a good candidate for the shaker rat phenotype based on genetic criteria, but cannot yet be considered a definite pathogenic variant owing to lack of functional changes. PMID:27013529

  19. Physiological prion and activity of plasma membrane Na(+,K(+- and Ca(2+-ATPase in the medulla oblongata of rats of different ages

    Directory of Open Access Journals (Sweden)

    M. V. Kushkevych

    2013-04-01

    Full Text Available Based on the results of immunohistochemical analysis of the rat medulla tissue the localization of physiological prion has been established. Specifically, in rats aged one month they are placed in the gray matter near the bodies of neurons and mikrohliocytes and in animals of six and thirty months – in olive kernel core and upward path bodies. Physiological prion is localized along the nerve processes and is absent in the neuron bodies­. In the medulla oblongata of animals aged six months its amount is the highest compared to animals of other age. The activity of plasma membrane ATPases in this tissue decreases with age, the content of sodium and calcium ions increases, while that of potassium is almost unchanged.

  20. Purification, crystallization and preliminary X-ray diffraction analysis of the non-ATPase subunit Nas6 in complex with the ATPase subunit Rpt3 of the 26S proteasome from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Nakamura, Yoshihiro; Umehara, Takashi; Tanaka, Akiko; Horikoshi, Masami; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

    2007-01-01

    The complex of the non-ATPase subunit Nas6 with the C-terminal domain of the ATPase subunit Rpt3 of the 26S proteasome from S. cerevisiae was co-expressed in E. coli and purified to homogeneity. The crystals obtained from the protein complex diffracted to a resolution of 2.2 Å. The non-ATPase subunit Nas6, which is the human orthologue of gankyrin, was co-expressed with the C-terminal domain of the ATPase subunit Rpt3 of the yeast 26S proteasome in Escherichia coli, purified to near-homogeneity and crystallized using the hanging-drop vapour-diffusion method. The protein crystallized in space group P2 1 , with unit-cell parameters a = 60.38, b = 100.22, c = 72.20 Å, β = 94.70° and with three Nas6–Rpt3C molecules per asymmetric unit. The crystal diffracted to beyond 2.2 Å resolution using synchrotron radiation

  1. Na,K-ATPase activity modulates Src activation: A role for ATP/ADP ratio.

    NARCIS (Netherlands)

    Weigand, K.M.; Swarts, H.G.P.; Fedosova, N.U.; Russel, F.G.M.; Koenderink, J.B.

    2012-01-01

    Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target

  2. Relationship between serum leptin levels, ATPase activity of erythrocyte membrance and development of diabetic nephropathy in patients with DM2

    International Nuclear Information System (INIS)

    Wang Yuming

    2009-01-01

    Objective: To study the possible mechanism of development of nephrosis affected by changes of serum leptin levels and alteration of activities of Na + K + -ATPase and Ca 2+ Mg 2+ -ATPase of erythrocyte membrane in patients with type 2 diabetes(DM2). Methods: Serum leptin levels (with RIA) and erythrocyte membrane Na + K + -ATPase and Ca 2+ Mg 2+ -ATPase activitities (with Reinila method) were determined in 40 DM2 patients without nephropathy, 32 DM2 patients with nephropathy and 35 controls. Results Serum leptin levels were significantly higher in the diabetics as a whole than those in controls (P + K + -ATPase and Ca 2+ Mg 2+ -ATPase activities were significantly lower (P<0.01). Among the diabetic patients, the serum leptin levels in patients without nephrosis (P<0.05), but the RBC membrance ATPase activities were significantly lower(P<0.05). Conclusion: Development of type 2 diabetes nephrosis might be correlated with the high serum leptin level and decreased ATPase activities of erythrocite membrane. (authors)

  3. Density gradient localization of vanadate- and NO-3-sensitive ATPase from sterile cultures of Spirodela polyrrhiza (L. Schleiden

    Directory of Open Access Journals (Sweden)

    Józef Buczek

    2014-01-01

    Full Text Available The present work deals with the separation and some characteristics of ATPase activities bound with plant membanes prepared from sterile cultures of Spirodela polyrrhiza. The membrane-bound ATPases were separated on sucrose gradients and distinguished by membrane density and sensitivity to several inhibitors. The results showed that N0-3-sensitive ATPase activity associated with the tonoplast was localized at a sucrose density between 1.095-1.117 g•cm-3. The vanadate-sensitive ATPase activity bound with the plasma membrane showed a density between 1.127-1.151 g•cm-3. Both ATPases were insensitive to azide and oligomycin and were separable from markers for mitochondria.

  4. Nonspecific nature of the vanadate inhibition of rat ileal (Na, K)-ATPase

    International Nuclear Information System (INIS)

    Hajjar, J.J.; Rowe, W.A.; Tomicic, T.K.

    1988-01-01

    Vanadate has been suggested as an intracellular regulator of (Na+ + K+)-ATPase. To test this hypothesis the authors examined the stimulatory and inhibitory effects of vanadate on 86 Rb efflux and influx (measurements of the activity of the Na-pump) in rat ileum under conditions of normal, reduced and increased (Na+ + K+)-ATPase activity. The half maximal inhibition of the Rb efflux and the half maximal inhibition of the Rb influx were not different in the three conditions tested. This suggests that vanadate does not have a regulatory effect on the activity of the Na-K-transport enzyme. The vanadate effect seem rather, to be nonspecific in terms of being unrelated, on a mole per mole basis, to the activity of the (Na+ + K+)-ATPase enzyme

  5. Nonspecific nature of the vanadate inhibition of rat ileal (Na, K)-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Hajjar, J.J.; Rowe, W.A.; Tomicic, T.K.

    1988-01-01

    Vanadate has been suggested as an intracellular regulator of (Na+ + K+)-ATPase. To test this hypothesis the authors examined the stimulatory and inhibitory effects of vanadate on /sup 86/Rb efflux and influx (measurements of the activity of the Na-pump) in rat ileum under conditions of normal, reduced and increased (Na+ + K+)-ATPase activity. The half maximal inhibition of the Rb efflux and the half maximal inhibition of the Rb influx were not different in the three conditions tested. This suggests that vanadate does not have a regulatory effect on the activity of the Na-K-transport enzyme. The vanadate effect seem rather, to be nonspecific in terms of being unrelated, on a mole per mole basis, to the activity of the (Na+ + K+)-ATPase enzyme.

  6. Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

    Directory of Open Access Journals (Sweden)

    H.L. Santos

    2002-03-01

    Full Text Available SDS, C12E8, CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C12E8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,K-ATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C12E8/ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K0.5 = 4.0 µM and low (K0.5 = 1.4 mM affinity sites for ATP, with negative cooperativity. Ouabain (5 mM, oligomycin (1 µg/ml and sodium vanadate (3 µM inhibited the ATPase activity of C12E8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C12E8 can be purified and retains its activity. The activity is consistent with the form of (alphaß2 association.

  7. Na+-stimulated ATPase of alkaliphilic halotolerant cyanobacterium Aphanothece halophytica translocates Na+ into proteoliposomes via Na+ uniport mechanism

    Directory of Open Access Journals (Sweden)

    Soontharapirakkul Kanteera

    2010-08-01

    Full Text Available Abstract Background When cells are exposed to high salinity conditions, they develop a mechanism to extrude excess Na+ from cells to maintain the cytoplasmic Na+ concentration. Until now, the ATPase involved in Na+ transport in cyanobacteria has not been characterized. Here, the characterization of ATPase and its role in Na+ transport of alkaliphilic halotolerant Aphanothece halophytica were investigated to understand the survival mechanism of A. halophytica under high salinity conditions. Results The purified enzyme catalyzed the hydrolysis of ATP in the presence of Na+ but not K+, Li+ and Ca2+. The apparent Km values for Na+ and ATP were 2.0 and 1.2 mM, respectively. The enzyme is likely the F1F0-ATPase based on the usual subunit pattern and the protection against N,N'-dicyclohexylcarbodiimide inhibition of ATPase activity by Na+ in a pH-dependent manner. Proteoliposomes reconstituted with the purified enzyme could take up Na+ upon the addition of ATP. The apparent Km values for this uptake were 3.3 and 0.5 mM for Na+ and ATP, respectively. The mechanism of Na+ transport mediated by Na+-stimulated ATPase in A. halophytica was revealed. Using acridine orange as a probe, alkalization of the lumen of proteoliposomes reconstituted with Na+-stimulated ATPase was observed upon the addition of ATP with Na+ but not with K+, Li+ and Ca2+. The Na+- and ATP-dependent alkalization of the proteoliposome lumen was stimulated by carbonyl cyanide m - chlorophenylhydrazone (CCCP but was inhibited by a permeant anion nitrate. The proteoliposomes showed both ATPase activity and ATP-dependent Na+ uptake activity. The uptake of Na+ was enhanced by CCCP and nitrate. On the other hand, both CCCP and nitrate were shown to dissipate the preformed electric potential generated by Na+-stimulated ATPase of the proteoliposomes. Conclusion The data demonstrate that Na+-stimulated ATPase from A. halophytica, a likely member of F-type ATPase, functions as an electrogenic Na

  8. Towards defining the substrate of orphan P5A-ATPases

    DEFF Research Database (Denmark)

    Sørensen, Danny Mollerup; Holen, Henrik Waldal; Holemans, Tine

    2015-01-01

    leads to broad and unspecific phenotypes related to the impairment of basic ER functions such as protein folding and processing. Genetic interactions in Saccharomyces cerevisiae point to a role of the endogenous P5A-ATPase Spf1p in separation of charges in the ER, in sterol metabolism, and in insertion...... 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....

  9. Structure and Function of Cu(I)- and Zn(II)-ATPases

    DEFF Research Database (Denmark)

    Sitsel, Oleg; Grønberg, Christina; Autzen, Henriette Elisabeth

    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...

  10. Autoradiographic localization of Na+-K+-ATPase with 3H-ouabain

    International Nuclear Information System (INIS)

    Dormans, J.A.M.A.

    1976-01-01

    Using 3 H-ouabain as an inhibitor, the site of the Na + -K + -ATPase system in cells was determined autoradiographically. Experiments were performed woth guinea pig's kidney tissue. The application of light microscopical autoradiography to freeze-dried tissue showed that especially the distal tubule, and to a smaller extent the proximal tubule and the collecting tubule have Na + -K + -ATPase. Electron microscopical autoradiography showed that this activity is restricted to the baso-lateral plasmamembranes. The quantity of specific bound ouabain turns out to be correlated to the quantity of baso-lateral plasmamembrane's surface

  11. Arabidopsis calmodulin-like protein CML36 is a calcium (Ca2+) sensor that interacts with the plasma membrane Ca2+-ATPase isoform ACA8 and stimulates its activity.

    Science.gov (United States)

    Astegno, Alessandra; Bonza, Maria Cristina; Vallone, Rosario; La Verde, Valentina; D'Onofrio, Mariapina; Luoni, Laura; Molesini, Barbara; Dominici, Paola

    2017-09-08

    Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca 2+ )-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca 2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana We analyzed Ca 2+ and Mg 2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca 2+ /Mg 2+ mixed binding sites and two low-affinity Ca 2+ -specific sites. Binding of Ca 2+ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca 2+ or Mg 2+ , stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca 2+ -ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca 2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca 2+ pumps. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

    OpenAIRE

    Ogawa, Haruo; Shinoda, Takehiro; Cornelius, Flemming; Toyoshima, Chikashi

    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. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na+,K+-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analog...

  13. Characterization of ATPase activity of the AAA ARC from Bifidobacterium longum subsp. infantis.

    Science.gov (United States)

    Guzmán-Rodríguez, Mabel; de la Rosa, Ana Paulina Barba; Santos, Leticia

    2015-01-01

    Bifidobacteria are considered to be probiotics that exist in the large intestine and are helpful to maintain human health. Oral administration of bifidobacteria may be effective in improving the intestinal flora and environment, stimulating the immune response and possibly preventing cancer. However, for consistent and positive results, further well-controlled studies are urgently needed to describe the basic mechanisms of this microorganism. Analysis of the proteasome-lacking Bifidobacterium longum genome reveals that it possesses a gene, IPR003593 AAA ATPase core, which codes a 56 kDa protein containing one AAA ATPase domain. Phylogenetic classification made by CLANS, positioned this sequence into the ARC divergent branch of the AAA ATPase family of proteins. N-terminal analysis of the sequence indicates this protein is closely related to other ATPases such as the Rhodococcus erythropolis ARC, Archaeoglobus fulgidus PAN, Mycobacterium tuberculosis Mpa and the human proteasomal Rpt1 subunit. This gene was cloned, the full-length recombinant protein was overexpressed in Escherichia coli, purified as a high-molecular size complex and named Bl-ARC. Enzymatic characterization showed that Bl-ARC ATPase is active, Mg(+2)-dependent and sensitive to N-ethylmaleimide. Gene organization positions bl-arc in a region flanked by a cluster of genes that includes pup, dop and pafA genes. These findings point to a possible function as a chaperone in the degradation pathway via pupylation.

  14. 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.

  15. Critical role of γ-phosphate in structural transition of Na,K-ATPase upon ATP binding

    Science.gov (United States)

    Petrushanko, Irina Yu.; Mitkevich, Vladimir A.; Anashkina, Anastasia A.; Klimanova, Elizaveta A.; Dergousova, Elena A.; Lopina, Olga D.; Makarov, Alexander A.

    2014-06-01

    Active transport of sodium and potassium ions by Na,K-ATPase is accompanied by the enzyme conformational transition between E1 and E2 states. ATP and ADP bind to Na,K-ATPase in the E1 conformation with similar affinity but the properties of enzyme in complexes with these nucleotides are different. We have studied thermodynamics of Na,K-ATPase binding with adenine nucleotides at different temperatures using isothermal titration calorimetry. Our data indicate that β-phosphate is involved in complex formation by increasing the affinity of adenine nucleotides to Na,K-ATPase by an order of magnitude, while γ-phosphate does not affect it. ATP binding to Na,K-ATPase in contrast to ADP binding generates a structural transition in the enzyme, which is consistent with the movement of a significant portion of the surface area to a solvent-protected state. We propose that ATP binding leads to convergence of the nucleotide-binding and phosphorylation domains transferring the enzyme from the ``E1-open'' to ``E1-closed'' conformation ready for phosphorylation.

  16. Effects of dietary supplementation of arginine-silicate-inositol complex on absorption and metabolism of calcium of laying hens.

    Directory of Open Access Journals (Sweden)

    Kazim Sahin

    Full Text Available The effects of supplementation of arginine-silicate-inositol complex (ASI; 49.5-8.2-25 g/kg, respectively to laying hens were investigated with respect to eggshell quality, calcium (Ca balance, and expression of duodenal proteins related to Ca metabolism (calbindin and tight junction proteins. A total of 360 laying hens, 25 weeks old, were divided into 3 groups consisting of 6 replicate of cages, 20 birds per cage. The groups were fed a basal diet and the basal diet supplemented with 500 or 1000 mg ASI complex per kilogram for 90 days. Data were analyzed by ANCOVA using data during the first week of the adaptation period as covariates. As the ASI complex supplementation level increased, there were increases in feed intake (P < 0.0001, egg production (P < 0.001, egg weight (P < 0.0001 and eggshell weight (P < 0.001 weight, and shell thickness (P < 0.001 and decreases in feed conversion ratio and cracked egg percentage (P < 0.0001 for both. Concentrations of serum osteocalcin (P < 0.0001, vitamin D (P < 0.0001, calcium (P < 0.001, phosphorus (P < 0.001, and alkaline phosphatase (P < 0.008 as well as amounts of calcium retention (P < 0.0001 and eggshell calcium deposition (P < 0.001, and Ca balance (P < 0.0001 increased, whereas amount of calcium excretion (P < 0.001 decreased linearly in a dose-dependent manner. The ASI complex supplementation increased expressions of calcium transporters (calbindin-D28k, N sodium-calcium exchanger, plasma membrane calcium ATPase, and vitamin D receptor and tight junction proteins (zonula occludens-1 and occludin in the duodenum in a linear fashion (P < 0.0001 for all. In conclusion, provision of dietary ASI complex to laying hens during the peak laying period improved eggshell quality through improving calcium utilization as reflected by upregulation of genes related to the calcium metabolism. Further studies are needed to elucidate the contribution of each of the ASI complex ingredients.

  17. Temperature-specific inhibition of human red cell (Na/sup +//K/sup +/) ATPase by 2450-MHz microwave radiation

    Energy Technology Data Exchange (ETDEWEB)

    Allis, J.W.; Sinha-Robinson, B.L.

    1987-01-01

    The ATPase activity in human red blood cell membranes was investigated in vitro as a function of temperature and exposure to 2450-MHz (CW) microwave radiation. Assays were conducted spectrophotometrically during microwave exposure with a custom-made spectrophotometer-waveguide apparatus. Temperature profiles of total ATPase and Ca+2 ATPase (ouabain-inhibited) activity between 17 and 31 C were graphed as an Arrhenius plot. Each data set was fitted to two straight lines which intersected between 23 and 24 C. The difference between the total and Ca+2 ATPase activities, which represented the Na+/K+ ATPase activity, was also plotted and treated similarly to yield an intersection near 25 C. Exposure of membrane suspensions to a 6 W/kg dose rate at 1 C intervals between 23 and 27 C, resulted in an activity change only for the Na+/K+ ATPase at 25 C. The activity decreased by approximately 35% compared to sham-irradiated samples. An hypothesis based on the interaction of microwave radiation with enzyme structure during a conformational rearrangement is proposed as an explanation for the effect.

  18. 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.

  19. 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.

  20. Characterization of detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase by high-performance liquid chromatography

    International Nuclear Information System (INIS)

    Andersen, J.P.; Vilsen, B.; Nielsen, H.; Moller, J.V.

    1986-01-01

    Sarcoplasmic reticulum Ca 2+ -ATPase solubilized by the nonionic detergent octaethylene glycol monododecyl ether was studied by molecular sieve high-performance liquid chromatography (HPLC) and analytical ultracentrifugation. Significant irreversible aggregation of soluble Ca 2+ -ATPase occurred within a few hours in the presence of ≤ 50 μM Ca 2+ . The aggregates were inactive and were primarily held together by hydrophobic forces. In the absence of reducing agent, secondary formation of disulfide bonds occurred. The stability of the inactive dimer upon dilution permitted unambiguous assignment of its elution position and sedimentation coefficient. At high 45 Ca 2+ concentration (500 μM), monomeric Ca 2+ -ATPase was stable for several house. Reversible self-association induced by variation in protein, detergent, and lipid concentrations was studied by large-zone HPLC. The association constant for dimerization of active Ca 2+ -ATPase was found to be 10 5 -10 6 M -1 depending on the detergent concentration. More detergent was bound to monomeric than to dimeric Ca 2+ -ATPase, even above the critical micellar concentration of the detergent. Binding of Ca 2+ and 48 V vanadate as well as ATP-dependent phosphorylation was studied in monomeric and in reversibly associated dimeric preparations. In both forms, two high-affinity Ca 2+ binding sites per phosphorylation site existed. The delipidated monomer purified by HPLC was able to form ADP-insensitive phosphoenzyme and to bind ATP and vanadate simultaneously. The results suggest that formation of Ca 2+ -ATPase oligomers in the membrane is governed by nonspecific forces (low affinity) and that each polypeptide chain constitutes a functional unit