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  • Analysis of subunit assembly of the Na-K-ATPase. 8166221

    The Na-K-ATPase, or sodium pump, is comprised of two subunits, alpha and beta. Each subunit spans the lipid bilayer of the cell membrane. This review summarizes our efforts to determine how the two subunits interact to form the functional ion transporter. Our major approach has been to observe the potential for subunit assembly when one or both subunits are truncated or present as chimeras that retain only a limited region of the Na-K-ATPase. DNAs encoding these altered subunit forms of the avian Na-K-ATPase are expressed in mammalian cells. Monoclonal antibodies specific for the avian beta-subunit are then used to purify newly synthesized avian beta-subunits, and the presence of accompanying alpha-subunits indicates that subunit assembly has occurred. The ectodomain of the beta-subunit (approximately residues 62-304) is sufficient for assembly with the alpha-subunit, and a COOH-terminal truncation of the beta-subunit that lacks aminoacyl residues beyond 162 will assemble inefficiently. A maximum of 26 aminoacyl residues of the alpha-subunit are necessary for robust assembly with the beta-subunit, when this sequence replaces the COOH-terminal half of the loop between membrane spans 7 and 8 in the SERCA1 Ca-ATPase. This region of the Ca-ATPase faces the lumen of the endoplasmic reticulum. These findings encourage study of other related questions, including whether there is preferential assembly of certain subunit isoforms and how various P-type ATPases are targeted to their appropriate subcellular compartments.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-382
    Nombre del producto:
    Anti-Na+/K+ ATPase β-1 Antibody, clone C464.8

  • Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli. 19835592

    Efflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AG100
    Nombre del producto:
    Human IgG Fc Control Protein, recombinant protein

  • Na+-K+--ATPase-mediated signal transduction: from protein interaction to cellular function. 14993422

    The Na+-K+--ATPase, or Na+ pump, is a member of the P-type ATPase superfamily. In addition to pumping ions, Na+-K+--ATPase is engaged in assembly of multiple protein complexes that transmit signals to different intracellular compartments. The signaling function of the enzyme appears to have been acquired through the evolutionary incorporation of many specific binding motifs that interact with proteins and ligands. In some cell types the signaling Na+ --ATPase and its protein partners are compartmentalized in coated pits (i.e., caveolae) the plasma membrane. Binding of ouabain to the signaling Na+-K+--ATPase activates the cytoplasmic tyrosine kinase Src, resulting in the formation of an active "binary receptor" that phosphorylates and assembles other proteins into different signaling modules. This in turn activates multiple protein kinase cascades including mitogen-activated protein kinases and protein kinase C isozymes in a cell-specific manner. It also increases mitochondrial production of reactive oxygen species (ROS)and regulates intracellular calcium concentration. Crosstalk among the activated pathways eventually results in changes in the expression of a number of genes. Although ouabain stimulates hypertrophic growth in cardiac myocytes and proliferation in smooth muscle cells, it also induces apoptosis in many malignant cells. Finally, the signaling function of the enzyme is also pivotal to ouabain-induced nongenomic effects on cardiac myocytes.
    Tipo de documento:
    Referencia
    Referencia del producto:
    16-243
    Nombre del producto:
    Anti-Na+/K+ ATPase α-1 Antibody, clone C464.6, Alexa Fluor® 488

  • ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate. 19270533

    The efflux of Hoechst 33342 by ATP-binding cassette protein G2 (ABCG2) membrane pump allows reproducible identification of a subpopulation of cells by flow cytometric analysis termed the side population (SP). The SP identified by constitutive Hoechst efflux contains the stem/progenitor cell population from bone marrow and many solid organs, including prostate. DyeCycle Violet (DCV) is a cell membrane permeable, fluorescent vital dye that intercalates into DNA and is a substrate for ABCG2-mediated efflux. Therefore, DCV was evaluated in this study as a tool for identification of the SP from prostate cancer cell lines and from freshly harvested human prostate tissue. SPs that demonstrated ABCG2-mediated efflux of DCV were identified in the human prostate cancer cell lines CWR-R1, DU-145 and RWPE-1, but not in the BPH-1, LAPC-4 or PC-3 cell lines. Additionally, a SP was identified in enzymatically disaggregated prostate tumors from Transgenic Adenocarcinoma of Mouse Prostate (TRAMP), human benign prostate tissue and human prostate cancer tissue. The causal role of ABCG2-mediated efflux of DCV in the identification of the SP was confirmed by loss of the SP by incubation with the specific inhibitor of ABCG2, Fumitremorgin C. Expression of ABCG2 in the SP cells was confirmed by qRT-PCR and immunofluorescence analysis. Consequently, DCV represents an important new tool for isolation of viable candidate stem cells/cancer stem cells as a SP from cultured prostate cell lines, and prostate tissue specimens, without the requirement for instrumentation with ultra-violet excitation capability and minimizing the risk of damage to DNA in the sorted population.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB4146

  • Reciprocal modulation of function between the D1 and D2 dopamine receptors and the Na+,K+-ATPase. 18984584

    It is well documented that dopamine can increase or decrease the activity of the Na+,K+-ATPase (NKA, sodium pump) in an organ-specific fashion. This regulation can occur, at least partially, via receptor-mediated second messenger activation and can promote NKA insertion or removal from the plasma membrane. Using co-immunoprecipitation and mass spectrometry, we now show that, in both brain and HEK293T cells, D1 and D2 dopamine receptors (DARs) can exist in a complex with the sodium pump. To determine the impact of NKA on DAR function, biological assays were conducted with NKA and DARs co-expressed in HEK293T cells. In this system, expression of NKA dramatically decreased D1 and D2 DAR densities with a concomitant functional decrease in DAR-mediated regulation of cAMP levels. Interestingly, pharmacological inhibition of endogenous or overexpressed NKA enhanced DAR function without altering receptor number or localization. Similarly, DAR function was also augmented by small interfering RNA reduction of the endogenous NKA. These data suggest that, under basal conditions, NKA negatively regulates DAR function via protein-protein interactions. In reciprocal fashion, expression of DARs decreases endogenous NKA function in the absence of dopamine, implicating DAR proteins as regulators of NKA activity. Notably, dopamine stimulation or pertussis toxin inhibition of D2 receptor signaling did not alter NKA activity, indicating that the D2-mediated decrease in NKA function is dependent upon protein-protein interactions rather than signaling molecules. This evidence for reciprocal regulation between DARs and NKA provides a novel control mechanism for both DAR signaling and cellular ion balance.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • GABAergic pump cells of solitary tract nucleus innervate retrotrapezoid nucleus chemoreceptors. 17460107

    The retrotrapezoid nucleus (RTN) contains central respiratory chemoreceptors that are inhibited by activation of slowly adapting pulmonary stretch receptors (SARs). Here we examine whether RTN inhibition by lung inflation could be mediated by a direct projection from SAR second-order neurons (pump cells). Pump cells (n = 56 neurons, 13 rats) were recorded in the nucleus of solitary tract (NTS) of halothane-anesthetized rats with intact vagus nerves. Pump cells had discharges that coincided with lung inflation as monitored by the tracheal pressure. Their activity increased when end-expiratory pressure was raised and stopped instantly when ventilation was interrupted in expiration. Many pump cells could be antidromically activated from RTN (12/36). Nine of those were labeled with biotinamide. Of these nine cells, eight contained glutamic acid decarboxylase 67 (GAD67) mRNA and seven were found to reside in the lower half of the interstitial subnucleus of NTS (iNTS). Using the retrograde tracer cholera toxin-B, we confirmed that neurons located in or close to iNTS innervate RTN (two rats). Many such neurons contained GAD67 mRNA and a few contained glycine transporter2 (GLYT2) mRNA. Anterograde tract tracing with biotinylated dextranamide (four rats) applied to iNTS also confirmed that this region innervates RTN by a predominantly GABAergic projection. This work confirms that many rat NTS pump cells are located in and around the interstitial subnucleus at area postrema level. We demonstrate that a GABAergic subset of these pump cells innervates the RTN region. We conclude that these inhibitory neurons probably contact RTN chemoreceptors and mediate their inhibition by lung inflation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1511

  • Skeletal muscle Na,K-ATPase alpha and beta subunit protein levels respond to hypokalemic challenge with isoform and muscle type specificity. 8955095

    During potassium deprivation, skeletal muscle loses K+ to buffer the fall in extracellular K+. Decreased active K+ uptake via the sodium pump, Na,K-ATPase, contributes to the adjustment. Skeletal muscle expresses alpha1, alpha2, beta1, and beta2 isoforms of the Na, K-ATPase alphabeta heterodimer. This study was directed at testing the hypothesis that K+ loss from muscle during K+ deprivation is a function of decreased expression of specific isoforms expressed in a muscle type-specific pattern. Isoform abundance was measured in soleus, red and white gastrocnemius, extensor digitorum longus, and diaphragm by immunoblot. alpha2 expression was uniform across control muscles, whereas alpha1 and beta1 were twice as high in oxidative (soleus and diaphragm) as in fast glycolytic (white gastrocnemius) muscles, and beta2 expression was reciprocal: highest in white gastrocnemius and barely detectable in soleus and diaphragm. Following 10 days of potassium deprivation plasma K+ fell from 4.0 to 2.3 mM, and there were distinct responses in glycolytic versus oxidative muscles. In glycolytic white gastrocnemius alpha2 and beta2 fell 94 and 70%, respectively; in mixed red gastrocnemius and extensor digitorum longus both fell 60%, and beta1 fell 25%. In oxidative soleus and diaphragm alpha2 fell 55 and 30%, respectively, with only minor changes in beta1. Although decreases in alpha2 and beta2 expression are much greater in glycolytic than oxidative muscles during K+ deprivation, both types of muscle lose tissue K+ to the same extent, a 20% decrease, suggesting that multiple mechanisms are in place to regulate the release of skeletal muscle cell K+.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Effect of 2-wk intensified training and inactivity on muscle Na+-K+ pump expression, phospholemman (FXYD1) phosphorylation, and performance in soccer players. 20133439

    The present study examined muscle adaptations and alterations in performance of highly trained soccer players with intensified training or training cessation. Eighteen elite soccer players were, for a 2-wk period, assigned to either a group that performed high-intensity training with a reduction in the amount of training (HI, n = 7), or an inactivity group without training (IN, n = 11). HI improved (P less than 0.05) performance of the 4th, 6th, and 10th sprint in a repeated 20-m sprint test, and IN reduced (P less than 0.05) performance in the 5th to the 10th sprints after the 2-wk intervention period. In addition, the Yo-Yo intermittent recovery level 2 test performance of IN was lowered from 845 +/- 48 to 654 +/- 30 m. In HI, the protein expression of the Na(+)-K(+) pump alpha(2)-isoform was 15% higher (P less than 0.05) after the intervention period, whereas no changes were observed in alpha(1)- and beta(1)-isoform expression. In IN, Na(+)-K(+) pump expression was not changed. In HI, the FXYD1ser68-to-FXYD1 ratio was 27% higher (P less than 0.01) after the intervention period, and, in IN, the AB_FXYD1ser68 signal was 18% lower (P less than 0.05) after inactivity. The change in FXYD1ser68-to-FXYD1 ratio was correlated (r(2) = 0.35; P less than 0.05) with change in performance in repeated sprint test. The present data suggest that short-term intensified training, even for trained soccer players, can increase muscle Na(+)-K(+) pump alpha(2)-isoform expression, and that cessation of training for 2 wk does not affect the expression of Na(+)-K(+) pump isoforms. Resting phosphorylation status of the Na(+)-K(+) pump is changed by training and inactivity and may play a role in performance during repeated, intense exercise.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Exercise-induced regulation of muscular Na+-K+ pump, FXYD1, and NHE1 mRNA and protein expression: importance of training status, intensity, and muscle type. 21325644

    It is investigated if exercise-induced mRNA changes cause similar protein expression changes of Na(+)-K(+) pump isoforms (α(1), α(2), β(1), β(2)), FXYD1, and Na(+)/K(+) exchanger (NHE1) in rat skeletal muscle. Expression was evaluated (n = 8 per group) in soleus and extensor digutorum longus after 1 day, 3 days, and 3 wk (5 sessions/wk) of either sprint (4 × 3-min sprint + 1-min rest) or endurance (20 min) running. Two hours after exercise on day 1, no change in protein expression was apparent in either training group or muscle, whereas sprint exercise increased the mRNA of soleus α(2) (4.9 ± 0.8-fold; P less than 0.05), β(2) (13.2 ± 4.4-fold; P less than 0.001), and NHE1 (12.0 ± 3.1-fold; P less than 0.01). Two hours after sprint exercise, protein expression normalized to control samples was higher on day 3 than day 1 for soleus α(1) (41 ± 18% increase vs. 15 ± 8% reduction; P less than 0.05), α(2) (64 ± 35% increase vs. 37 ± 12% reduction; P less than 0.05), β(1) (17 ± 21% increase vs. 14 ± 29% reduction; P less than 0.05), and FXYD1 (35 ± 16% increase vs. 13 ± 10% reduction; P less than 0.05). In contrast, on day 3, soleus α(1) (0.1 ± 0.1-fold; P less than 0.001), α(2) (0.2 ± 0.1-fold; P less than 0.001), β(1) (0.4 ± 0.1-fold; P less than 0.05), and β(2)-mRNA (2.9 ± 1.7-fold; P less than 0.001) expression was lower than after exercise on day 1. After 3 wk of training, no change in protein expression relative to control existed. In conclusion, increased expression of Na(+)-K(+) pump subunits, FXYD1 and NHE1 after 3 days exercise training does not appear to be an effect of increased constitutive mRNA levels. Importantly, sprint exercise can reduce mRNA expression concomitant with increased protein expression.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Sodium pump activity in the yolk syncytial layer regulates zebrafish heart tube morphogenesis. 22182522

    Na(+),K(+) ATPase pumps Na(+) out of and K(+) into the cytosol, maintaining a resting potential that is essential for the function of excitable tissues like cardiac muscle. In addition to its well-characterized physiological role in the heart, Na(+),K(+) ATPase also regulates the morphogenesis of the embryonic zebrafish heart via an as yet unknown mechanism. Here, we describe a novel non-cell autonomous function of Na(+),K(+) ATPase/Atp1a1 in the elongation of the zebrafish heart tube. Embryos lacking Atp1a1 function exhibit abnormal migration behavior of cardiac precursors, defects in the elongation of the heart tube, and a severe reduction in ECM/Fibronectin deposition around the myocardium, despite the presence of normal cell polarity and junctions in the myocardial epithelium prior to the timeframe of heart tube elongation. Interestingly, we found that Atp1a1 is not present in the myocardium at the time when cardiac morphogenesis defects first become apparent, but is expressed in an extra-embryonic tissue, the yolk syncytial layer (YSL), at earlier stages. Knockdown of Atp1a1 activity specifically in the YSL using morpholino oligonucleotides produced heart tube elongation defects like those found in atp1a1 mutants, indicating that Atp1a1 function in the YSL is necessary for heart tube elongation. Furthermore, atp1a1 expression in the YSL was regulated by the homeobox transcription factor mxtx1. Together, these data reveal a new non-cell autonomous role for Atp1a1 in cardiac morphogenesis and establish Na(+),K(+) ATPase as a major player in the genetic pathway by which the YSL regulates embryonic ECM deposition.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-419
    Nombre del producto:
    Anti-Myc Tag Antibody, clone 9E10