Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ankyrin has emerged as a ubiquitous protein linking integral membrane transport proteins such as Na,K-ATPase to an underlying spectrin cytoskeleton. This interaction is mediated by the alpha subunit of Na,K-ATPase; however, the nature of the ankyrin binding site in Na,K-ATPase is unknown. As a step to determine the mechanism of this interaction, the ankyrin binding region of human erythrocyte spectrin and each of five putative cytoplasmic domains of the Na,K-ATPase alpha subunit have been prepared as recombinant fusion proteins in bacteria and analyzed for their interaction with erythrocyte and kidney ankyrin (Ank1 and Ank3, respectively) in vitro. Spectrin binds both Ank1 and Ank3 avidly, as expected. Two of the Na,K-ATPase domains, immobilized on a bioaffinity column, also interact specifically with both of these ankyrins. These ATPase domains are encoded by codons 140-290 (domain II) and 345-784 (domain III), with domain II displaying the greatest apparent affinity. Sequences in domain II are highly conserved between species and isoforms of Na,K-ATPase and are homologous to a cytoplasmic domain in H,K-ATPase and to a limited region of sequence in Ca-ATPase. Conversely, domain II shares no significant homology with other ankyrin binding proteins such as band 3 and Na(+)-channel proteins. These results identify a clear function for a conserved but previously not understood region of the alpha subunit of Na,K-ATPase and suggest that the interaction of ankyrin with membrane transport proteins may involve complex tertiary structural determinants not easily deduced from the primary sequence.
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PMID:Ankyrin binds to two distinct cytoplasmic domains of Na,K-ATPase alpha subunit. 815 88

We have analyzed the expression pattern of Na,K-ATPase alpha and beta subunit isoforms within the rodent and primate central nervous system. Membrane fractions prepared from rat cerebral cortical type-1 astrocytes and rat cerebellar granule and hippocampal neurons were characterized by immunoblot analyses using a panel of alpha and beta subunit isoform-specific antisera. Each cell type was found to express the alpha 1 isoform but showed differences in the expression of other subunits. Cortical astrocytes displayed alpha 2 and beta 2 subunits, whereas cerebellar granule neurons showed expression of alpha 3 and beta 1 subunits. All three alpha subunit isotypes were detected in hippocampal neurons. A survey of the immunofluorescent staining pattern of the alpha 3 subunit in rat and monkey brain confirmed that expression of this Na,K-ATPase alpha subunit isoform was restricted exclusively to neurons. These results suggest that both neurons and astrocytes express multiple, yet distinct, Na,K-ATPase isoenzymes. The identification of cell types expressing limited combinations of alpha and beta subunits should provide a framework for understanding the physiological significance of Na,K-ATPase isoenzyme diversity and may provide useful tools for the analysis of cell lineage in the mammalian central nervous system.
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PMID:Neurons and astroglia express distinct subsets of Na,K-ATPase alpha and beta subunits. 817 Mar 54

Site-directed mutagenesis of the Na,K-ATPase has provided a rational approach to identify several amino acids that appear to be involved in ouabain sensitivity. In order to identify additional amino acids that play a role in ouabain binding, we used formic acid to randomly mutagenize a cDNA cassette encoding amino acids 691-946 of the sheep Na,K-ATPase alpha subunit. This was then used to replace the wild type cDNA cassette in the full-length cDNA, and pools of mutants were electroporated into HeLa cells. Ouabain-resistant cells were selected in 0.5 microM ouabain, and the polymerase chain reaction was used to amplify the region corresponding to the mutagenized cassette from the genomic DNA of the resistant cells. Sequence analysis of the polymerase chain reaction product revealed a single amino acid substitution, T797N. Site-directed mutagenesis was used to reproduce this change in the wild type sheep alpha subunit, and this construct was able to confer resistance to HeLa cells, verifying the role of the mutation in ouabain resistance. The mutant sheep enzyme was found to be as resistant to ouabain as is the rat Na,K-ATPase. These data suggest that T797N, predicted to be in the fifth putative transmembrane domain, is involved in the interaction between ouabain and the Na,K-ATPase.
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PMID:Random mutagenesis of the sheep Na,K-ATPase alpha-1 subunit generates a novel T797N mutation that results in a ouabain-resistant enzyme. 824 98

The ion-transporting H,K-ATPase and Na,K-ATPase enzymes are each composed of an alpha and a beta subunit. It is known that assembly of the alpha and beta subunits of the Na,K-ATPase is necessary for the cell-surface delivery of the active enzyme. We have examined the molecular domains involved in the assembly of the H,K-ATPase and Na,K-ATPase alpha and beta subunits by expressing individual subunits and subunit chimeras in transiently transfected COS-1 cells. Our results demonstrate that the H,K-ATPase alpha subunit requires its beta subunit for efficient cell-surface expression, as determined by indirect immunofluorescence. The H,K-ATPase beta protein appears to be able to get to the cell surface unaccompanied by any alpha subunit and appears to localize as well to a population of intracellular vesicles. We find that a transfected chimera encoding the NH2-terminal half of the H,K-ATPase alpha subunit and the COOH-terminal half of the Na,K-ATPase alpha subunit appears to assemble with the endogenous Na,K-ATPase beta subunit and to reach the plasmalemma. Transfection of the complementary alpha chimera requires coexpression with the H,K-ATPase beta subunit in order to attain surface delivery. Thus, it is the COOH-terminal half of the alpha subunit that specifies assembly with a particular beta subunit.
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PMID:Molecular requirements for the cell-surface expression of multisubunit ion-transporting ATPases. Identification of protein domains that participate in Na,K-ATPase and H,K-ATPase subunit assembly. 839 Sep 91

In a highly ouabain-resistant clone from the Madin-Darby canine kidney cell line (Ki > 4 mM), we have previously identified mutations (C113 to Y or C113 to F) in the first transmembrane helix (H1) of the Na,K-ATPase alpha subunit that increase the Ki of a ouabain-sensitive Na,K pump by 1000-fold. Here we report another mutation (Y317 to C) located in the extracellular segment that joins the third and fourth transmembrane domains (H3-H4 ectodomain) of the alpha subunit that also changes ouabain sensitivity of the Na pump. When this mutation (Y317C) was introduced into the Na,K-ATPase alpha 1 subunit of Xenopus laevis, the ouabain inhibition constant increased by a factor of 5, from 130 (wild type) to 800 nM (mutant). However, the expression of double mutants (C113Y + Y317C) in Xenopus oocytes resulted in highly ouabain-resistant Na,K pumps (Ki approximately 7 mM), reproducing the phenotype of the original Madin-Darby canine kidney cell line. When a more conservative change (Y317F) was introduced into the Na,K-ATPase alpha 1 subunit of X. laevis, the ouabain koff increased and expression of double mutants (C113Y + Y317F) resulted in an intermediate ouabain-resistant Na,K pump (Ki approximately 500 microM). We propose that, in addition to the previously identified H1-H2 ectodomain of Na,K-ATPase alpha subunit, the H3-H4 ectodomain also participates in the structure and/or the function of the ouabain binding site. In this respect, the Y317 plays a critical role since the most conservative change Y313 to F is sufficient to significantly affect ouabain binding.
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PMID:Mutation of a tyrosine in the H3-H4 ectodomain of Na,K-ATPase alpha subunit confers ouabain resistance. 839 48

The intramembrane Glu781 residue of the Na,K-ATPase alpha subunit has been postulated to have a role in the binding and/or occlusion of cations. To ascertain the role of Glu781, the residue was substituted with an aspartate, alanine, or lysine residue and the mutant Na,K-ATPases were coexpressed with the native beta 1 subunit in Sf9 insect cells using the baculovirus expression system. All alpha mutants are able to efficiently assemble with the beta 1 subunit and produce catalytically competent Na,K-ATPase molecules with hydrolytic activities comparable to that of the wild-type enzyme. Analysis of the kinetic properties of the mutated enzymes showed a decrease in apparent affinity for K+ compared to wild-type Na,K-ATPase, with the lysine and alanine substitutions displaying the greatest reduction. All Na,K-ATPase mutants demonstrated a significant increase in apparent affinity for ATP compared to wild-type Na,K-ATPase, while the sensitivity to the cardiotonic inhibitor, ouabain, was unchanged. The dependence on Na+, however, differs among the mutant enzymes with both the Glu781-->Asp and Glu781-->Ala mutants displaying a decrease in the apparent affinity for the cation, while the Glu781-->Lys mutant exhibits a modest increase. Furthermore, in the absence of K+, the Glu781-->Ala mutant displays a Na(+)-ATPase activity and a cellular Na+ influx suggesting that Na+ is substituting for K+ at the extracellular binding sites. The observation that trypsin digestion of the Glu781-->Ala mutant in Na+ medium produces a K(+)-stabilized tryptic fragment also intimates a decreased capacity of the mutant to discriminate between Na+ and K+ at the extracellular loading sites. All together, these data implicate Glu781 of the Na,K-ATPase alpha subunit as an important coordinate of cation selectivity and activation, although the modest effect of Glu781-->Lys substitution seemingly precludes direct involvement of the residue in the cation binding process. In addition, the fifth membrane segment is proposed to represent an important communicative link between the extramembraneous ATP binding domain and the cation transport regions of the Na,K-ATPase.
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PMID:Substitutions of glutamate 781 in the Na,K-ATPase alpha subunit demonstrate reduced cation selectivity and an increased affinity for ATP. 857

Na,K-ATPase in lens epithelium plays a key role in conducting sodium-potassium transport. The purpose of this study was to test whether epithelium or fiber cells can synthesize new Na,K-ATPase protein in response to an increase of membrane permeability. Western blot methodology was used to identify Na,K-ATPase alpha subunit polypeptides in membrane material isolated from lens cells. As judged by immunoblot density, epithelial cell membrane material isolated from porcine lenses cultured 24 h with 1 microM amphotericin B contained more Na,K-ATPase alpha subunit polypeptide than epithelial material isolated from control lenses. This increase stemmed from the apparent synthesis of Na,K-ATPase alpha 2 isoform polypeptide by the epithelium; Na,K-ATPase alpha 1 isoform polypeptide abundance was not detectably altered. The apparent amphotericin B-induced expression of Na,K-ATPase alpha 2 was seen in lens epithelial cells but not fiber cells. This study suggests that the epithelium of the adult porcine lens may be capable of expressing additional sodium pump molecules of the alpha 2-subtype when membrane permeability is increased.
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PMID:Differential expression of sodium pump catalytic subunits in the lens epithelium and fibers. 872 71

The effects of changing Glu-779, located in the fifth transmembrane segment of the Na,K-ATPase alpha subunit, on the phosphorylation characteristics and ion transport properties of the enzyme were investigated. HeLa cells were transfected with cDNA coding the E779A substitution in an ouabain-resistant sheep alpha1 subunit (RD). Steady state phosphorylation stimulated by Na+ concentrations less than 20 mM or by imidazole were similar for RD and E779A enzymes, an indication that phosphorylation and Na+ occlusion were not altered by this mutation. With E779A enzyme, higher Na+ concentrations reduced the level of phosphoenzyme and stimulated Na-ATPase activity in the absence of K+. These effects were a consequence of Na+ increasing the rate of protein dephosphorylation. In voltage-clamped HeLa cells expressing E779A enzyme, a prominent electrogenic Na+-Na+ exchange was observed in the absence of extracellular K+. Thus, increased Na-ATPase activity and Na+-dependent dephosphorylation result from Na+ acting as a K+ congener with low affinity at extracellular binding sites. These data suggest that E779A does not directly participate in ion binding but does affect the connection between extracellular ion binding and intracellular enzyme dephosphorylation. In cells expressing control RD enzyme, Na,K-pump current was dependent on membrane potential and extracellular K+ concentration. However, Na,K-pump current in cells expressing E779A enzyme was voltage independent at all extracellular K+ tested. These results indicate that Glu-779 may be part of the access channel determining the voltage dependence of ion transport by the Na, K-ATPase.
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PMID:Substitution of glutamic 779 with alanine in the Na,K-ATPase alpha subunit removes voltage dependence of ion transport. 879 26

The functional roles of Asp804 and Asp808, located in the sixth transmembrane segment of the Na,K-ATPase alpha subunit, were examined. Nonconservative replacement of these residues yielded enzymes unable to support cell viability. Only the conservative substitution, Ala808 --> Glu, was able to maintain the essential cation gradients (Van Huysse, J. W., Kuntzweiler, T. A., and Lingrel, J. B (1996) FEBS Lett. 389, 179-185). Asp804 and Asp808 were replaced by Ala, Asn, and Glu in the sheep alpha1 subunit and expressed in a mouse cell line where [3H]ouabain binding was utilized to probe the exogenous proteins. All of the heterologous proteins were targeted into the plasma membrane, bound ouabain and nucleotides, and adopted E1Na, E1ATP, and E2P conformations. K+ competition of ouabain binding to sheep alpha1 and Asp808 --> Glu enzymes displayed IC50 values of 4.11 mM (nHill = 1.4) and 23.8 mM (nHill = 1.6), respectively. All other substituted proteins lacked this K+-ouabain antagonism, e.g. 150 mM KCl did not inhibit ouabain binding. Na+ antagonized ouabain binding to all the expressed isoforms, however, the proteins carrying nonconservative substitutions displayed reduced Hill coefficients (nHill </= 2.0) compared to the control (nHill </= 2.8). Therefore, Asp804 and Asp808 of the Na,K-ATPase are required for normal Na+ and K+ transport, possibly coordinating these cations during transport.
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PMID:Asp804 and Asp808 in the transmembrane domain of the Na,K-ATPase alpha subunit are cation coordinating residues. 893 1

Na,K-ATPase, an essential transporter of mammalian cells, is an oligomeric transmembrane protein composed of two subunits, alpha and beta, of which there are several isoforms. In this study, we demonstrate that the alpha1 and alpha2 isoforms of the Na,K-ATPase alpha subunit are modified by the covalent attachment of ubiquitin polymers in COS-7 cells. We propose that polyubiquitination of the Na,K-ATPase alpha subunit may play a role in regulating its degradation.
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PMID:Ubiquitination of Na,K-ATPase alpha1 and alpha2 subunits. 910 5


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